WO2026064512A1

WO2026064512A1 - Polyglycerol-conjugated lipids and lipid nanoparticle compositions comprising the same

Info

Publication number: WO2026064512A1
Application number: PCT/US2025/046973
Authority: WO
Inventors: Matthew G. Stanton; Douglas A. ROSE; Sachit SHAH; Viktoriya SYROVATKINA; Luke S. HAMM; Julie ROSENFELD
Original assignee: Generation Bio Co
Current assignee: Generation Bio Co
Priority date: 2024-09-18
Filing date: 2025-09-18
Publication date: 2026-03-26
Anticipated expiration: 2027-03-18

Abstract

The present disclosure provides novel polymer-conjugated lipids conjugated to a polyglycerol or a polyglycerol derivative. The present disclosure also provides lipid nanoparticles (LNPs) formulation using the polymer-conjugated lipids and methods of treating a disease by administering the LNP formulations, including multiple doses of the LNP formulations.

Description

131698-30520

POLYGLYCEROL-CONJUGATED LIPIDS AND LIPID NANOPARTICLE

COMPOSITIONS COMPRISING THE SAME

RELATED APPLICATIONS

The instant application claims priority to U.S. Provisional Application No. 63/696,269, filed on September 18, 2024; U.S. Provisional Application No. 63/758,723, filed on February 14, 2025; U.S. Provisional Application No. 63/805,093, filed on May 13, 2025; U.S. Provisional Application No. 63/836,262, filed on June 30, 2025; and 63/861,836, filed on August 11, 2025; and is a continuation-in-part of PCT Application No. PCT/US2024/052650, filed October 23, 2024. The entire contents of each of the foregoing applications are expressly incorporated herein by reference in their entireties.

TECHNICAL FIELD

The present disclosure relates to the field of gene and nucleic acid therapy, including polymer-conjugated lipids conjugated to a polyglycerol or a polyglycerol derivative and methods of treating a disease by administering the LNP formulations, including multiple doses of the LNP formulations.

BACKGROUND

Lipid-based nanoparticles have played a pivotal role in the successes of COVID- 19 vaccines and many other nanomedicines, such as DOXIL® and ONPATTTRO®, and have therefore been considered as a frontrunner among nanoscale drug delivery systems. However, effective targeted delivery of biologically active substances, such as therapeutic nucleic acids, represents a continuing medical challenge. This has severely limited broad applications of nucleic acids such as mRNA and DNA in protein replacement therapy, gene therapy, gene editing, and vaccination.

Lack of effective methods and vehicles for intracellular delivery represents a major barrier to a broad use of nucleic acid therapeutics. Generally, intracellular delivery of mRNA or DNA is more challenging than intracellular delivery of small oligonucleotides, in part due to the fact that mRNA and DNA molecules (which typically range from 300 kDa to 5,000 kDa, or ~ 1-15 kb) are significantly larger than other types of RNAs, such as small interfering RNAs (siRNA, which are typically ~14 kDa) or antisense oligonucleotides (ASOs), which typically range from 4 kDa to 10 kDa).

Furthermore, intracellular delivery of nucleic acid therapeutics to targeted cells is hindered by the activation of the innate and/or adaptive immune responses. Whereas it is possible to avoid RNA sensing by myeloid dendritic cells (MDCs) by chemically modifying RNA cargo (e.g., with Im . 2’OMe, ete.), there are no known chemical modifications to a DNA cargo that can limit pattern recognition receptor (PRR) sensing and still maintain transcriptional activity.

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MEl\57916143.vl 131698-30520

An alternative approach to gene therapy is the recombinant adeno-associated virus (rAAV) vector platform that packages heterologous DNA in a viral capsid. However, there are several major disadvantages to using rAAV vectors as a gene delivery vector. One major drawback associated with rAAV is its limited viral packaging capacity of about 4.5 kb of heterologous DNA. Another major drawback is capsid immunogenicity that prevents re-administration to patients.

Thus, there remains a need for effective delivery vehicles that enable safe and effective delivery of nucleic acid therapeutics to desired cell populations.

SUMMARY

The present disclosure overcomes some of the obstacles to intracellular delivery of nucleic acids (e.g., therapeutic nucleic acids). It has been surprisingly discovered that the novel LNPs disclosed herein comprising polymer-conjugated lipids are characterized by low levels of undesirable opsonization-driven uptake of LNPs into non-target cells, and are balanced with desirable levels of endosomal escape, thereby achieving advantageous stealth/endosomal escape tradeoff, as described herein. Further, the novel LNPs described herein can surprisingly be administered in multiple doses without inducing antibody-mediated clearance of the LNPs from the blood. Thus, the present disclosure provides extended half-life LNPs with a prolonged duration of action, e.g. due to a prolonged half-life in circulation, which advantageously allows for dosing at longer intervals. It is a finding of the present disclosure that repeat dosing of LNPs comprising a polyglycerol (PG) or a PG derivative conjugated lipid maintained an extended blood circulation profile (e.g., increased blood ti/2) and were not rapidly cleared when compared to LNPs comprising a polyethylene glycol (PEG) conjugated lipid.

Accordingly, in some aspects, the present disclosure provides a method of treating a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject at least a first dose and at least a second dose of an effective amount of a lipid nanoparticle (LNP) comprising a therapeutic nucleic acid (TNA), a first lipid-anchored polymer, and a second lipid-anchored polymer, wherein the first lipid-anchored polymer comprises: (i) a first polymer, wherein the first polymer comprises a first polyglycerol (PG) or PG derivative; (ii) a first lipid moiety; and (iii) an optional first linker conjugating the first PG or PG derivative to the first lipid moiety, and wherein the second lipid- anchored polymer comprises: (i) a second polymer; (ii) a second lipid moiety; (iii) an optional second linker, wherein the second polymer is conjugated to the second lipid moiety via the second linker; and (iv) a reactive species conjugated to the second polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety.

In some aspects, the present disclosure provides a method of inhibiting gene expression in an immune cell, the method comprising: administering to a subject at least a first dose and at least a second dose of an effective amount of a lipid nanoparticle (LNP) comprising a therapeutic nucleic acid (TNA), a first lipid-anchored polymer, and a second lipid-anchored polymer, wherein the first

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MEl\57916143.vl 131698-30520 lipid-anchored polymer comprises: (i) a first polymer, wherein the first polymer comprises a first polyglycerol (PG) or PG derivative; (ii) a first lipid moiety; and (iii) an optional first linker conjugating the first PG or PG derivative to the first lipid moiety, and wherein the second lipid- anchored polymer comprises: (i) a second polymer; (ii) a second lipid moiety; (iii) an optional second linker, wherein the second polymer is conjugated to the second lipid moiety via the second linker; and (iv) a reactive species conjugated to the second polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety, thereby inhibiting expression of the gene in the immune cell.

In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a naive T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is an autologous T cell. In some embodiments, the T cell is an allogeneic T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the gene is a target gene selected from a target gene in Table 27.

In some embodiments, the gene expression is inhibited by at least about 50%, about 60%, 70%, about 80%, about 90%, about 95%, about 98%, or about 100%. In some embodiments, the cell is in a subject. In some embodiments, the subject is a human. In some embodiments, the subject is suffering from an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is selected from the group consisting of rheumatoid arthritis juvenile idiopathic arthritis, autoimmune hepatitis, sarcoidosis, giant cell arteritis, Sjogren’s syndrome, systemic sclerosis, systemic lupus erythematosus, inflammatory bowel disease, primary biliary cirrhosis, dermatomyositis, multiple sclerosis, type I diabetes, psoriasis, psoriatic arthritis, Crohn’s disease, Celiac disease, ulcerative colitis, Graves’ disease, Hashimoto’s thyroiditis, Addison’s disease, dermatomyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome, myasthenia gravis, autoimmune vasculitis, pernicious anemia, vitiligo, systemic sclerosis, scleroderma, diffuse scleroderma, limited scleroderma, linear scleroderma, localized scleroderma, hemolytic anemia, achantholysis, acute disseminated encephalomyelitis, adult-onset Still disease, allergic glomerulonephritis, ANCA associated vasculitis, ankylosing spondylitis, anti-glomerular basement membrane disease, anti-N-methyl-D-aspartate receptor encephalitis, antiphospholipid syndrome, antisynthetase syndrome, autoimmune gastritis, autoimmune hypophysitis, autoimmune liver disease, autoimmune lung disease, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, autoimmune skin disease, autoimmune thyroiditis, autoimmune uveitis, Behcet disease, bullous pemphigoid, Churg Strauss syndrome, dermatitis herpetiformis, endocrine ophthalmopathy, erythematous pemphigus, Felty syndrome, Giant cell arteritis, IgA glomerulonephritis, membranous glomerulonephritis, Goodpasture syndrome, Granulomatosis with polyangiitis, Graves’ ophthalmopathy, Hailey syndrome, heparin induced thrombocytopenia, autoimmune hepatitis, idiopathic hypoparathryroidism,

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MEl\57916143.vl 131698-30520 idiopathic thrombocytopenic purpura, IgA pemphigus, immune complex nephritis, immunoglobulin A nephropathy, juvenile rheumatoid arthritis, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, linear IgA bullous dermatosis, lupus nephritis, lupus vasculitis, microscopic polyangiitis, morphea, mucous membrane phemphigoid, myasthenia gravis, nonarticular rheumatism, sympathetic ophthalmia, opsoclonous myoclonus syndrome, paraneoplastic pemphigus, pemphigoid, pemphigoid gestationis, pemphigus, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, autoimmune polyendocrinopathies, polyradiculoneuropathy, postpartum thyroiditis, rheumatic disease, rheumatic heart disease, rheumatoid polymyalgia, rheumatoid nodule, rheumatoid vasculitis, stiff-person syndrome, CREST syndrome, sarcoidosis, and uveomeningoencephalitic syndrome. In some embodiments, the autoimmune disease or disorder is selected from the group consisting of: autoimmune hepatitis juvenile idiopathic arthritis, sarcoidosis, giant cell arteritis, Sjogren’s Syndrome, systemic sclerosis, systemic lupus erythematosus, inflammatory bowel disease, primary biliary cirrhosis, and dermatomyositis. In some embodiments, the juvenile idiopathic arthritis (JIA) is systemic onset JIA, oligoarticular JIA, polyarticular JIA, enthesitis-related JIA, or psoriatic arthritis. In some embodiments, the cell is in vitro, ex vivo, or in vivo.

In some aspects, the present disclosure provides a method of modulating T cell activation or activity, the method comprising: administering to the subject at least a first dose and at least a second dose of an effective amount of a lipid nanoparticle (LNP) comprising a therapeutic nucleic acid (TNA), a first lipid-anchored polymer, and a second lipid-anchored polymer, wherein the first lipid- anchored polymer comprises: (i) a first polymer, wherein the first polymer comprises a first polyglycerol (PG) or PG derivative; (ii) a first lipid moiety; and (iii) an optional first linker conjugating the first PG or PG derivative to the first lipid moiety, and wherein the second lipid- anchored polymer comprises: (i) a second polymer; (ii) a second lipid moiety; (iii) an optional second linker, wherein the second polymer is conjugated to the second lipid moiety via the second linker; and (iv) a reactive species conjugated to the second polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety., thereby inhibiting expression of the gene in the immune cell, thereby modulating T cell activation or activity.

In some embodiments, the decrease is by at least 50%, at least 60%, at least 70% or at least 80% at day 15 compared to a T cell contacted with a control TNA.

In some embodiments, the method further comprises administering to the subject at least a third dose of an effective amount of the LNP. In some embodiments, the method further comprises administering to the subject at least a fourth, fifth, sixth, seventh, eighth, ninth, tenth, or subsequent dose of an effective amount of the LNP. In some embodiments, each dose is formulated as a pharmaceutical composition comprising the LNP and a pharmaceutically effective carrier.

In some embodiments, the second polymer is a second PG or PG derivative. In some embodiments, the first PG derivative is a carboxylated PG. In some embodiments, the carboxylated PG is a glutarylated PG. In some embodiments, the glutarylated PG is 3 -methyl glutarylated PG. In

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MEl\57916143.vl 131698-30520 some embodiments, the carboxylated PG is 2-carboxycyclohexane-l-carboxylated PG. In some embodiments, the first PG or PG derivative is linear or branched.

In some embodiments, the first lipid moiety is represented by Formula (I): or a pharmaceutically acceptable salt thereof, wherein: R¹ is absent, hydrogen, Ci-Ce alkyl, or a hydrophobic tail comprising 10-30 carbon atoms; R² is absent, hydrogen, Ci-Ce alkyl, or a hydrophobic tail comprising 10-30 carbon atoms; wherein, when R¹ and R² are each hydrogen, Ci-Ce alkyl, or a hydrophobic tail comprising 10-30 carbon atoms, N is positively charged; and R³ is a hydrophobic tail comprising 10-30 carbon atoms, wherein ‘'wv in Formula (I) is a bond conjugating the lipid moiety and the linker, when present. In some embodiments, R¹ is absent, and wherein R² and R³ are each independently a hydrophobic tail comprising 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In some embodiments, R² and R³ are each independently a hydrophobic tail comprising 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In some embodiments, R² and R³ are each independently a hydrophobic tail comprising 18 carbon atoms, and wherein the lipid moiety is dioctadecylamine (DODA).

In some embodiments, the first lipid moiety conjugated to the first linker is represented by the following structure:

In some embodiments, the first lipid moiety is selected from the group consisting of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 -palmitoyl -2 -oleoyl -glycero-3 -phosphocholine (POPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), 1 -palmitoyl -2 -oleoyl-sn- glycero-3-phospho-(l'-rac-glycerol) (POPG), l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), l,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn- phosphatidylethanolamine (DEPE), 1 -stearoyl -2 -oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE), l,2-dioleoyl-sn-glycero-3 -phosphoglycerol (DOPG), l,2-dipalmitoyl-sn-glycero-3 -phosphoglycerol (DPPG), 18-1-trans PE, l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn- glycero-3 -phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), dihexadecylamine, distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-O-dioctadecyl-sn-glycerol (DODG), and a derivative of any of the foregoing. In some embodiments, the first lipid moiety is selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, DODG, and a derivative of any of the foregoing.

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MEl\57916143.vl 131698-30520

In some embodiments, the first PG or PG derivative comprises an average of about 5-100 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 30-80 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 5-100, about 10-100, about 15-100, about 20-100, about 25-100, about 27-100, about 30-100, about 34-100, about 35-100, about 39-100, about 40-100, about 45-100, about 46-100, about 50-100, about 55-100, about 58-100, about 60-100, about 65-100, about 68-100, about 70-100, about 75-100, about 80-100, about 85-100, about 90-100, or about 95-100 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of at least about 20, at least about 25, at least about 27, at least about 30, at least about 34, at least about 35, at least about 39, at least about 40, at least about 45, at least about 46, at least about 50, at least about 55, at least about 58, at least about 60, at least about 65, at least about 68, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35- 40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 5-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 5-25, 25-50, 50-75, or 75- 100 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,

32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,

59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 8, 27, 34, 39, 45, 46, 50, 58, or 68 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 8 monomer units. In some embodiments, the first PG or PG derivative comprises an average of about 27 monomer units. In some embodiments, the first PG or PG derivative comprises an average of about 34 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 39 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 45 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 46 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 50 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 58 monomeric units. In some embodiments, the first PG or PG derivative comprises an average of about 68 monomeric units.

In some embodiments, the first linker is absent. In some embodiments, the first linker, when present, is selected from the group consisting of an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker, an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a

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MEl\57916143.vl 131698-30520 carbamate linker, a diamide alkyl linker, a cleavable linker, and any combination thereof. In some embodiments, the first linker, when present, is selected from the group consisting of -(CH2)_n-, - C(O)(CH₂)n-, -C(O)O(CH₂)n, -OC(O)(CH₂)nC(O)O-, and -NH(CH₂)_nC(O)O-, wherein n is an integer ranging from 1 to 20. In some embodiments, the first linker, when present, is a glutaryl linker or a succinyl linker. In some embodiments, the first linker, when present, is -C(O)(CH₂)_n-, and wherein n is 2, 3, 4, 5, or 6. In some embodiments, n is 4.

In some embodiments, the first lipid-anchored polymer is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

In some embodiments, the first lipid-anchored polymer and/or the second lipid-anchored polymer are represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

In some embodiments, the first lipid-anchored polymer and/or the second lipid-anchored polymer are represented by the following structure:

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MEl\57916143.vl 131698-30520 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

In some embodiments, the reactive species is a click chemistry reagent, maleimide, or thiol. In some embodiments, the reactive species is maleimide or thiol. In some embodiments, the click chemistry reagent is selected from the group consisting of a dibenzocyclooctyne (DBCO) reagent, a transcylooctene (TCO) reagent, a tetrazine (Tz) reagent, an alkyne reagent, and an azide reagent.

In some embodiments, the LNP further comprises a targeting moiety conjugated to the second polymer derivative via the reactive species. In some embodiments, the targeting moiety is conjugated to the second polymer via a dibenzocyclooctyne (DBCO)-azide conjugation, an azide-alkyne conjugation, a TCO-Tz conjugation, or a thiol-maleimide conjugation. In some embodiments, the targeting moiety is conjugated to the second polymer via a thiol-maleimide conjugation. In some embodiments, the targeting moiety is capable of binding to a liver cell. In some embodiments, the liver cell is a hepatocyte. In some embodiments, the targeting moiety is N-acetyl galactosamine

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MEl\57916143.vl 131698-30520

(GalNAc) or a GalNAc derivative. In some embodiments, the targeting moiety is a tri-antennary GalNAc conjugate or a tetra-antennary GalNAc conjugate. In some embodiments, the targeting moiety is selected from the group consisting of an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, and a fragment or a variant thereof. In some embodiments, the targeting moiety is an antibody or an antibody fragment. In some embodiments, the antibody or the antibody fragment is capable of specifically binding an antigen present on the surface of a cell. In some embodiments, the antibody or the antibody fragment is a monoclonal antibody (mAb), a single chain variable fragment (scF_v), a heavy chain antibody (hcAb), a nanobody (Nb), a heavy-chain-only immunoglobulin (HCIg), an immunoglobulin new antigen receptor (IgNAR), variable domain of immunoglobulin new antigen receptor (VNAR), a single-domain antibody, or a variable heavy chain- only antibody (VHH). In some embodiments, the antibody or antibody fragment is a VHH. In some embodiments, the antibody or antibody fragment is an scF_v.

In some embodiments, the second lipid-anchored polymer is represented by the following structure: wherein n is 65, 66, 67, 68, 69, or 70; or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof. In some embodiments, n is 68.

In some embodiments, the targeting moiety is capable of binding to an antigen on a cell or cell fragment selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a macrophage, a red blood cell, a platelet, a megakaryocyte, a hematopoietic stem cell (HSC), a hematopoietic stem or progenitor cell (HSPC), a CD34+ cell, a muscle cell, a brain cell, a nerve cell, a lung cell, an endothelial cell, an epithelial cell, a kidney cell, a spleen cell, an ovarian cell, a testicular cell, a uterine cell, a placental cell, a vascular cell, a skin cell, and an endocrine cell. In some embodiments, the cell is a T cell. In some embodiments, the T cell is a naive T cell. In some embodiments, the T cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the targeting moiety binds to an antigen expressed on a T cell selected from the group consisting of a CD4+ T cellspecific antigen, a CD8+ T cell-specific antigen, and a CD3+ T cell-specific antigen. In some embodiments, the targeting moiety binds to an antigen expressed on a T cell selected from the group consisting of CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, PD-1, and TCR. In some embodiments, the targeting moiety binds to CD7. In some embodiments, the cell is an NK cell. In some embodiments, the cell is a hematopoietic stem cell (HSC), a hematopoietic stem or progenitor cell (HSPC), or a CD34+ cell.

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In some embodiments, the LNP further comprises: (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid; and (iii) a sterol.

In some embodiments, the LNP further comprises a helper lipid. In some embodiments, the helper lipid comprises a phospholipid. In some embodiments, the helper lipid is selected from the group consisting of l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), hydrogenated soybean PC

(HSPC), phosphatidylserine (PS), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phosphocholine (POPC), l,2-dilauroyl-sn-glycero-3 -phosphocholine (DLPC), l-margaroyl-2-oleoyl-sn-glycero-3- phosphocholine (MOPC), 1 -palmitoyl -2 -linoleoyl-sn-glycero-3 -phosphocholine (PLPC), 1 -stearoyl -2- myristoyl-sn-glycero-3-phosphocholine (SMPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine

(DMPC), l,2-dihexanoyl-sn-glycero-3 -phosphocholine (DHPC), l,2-dierucoyl-sn-glycero-3- phosphocholine (DEPC), 1 -palmitoyl -2 -oleoyl -glycero-3 -phosphocholine (POPC), and 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the helper lipid is DSPC. In some embodiments, the helper lipid is represented by Formula (II): or a pharmaceutically acceptable salt or an ester thereof, wherein: '' is a single bond or a double bond; R¹ is C1-C17 alkyl or C2-C17 alkenyl; R² is C1-C22 alkyl or C2-C22 alkenyl; R³ is hydrogen or C1-C2 alkyl; and R⁴ is hydrogen or C1-C2 alkyl. In some embodiments, '' is a double bond. In some embodiments, R¹ is C10-C20 alkenyl, R² is C10-C20 alkyl and R³ is hydrogen.

In some embodiments, the helper lipid represented by Formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

In some embodiments, the helper lipid represented by Formula (II) is:

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In some embodiments, the helper lipid represented by Formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof. In some embodiments, the helper lipid represented by Formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

In some embodiments, the helper lipid represented by Formula (II) is:

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In some embodiments, the sterol is selected from the group consisting of cholesterol, betasitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and a derivative thereof. In some embodiments, the sterol is cholesterol.

In some embodiments, the ionizable lipid is represented by: a) Formula (A):

Formula (A), or a pharmaceutically acceptable salt thereof, wherein: R¹ and R¹ are each independently optionally substituted linear or branched C1-3 alkylene; R² and R² are each independently optionally substituted linear or branched Ci-e alkylene; R³ and R³ are each independently optionally substituted linear or branched Ci-e alkyl; or alternatively, when R² is optionally substituted branched C1-6 alkylene, R² and R³, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R² is optionally substituted branched Ci-e alkylene, R² and R³ , taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; R⁴ and R⁴ are each independently -CR^a, -C(R^a)2CR^a, or -[C(R^a)2]2CR^a; R^a, for each occurrence, is independently H or C1-3 alkyl; or alternatively, when R⁴is -C(R^a)2CR^a, or -[C(R^a)2]2CR^a and when R^a is C1-3 alkyl, R³ and R⁴, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R⁴ is -C(R^a)2CR^a, or -[C(R^a)2]2CR^a and when R^a is C1-3 alkyl, R³ and R⁴ , taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; R⁵ and R⁵ are each independently hydrogen, C1-20 alkylene or C2-20 alkenylene; R⁶ and R⁶ , for each occurrence, are independently C1-20 alkylene, C3-20 cycloalkylene, or C2-20 alkenylene; and m and n are each independently an integer selected from 1, 2, 3, 4, and 5; b) Formula (B):

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MEl\57916143.vl 131698-30520

Formula (B), or a pharmaceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10; R¹ is absent or is selected from (C2-C2o)alkenyl, -C(0)0(C2-C2o)alkyl, and cyclopropyl substituted with (C2-C2o)alkyl; and R² is (C2-C2o)alkyl; c) Formula (C):

Formula (C), or a pharmaceutically acceptable salt thereof, wherein: R¹ and R¹ are each independently (Ci- Ce)alkylene optionally substituted with one or more groups selected from R^a; R² and R² are each independently (Ci-C2)alkylene; R³ and R³ are each independently (Ci-Ce)alkyl optionally substituted with one or more groups selected from R^b; or alternatively, R² and R³ and/or R² and R³ are taken together with their intervening N atom to form a 4- to 7-membered heterocyclyl; R⁴ and R⁴’ are each a (C2-Ce)alkylene interrupted by -C(O)O-; R⁵ and R⁵’ are each independently a (C2-C3o)alkyl or (C2- C3o)alkenyl, each of which are optionally interrupted with -C(O)O- or (C3-Ce)cycloalkyl; and R^a and R^b are each halo or cyano; d) Formula (D):

Formula (D), or a pharmaceutically acceptable salt thereof, wherein: R’ is absent, hydrogen, or Ci-Ce alkyl; provided that when R’ is hydrogen or Ci-Ce alkyl, the nitrogen atom to which R’, R¹, and R² are all positively charged; R¹ and R² are each independently hydrogen, Ci-Ce alkyl, or C2-C6 alkenyl; R³ is C1-C12 alkylene or C2-C12 alkenylene; R⁴ is Ci-Cis unbranched alkyl, C2-C18 unbranched alkenyl, or

13

MEl\57916143.vl 131698-30520 wherein: R^4a and R^4b are each independently Ci-Cie unbranched alkyl or C2-C16 unbranched alkenyl; R⁵ is absent, Ci-Cs alkylene, or C2-C8 alkenylene; R^6a and R^6b are each independently C7-C16 alkyl or C7-C16 alkenyl; provided that the total number of carbon atoms in R^6a and R^6b as combined is greater than 15; X¹ and X² are each independently -OC(=O)-, -SC(=O)-, - OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(R^a)=N-, -N=C(R^a)-, -C(R^a)=NO-, -O-N=C(R^a)-, - C(=O)NR^a-, -NR^aC(=O)-, -NR^aC(=O)NR^a-, -OC(=O)O-, -OSi(R^a)₂O-, -C(=O)(CR^a ₂)C(=O)O-, or OC(=O)(CR^a2)C(=O)-; wherein: R^a, for each occurrence, is independently hydrogen or Ci-Ce alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6; e) Formula (E):

Formula (E), or a pharmaceutically acceptable salt thereof, wherein: R’ is absent, hydrogen, or C1-C3 alkyl; provided that when R’ is hydrogen or C1-C3 alkyl, the nitrogen atom to which R’, R¹, and R² are all attached is positively charged; R¹ and R² are each independently hydrogen or C1-C3 alkyl; R³ is C3-C10 alkylene or C3-C10 alkenylene; R⁴ is C1-C16 unbranched alkyl, C2-C16 unbranched alkenyl, or

R^4a and R^4b are each independently Ci-Cie unbranched alkyl or C2-C16 unbranched alkenyl; R⁵ is absent, Ci-Ce alkylene, or C2-C6 alkenylene; R^6a and R^6b are each independently C7-C14 alkyl or C7- C14 alkenyl; Xis -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(R^a)=N-, -N=C(R^a)-, -C(R^a)=NO-, -O-N=C(R^a)-, -C(=O)NR^a-, -NR^aC(=O)-, -NR^aC(=O)NR^a-, -OC(=O)O-, -OSi(R^a)₂O-, -C(=O)(CR^a)₂C(=O)O-, or OC(=O)(CR^a ₂)C(=O)-; wherein: R^a, for each occurrence, is independently hydrogen or Ci-Ce alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6; or f) an ionizable lipid selected from the group consisting of: any of the ionizable lipids in Table 1, 4, 5, 6, or 7.

In some embodiments, the ionizable lipid is Lipid 87, represented by the following structure:

In some embodiments, the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

In some embodiments, the ionizable lipid is represented by the following structure:

In some embodiments, the ionizable lipid is Lipid A, represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

In some embodiments, the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof. In some embodiments, the first lipid-anchored polymer and the second lipid-anchored polymer are the same. In some embodiments, the first lipid-anchored polymer and the second lipid- anchored polymer are different.

17

MEl\57916143.vl 131698-30520

In some embodiments, the second PG derivative is a carboxylated PG. In some embodiments, the carboxylated PG is a glutarylated PG or 2 -carboxycyclohexane- 1 -carboxylated PG. In some embodiments, the glutarylated PG is 3-methyl glutarylated PG. In some embodiments, the second PG or PG derivative is linear or branched. In some embodiments, the second PG or PG derivative comprises an average of about 5-100 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 30-80 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 5-100, about 10-100, about 15-100, about 20-100, about 25-100, about 27-100, about 30-100, about 34-100, about 35-100, about 39-100, about 40-100, about 45-100, about 46-100, about 50-100, about 55-100, about 58-100, about 60-100, about 65-100, about 68-100, about 70-100, about 75-100, about 80-100, about 85-100, about 90-100, or about 95- 100 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of at least about 20, at least about 25, at least about 27, at least about 30, at least about 34, at least about 35, at least about 39, at least about 40, at least about 45, at least about 46, at least about 50, at least about 55, at least about 58, at least about 60, at least about 65, at least about 68, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70- 75, 75-80, 80-85, 85-90, 90-95, or 95-100 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 5-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 5-25, 25-50, 50-75, or 75-100 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,

47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,

74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or

100 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 8, 27, 34, 39, 45, 46, 50, 58, or 68 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 8 monomer units. In some embodiments, the second PG or PG derivative comprises an average of about 27 monomer units. In some embodiments, the second PG or PG derivative comprises an average of about 34 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 39 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 45 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 46 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 50 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 58 monomeric units. In some embodiments, the second PG or PG derivative comprises an average of about 68 monomeric units. In some embodiments, the first PG or PG derivative comprises

18

MEl\57916143.vl 131698-30520 an average of about 39 monomeric units, and the second PG or PG derivative comprises an average of about 68 monomeric units.

In some embodiments, the LNP further comprises a targeting moiety conjugated to the second PG or PG derivative via the reactive species. In some embodiments, the targeting moiety is conjugated to the second PG or PG derivative via a dibenzocyclooctyne (DBCO)-azide conjugation, an azide-alkyne conjugation, a TCO-Tz conjugation, or a thiol-maleimide conjugation. In some embodiments, the targeting moiety is conjugated to the second PG or PG derivative via a thiol- maleimide conjugation. In some embodiments, the targeting moiety is capable of binding to a liver cell. In some embodiments, the liver cell is a hepatocyte. In some embodiments, the targeting moiety is N-acetyl galactosamine (GalNAc) or a GalNAc derivative. In some embodiments, the targeting moiety is a tri-antennary GalNAc conjugate or a tetra-antennary GalNAc conjugate. In some embodiments, the targeting moiety is selected from the group consisting of an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, and a fragment or a variant thereof. In some embodiments, the targeting moiety is an antibody or an antibody fragment. In some embodiments, the antibody or the antibody fragment is capable of specifically binding an antigen present on the surface of a cell. In some embodiments, the antigen on the surface of the cell is a T cell antigen. In some embodiments, the cell is positive for the cell surface marker CD7+. In some embodiments, the antibody or the antibody fragment is a monoclonal antibody (mAb), a single chain variable fragment (scF_v), a heavy chain antibody (hcAb), a nanobody (Nb), a heavy-chain-only immunoglobulin (HCIg), an immunoglobulin new antigen receptor (IgNAR), variable domain of immunoglobulin new antigen receptor (VNAR), a single-domain antibody, or a variable heavy chain-only antibody (VHH). In some embodiments, the antibody or antibody fragment is a VHH. In some embodiments, the antibody or antibody fragment is an scF_v.

In some embodiments, the targeting moiety is capable of binding to an antigen on a cell or cell fragment selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a macrophage, a red blood cell, a platelet, a megakaryocyte, a hematopoietic stem cell (HSC), a hematopoietic stem or progenitor cell (HSPC), a CD34+ cell, a muscle cell, a brain cell, a nerve cell, a lung cell, an endothelial cell, an epithelial cell, a kidney cell, a spleen cell, an ovarian cell, a testicular cell, a uterine cell, a placental cell, a vascular cell, a skin cell, and an endocrine cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a naive T cell. In some embodiments, the T cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a NK cell. In some embodiments, the cell is a hematopoietic stem cell (HSC), a hematopoietic stem or progenitor cell (HSPC), or a CD34+ cell.

In some embodiments, the first linker and the second linker are the same or are both absent. In some embodiments, the first linker and the second linker are different, wherein the first linker is absent and the second linker is present, or wherein the first linker is present and the second linker is absent. In some embodiments, the second linker, when present, is selected from the group consisting

19

MEl\57916143.vl 131698-30520 of an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker, an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, and any combination thereof. In some embodiments, the second linker, when present, is selected from the group consisting of -(CH2)_n-, -C(O)(CH2)_n-, -C(O)O(CH2)_n-, - OC(O)(CH2)_nC(O)O-, and -NH(CH2)_nC(O)O-, wherein n is an integer ranging from 1 to 20. In some embodiments, the second linker, when present, is a glutaryl linker or a succinyl linker. In some embodiments, the second linker, when present, is -C145O)(CH₂)n-, and wherein n is 2, 3, 4, 5, or 6. In some embodiments, n is 4.

In some embodiments, the second lipid moiety is represented by Formula (I) or a pharmaceutically acceptable salt thereof, wherein: R¹ is absent, hydrogen, Ci-Ce alkyl, or a hydrophobic tail comprising 10-30 carbon atoms; R² is absent, hydrogen, Ci-Ce alkyl, or a hydrophobic tail comprising 10-30 carbon atoms; wherein, when R¹ and R² are each hydrogen, Ci-Ce alkyl, or a hydrophobic tail comprising 10-30 carbon atoms, N is positively charged; and R³ is a hydrophobic tail comprising 10-30 carbon atoms, wherein i_n Formula (I) is a bond conjugating the lipid moiety, when present, and the linker. In some embodiments, R¹ is absent, and wherein R² and R³ are each independently a hydrophobic tail comprising 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In some embodiments, R² and R³ are each independently a hydrophobic tail comprising 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In some embodiments, R² and R³ are each independently a hydrophobic tail comprising 18 carbon atoms, and wherein the lipid moiety is dioctadecylamine (DODA).

In some embodiments, the second lipid moiety comprises a moiety selected from the group consisting of l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1 -palmitoyl -2 -oleoyl -glycero-3- phosphocholine (POPC), 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), 1- palmitoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (POPG), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), l,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dielaidoyl-sn-phosphatidylethanolamine (DEPE), 1 -stearoyl -2 -oleoyl-sn-glycero-3- phosphoethanolamine (SOPE), l,2-dioleoyl-sn-glycero-3 -phosphoglycerol (DOPG), 1,2-dipalmitoyl- sn-glycero-3 -phosphoglycerol (DPPG), 18-1-trans PE, l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), l,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), dihexadecylamine, distearoyl -rac -glycerol (DSG), 1,2-dipalmitoyl-rac -glycerol (DPG), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1,2-O-dioctadecyl-sn-glycerol (DODG), and a derivative thereof. In some embodiments, the second lipid moiety is selected from the group consisting of

20

MEl\57916143.vl 131698-30520

DOPE, DSPE, DSG, DODA, DPG, DODG, and a derivative of any of the foregoing. In some embodiments, the second lipid moiety comprises DODA. In some embodiments, the second lipid moiety comprises DSPE.

In some embodiments, the first lipid moiety and the second lipid moiety are the same. In some embodiments, the first lipid moiety and the second lipid moiety are different.

In some embodiments, the ionizable lipid is present in the LNP in an amount of about 20 mol% to about 60 mol% of the total lipid present in the LNP. In some embodiments, the ionizable lipid is present in the LNP in an amount of about 35 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the ionizable lipid is present in the LNP in an amount of about 40 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the ionizable lipid is present in the LNP in an amount of about 45 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the ionizable lipid is present in the LNP in an amount of about 47.5 mol% of the total lipid present in the LNP.

In some embodiments, the sterol is present in the LNP in an amount of about 20 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 30 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 38 mol% to about 42 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 39 mol% to about 40 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 39.5 mol% of the total lipid present in the LNP.

In some embodiments, the helper lipid is present in the LNP in an amount of about 1 mol% to about 40 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid is present in the LNP in an amount of about 5 mol% to about 15 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid is present in the LNP in an amount of about 7 mol% to about 13 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid is present in the LNP in an amount of about 9 mol% to about 11 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid is present in the LNP in an amount of about 10 mol% of the total lipid present in the LNP.

In some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 0.5 mol% to about 5 mol% of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 1.5 mol% to about 3 mol% of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 2 mol% to about 3 mol% of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 2.6 mol% to about 3 mol% of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 2.8 mol% of the total lipid present in the LNP.

21

MEl\57916143.vl 131698-30520

In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.05 mol% to about 5 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0. 1 mol% to about 1 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.15 mol% to about 0.5 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.15 mol% to about 0.3 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.2 mol% of the total lipid present in the LNP.

In some embodiments, the nanoparticle (LNP) comprises: (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid, wherein the ionizable lipid is heptadecan-9-yl 9-((4- (dimethylamino)butanoyl)oxy)hexadecanoate, having the following structure:

(iii) a sterol, wherein the sterol is cholesterol; (iv) a helper lipid, wherein the helper lipid is DSPC; (v) a first lipid-anchored polymer, wherein the first lipid-anchored polymer comprises DODA conjugated to a linear PG via a linker; and (vi) a second lipid-anchored polymer, wherein the second lipid- anchored polymer comprises DODA conjugated to PG, and further comprising a reactive species conjugated to the PG, wherein the reactive species is functionalized to be conjugated to a targeting moiety.

In some embodiments, the ionizable lipid is present in the LNP in an amount of about 35 mol% to about 50 mol% of the total lipid present in the LNP; the sterol is present in the LNP in an amount of about 30 mol% to about 45 mol% of the total lipid present in the LNP; the helper lipid is present in the LNP in an amount of about 5 mol% to about 15 mol% of the total lipid present in the LNP; the first lipid-anchored polymer is present in the LNP in an amount of about 1.5 mol% to about 3 mol% of the total lipid present in the LNP; and the second lipid-anchored polymer is present in the LNP in an amount of about 0.1 mol% to about 1 mol% of the total lipid present in the LNP.

In some embodiments, the TNA is selected from the group consisting of a minigene, a plasmid, a minicircle, a small interfering RNA (siRNA), a microRNA (miRNA), a guide RNA (gRNA), an antisense oligonucleotide (ASO), a ribozyme, a deoxyribozyme, a closed-ended DNA (ceDNA), a single-stranded DNA (ssDNA), a substantially single -stranded DNA, a ministring, a doggybone™, a protelomere closed ended DNA, a dumbbell linear DNA, a dicer-substrate dsRNA, a small hairpin RNA (shRNA), an asymmetrical interfering RNA (aiRNA), an mRNA, a tRNA, an rRNA, a gRNA, a DNA viral vector, a viral RNA vector, a non-viral vector, and a combination thereof. In some embodiments, the TNA is a single-stranded nucleic acid or a double-stranded

22

MEl\57916143.vl 131698-30520 nucleic acid. In some embodiments, the TNA is siRNA. In some embodiments, the siRNA comprises a sense strand and an antisense strand, wherein the antisense strand has a sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi). In some embodiments, the siRNA is sufficiently complementary to a target mRNA that specifies the amino acid sequence of a cellular protein involved or predicted to be involved in an autoimmune disease or disorder. In some embodiments, the siRNA is sufficiently complementary to a target selected from Table 27.

In some embodiments, the sense strand or the antisense strand is modified by the substitution of at least one nucleotide with a modified nucleotide, such that in vivo stability and/or target efficiency is enhanced as compared to a corresponding unmodified siRNA. In some embodiments, the modified nucleotide is an internal nucleotide. In some embodiments, substantially all of the nucleotides of the sense strand comprise a modification; substantially all of the nucleotides of the antisense strand comprise a modification; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise a modification. In some embodiments, all of the nucleotides of the sense strand comprise a modification; all of the nucleotides of the antisense strand comprise a modification; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In some embodiments, at least one of the nucleotide modifications is selected from the group consisting of a deoxy-nucleotide modification, a 3 ’-terminal deoxythimidine (dT) nucleotide modification, a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxy- nucleotide modification, a locked nucleotide modification, an unlocked nucleotide modification, a conformationally restricted nucleotide modification, a constrained ethyl nucleotide modification, an abasic nucleotide modification, a 2’-amino nucleotide modification, a 2’-O-allyl- nucleotide modification, 2’-C-alkyl- nucleotide modification, a 2 ’-methoxy ethyl nucleotide modification, a 2’-O- alkyl- nucleotide modification, a morpholino nucleotide modification, a phosphoramidate nucleotide modification, a non-natural base comprising nucleotide modification, a tetrahydropyran nucleotide modification, a 1,5-anhydrohexitol nucleotide modification, a cyclohexenyl nucleotide modification, a nucleotide comprising a phosphorothioate group nucleotide modification, a nucleotide comprising a methylphosphonate group nucleotide modification, a nucleotide comprising a 2’ -phosphate nucleotide modification, a nucleotide comprising a 5 ’-phosphate nucleotide modification, a nucleotide comprising a 5 ’-phosphate mimic nucleotide modification, a thermally destabilizing nucleotide modification, a glycol modified nucleotide (GNA) nucleotide modification, an unlocked nucleic acid (UNA) modification, a iAB modification, and a 2-O-(N-methylacetamide) nucleotide modification; and combinations thereof. In some embodiments, the modified nucleotide is: a sugar-modified nucleotide selected from the group consisting of 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro- adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-amino- guanosine and 2'-amino-butyryl-pyrene-uridine; a nucleobase-modified nucleotide selected from the

23

MEl\57916143.vl 131698-30520 group consisting of 5 -bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2- aminopurine, 5 -fluoro-cytidine, and 5 -fluoro-uridine, 2,6-diaminopurine, 4-thio-uridine; and 5-amino- allyl -uridine; a 2'-deoxy ribonucleotide and is present within the sense strand; a 2'-fluoro modified ribonucleotide; and/or selected from the group consisting of a 2'-fluoro, 2'-amino and 2'-thio modified ribonucleotide. In some embodiments, the modified nucleotide is a nucleotide analogue or artificial nucleotide base. In some embodiments, the nucleotide analogue or artificial nucleotide base comprises a 5'-vinylphosphonate modified nucleotide with a modification at a 5' hydroxyl group of the ribose moiety (5'-(E)-vinylphosphonate (5'-EVP) modified nucleotide).

In some embodiments, the TNA is an siRNA; the ionizable lipid is Lipid 87 and is present in the LNP in an amount of about 47.5 mol% of the total lipid present in the LNP; the helper lipid is DSPC and is present in the LNP in an amount of about 10 mol% of the total lipid present in the LNP; the sterol is cholesterol and is present in the LNP in an amount of about 39.5 mol% of the total lipid present in the LNP; the first lipid-anchored polymer is DODA-PG39 and is present in an amount of about 2.8 mol% of the total lipid present in the LNP; and the second lipid-anchored polymer is DODA-PG68 and is present in an amount of about 0.2 mol% of the total lipid present in the LNP.

In some embodiments, the TNA is mRNA. In some embodiments, the LNP does not comprise polyethylene glycol (PEG). In some embodiments, the LNP does not induce or minimally induces antibody-mediated clearance in the subject’s blood. In some embodiments, the LNP has a half-life (ti/2) in blood in vivo of about 3 hours to about 72 hours. In some embodiments, the LNP has a half-life (ti/2) in blood in vivo of greater than 3 hours. In some embodiments, the LNP has a half-life (ti/2) in blood in vivo of greater than 4 hours. In some embodiments, the LNP has a half-life (ti/2) in blood in vivo of greater than 3 hours, 4, hours, 5 hours, 6, hours, 8, hours, 10 hours, 12 hours, 14 hours, 15 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 25 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 35 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 45 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 55 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 65 hours, 66 hours, 68 hours, 70 hours, or 72 hours. In some embodiments, the LNP has an in vivo half-life (ti/2) that is prolonged in the subject’s blood as compared to the in vivo half-life of an LNP that does not comprise PG or a PG derivative. In some embodiments, the LNP has an in vivo half-life (ti/2) that is prolonged in the subject’s blood as compared to the in vivo half-life of an LNP that comprises polyethylene glycol (PEG). In some embodiments, the in vivo half-life of the LNP is increased by at least a factor of about two or more as compared to the in vivo half-life of an LNP that does not comprise PG or a PG derivative. In some embodiments, the in vivo half-life of the LNP is increased by at least a factor of about two or more as compared to the in vivo half-life of an LNP that comprises PEG. In some embodiments, the in vivo half-life of the LNP is increased by at least a factor of about three or more as compared to the in vivo half-life of an LNP that does not comprise PG or a PG derivative. In some embodiments, the in vivo half-life of the LNP is increased by at least a factor of about three or more as compared to the in vivo half-life of an LNP that comprises PEG.

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MEl\57916143.vl 131698-30520

In some embodiments, the LNP and/or the TNA persists in the subject’s blood at least 1, 2, 3,

4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 32, 34, 36, 38, 40,

42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 or more hours after the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or subsequent dose. In some embodiments, each dose is separated by a time period of at least about 6 hours, at least about 1 day, at least about 7 days, at least about 2 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 4 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 5 years, or at least about 10 years. In some embodiments, each dose is separated by a time period of about 6-24 hours, about 1-500 days, about 1-100 weeks, about 1-24 months, or about 1-10 years. In some embodiments, each dose is separated by a time period of about 7 days. In some embodiments, each dose is administered on a periodic schedule. In some embodiments, each dose is administered on a variable schedule. In some embodiments, each dose is about 0.01-10.0 mg/kg. In some embodiments, each dose is at least 0.01 mg/kg, at least 0.05 mg/kg, at least 0. 1 mg/kg, at least 0.5 mg/kg, at least 1.0 mg/kg, at least 1.5 mg/kg, at least 2.0 mg/kg, at least 2.5 mg/kg, at least 3.0 mg/kg, at least 4.0 mg/k, or at least 5.0 mg/kg. In some embodiments, each dose is about 0.1 mg/kg. In some embodiments, each dose is about 0.25 mg/kg. In some embodiments, each dose is about 0.5 mg/kg. In some embodiments, each dose is about 1.0 mg/kg. In some embodiments, each dose is about 1.5 mg/kg. In some embodiments, each dose is about 2.0 mg/kg. In some embodiments, each dose is about 2.5 mg/kg. In some embodiments, each dose is about 3.0 mg/kg. In some embodiments, each dose is about 4.0 mg/kg. In some embodiments, each dose is about 5.0 mg/kg. In some embodiments, each dose comprises the same amount of the LNP. In some embodiments, each dose comprises a different amount of the LNP.

In some embodiments, the subject is a human. In some embodiments, he disease, disorder or condition is related to abnormal expression of a gene product. In some embodiments, the disease, disorder, or condition is a blood disease, disorder, or condition. In some embodiments, the disease, disorder, or condition is an autoimmune disease, disorder, or condition. In some embodiments, the autoimmune disease, disorder, or condition is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, type I diabetes, systemic lupus erythematosus, psoriasis, psoriatic arthritis, Sjogren’s syndrome, Crohn’s disease, Celiac disease, ulcerative colitis, Graves’ disease, Hashimoto’s thyroiditis, Addison’s disease, dermatomyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome, myasthenia gravis, autoimmune vasculitis, pernicious anemia, vitiligo, systemic sclerosis, scleroderma, diffuse scleroderma, limited scleroderma, linear scleroderma, localized scleroderma, hemolytic anemia, inflammatory bowel disease (IBD), achantholysis, acute disseminated encephalomyelitis, adult-onset Still disease, allergic glomerulonephritis, ANCA associated vasculitis, ankylosing spondylitis, anti-glomerular basement membrane disease, anti-N- methyl-D-aspartate receptor encephalitis, antiphospholipid syndrome, antisynthetase syndrome, autoimmune gastritis, autoimmune hypophysitis, autoimmune liver disease, autoimmune lung disease,

25

MEl\57916143.vl 131698-30520 autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, autoimmune skin disease, autoimmune thyroiditis, autoimmune uveitis, Behcet diseae, bullous pemphigoid, Churg Strauss syndrome, dermatitis herpetiformis, endocrine ophthalmopathy, erythematous pemphigus, Felty syndrome, Giant cell arteritis, IgA glomerulonephritis, membranous glomerulonephritis, Goodpasture syndrome, Granulomatosis with polyangiitis, Graves’ ophthalmopathy, Hailey Hailey syndrome, heparin induced thrombocytopenia, autoimmune hepatitis, idiopathic hypoparathryroidism, idiopathic thrombocytopenic purpura, IgA pemphigus, immune complex nephritis, immunoglobulin A nephropathy, juvenile rheumatoid arthritis, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, linear IgA bullous dermatosis, lupus nephritis, lupus vasculitis, microscopic polyangiitis, morphea, mucous membrane phemphigoid, myasthenia gravis, nonarticular rheumatism, sympathetic ophthalmia, opsoclonous myoclonus syndrome, paraneoplastic pemphigus, pemphigoid, pemphigoid gestationis, pemphigus, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, autoimmune polyendocrinopathies, polyradiculoneuropathy, postpartum thyroiditis, primary biliary cirrhosis, rheumatic disease, rheumatic heart disease, rheumatoid polymyalgia, rheumatoid nodule, rheumatoid vasculitis, stiff-person syndrome, CREST syndrome, systemic juvenile idiopathic arthritis, sarcoidosis, and uveomeningoencephalitic syndrome.

In some embodiments, the disease, disorder, or condition is a genetic disease or disorder. In some embodiments, the genetic disease or disorder is selected from the group consisting of sickle cell disease, melanoma; hemophilia A (clotting factor VIII (FVIII) deficiency), hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR defect), familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson’s disease, phenylketonuria (PKU), congenital hepatic porphyria, an inherited disorder of hepatic metabolism; Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, a mucopolysaccharide storage disease, Niemann-Pick disease, Fabry disease, Schindler disease, GM2 -gangliosidosis Type II (Sandhoff Disease), Tay-Sachs disease, Metachromatic Leukodystrophy, Krabbe disease, mucolipidosis (ML), Sialidosis Type II, a glycogen storage disease (GSD), Gaucher disease, cystinosis, Batten disease, Aspartylglucosaminuria, Salla disease, Danon disease (LAMP -2 deficiency), Lysosomal Acid Lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (NCL), sphingolipidoses, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, spinocerebellar ataxia, spinal muscular atrophy (SMA), Friedreich’s ataxia, Duchenne muscular dystrophy (DMD), a Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber Congenital Amaurosis, Stargardt disease, wet macular degeneration (wet AMD), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha- 1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC), and Cathepsin A deficiency.

26

MEl\57916143.vl 131698-30520

In some embodiments, the method provides anti -tumor immunity to a subject in need thereof. In some embodiments, the disease, disorder or condition is associated with an elevated expression of a tumor antigen.

In some embodiments, the LNP further comprises a third lipid-anchored polymer, wherein the third lipid-anchored polymer comprises: (i) a third lipid moiety comprising at least one hydrophobic tail; (ii) a third polymer; (iii) an optional third linker, wherein the third polymer is conjugated to the third lipid moiety via the third linker; and (iv) a reactive species conjugated to the third polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety. In some embodiments, the third polymer is polyglycerol (PG) or a PG derivative. In some embodiments, the LNP further comprises a targeting moiety conjugated to the third polymer via the reactive moiety. In some embodiments, the targeting moiety conjugated to the third polymer is different from the targeting moiety conjugated to the second polymer. In some embodiments, the third lipid-anchored polymer is different from the second lipid-anchored polymer. In some embodiments, the targeting moiety conjugated to the third polymer and the targeting moiety conjugated to the second polymer bind to different antigens on the same tissue or cell type.

In some aspects, the present disclosure provides a method of synthesizing a linker-conjugated dioctadecylamine (DODA-1) of the following structure:

(DODA-1), said method comprising:

(a) reacting dioctadecylamine (DODA) with dihydro-2H-pyran-2,6(3H)-dione in the presence of a base to produce 5 -(dioctadecylamino) -5 -oxopentanoic acid (intermediate DODA-A1) of the following structure:

(DODA-A1);

(b) reacting DODA-A1 with N,O-dimethylhydroxylamine in the presence of a coupling reagent, a catalyst and a base to produce intermediate DODA-A2 of the following structure:

(DODA-A2); and

(c) reacting DODA-A2 with a reducing agent to produce DODA-1.

27

MEl\57916143.vl 131698-30520

In some embodiments, in (a) the base is N,N-Diisopropylethylamine (DIPEA). In some embodiments, in (b) the coupling reagent is l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). In some embodiments, in (b) the catalyst is 4-(dimethylamino)pyridine (DMAP). In some embodiments, in (b) the base is diisopropylethylamine (DIPEA). In some embodiments, in (c) the reducing agent is sodium borohydride. In some embodiments, the method further comprises: (dl) reacting DODA-1 with 2,3-epoxy-l-(l-ethoxyethoxy)propane (EEGE) in the presence of a base or an organocatalyst under argon atmosphere to produce DODA conjugated to a linker and polymerized EEGE (intermediate DODA-PG-OH) of the following structure:

(DODA-PG-OH), wherein n is an integer from 8 to 100; and

(el) subjecting DODA-PG-OH to acidic conditions to produce DODA conjugated to polyglycerol (DODA-PG) of the following structure:

(DODA-PG).

In some embodiments, in (dl) the base is a phosphazene base. In some embodiments, the base is P4-t-Bu. In some embodiments, in (dl) the organocatalyst is N-heterocyclic carbene (NHC) or an N-heterocyclic olefin (NHO). In some embodiments, in (el) the acidic conditions comprise a strong acid. In some embodiments, the strong acid is selected from the group consisting of hydrochloric acid (HC1), hydrobromic acid (HBr), hydroiodic acid (HI), perchloric acid (HCIO4), chloric acid (HCIO3), sulfuric acid (H2SO4), and nitric acid (HNO3). In some embodiments, the strong acid is HC1.

In some embodiments, the method further comprises:

(d2) reacting DODA-1 with 2,3-epoxy-l-(l-ethoxyethoxy)propane (EEGE) in the presence of a base or an organocatalyst under argon atmosphere to produce DODA conjugated to a linker and polymerized EEGE (intermediate DODA-PG-OH) of the following structure:

28

MEl\57916143.vl 131698-30520

(DODA-PG-OH), wherein n is an integer from 8 to 100; and

(e2) reacting DODA-PG-OH with tert-butyl (4-(bromomethyl)benzyl)carbamate in the presence of a reducing agent to produce intermediate DODA-PG-L1 of the following structure:

(DODA-PG-L1);

(f2) subjecting DODA-PG-L1 to acidic conditions to produce intermediate DODA-PG-L2 of the following structure:

(DODA-PG-L2); and

(g2) reacting DODA-PG-L2 with 2,5-dioxopyrrolidin-l-yl 4-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)butanoate in the presence of a base to produce DODA-PG-Maleimide of the following structure:

(DOD A-PG-Maleimide) .

In some embodiments, in (d2) the base is a phosphazene base. In some embodiments, the base is P4-t-Bu. In some embodiments, in (d2) the organocatalyst is N-heterocyclic carbene (NHC) or an N-heterocyclic olefin (NHO). In some embodiments, in (e2) the reducing agent is sodium hydride. In some embodiments, in (f2) the acidic conditions comprise a strong acid. In some embodiments, the

29

MEl\57916143.vl 131698-30520 strong acid is selected from the group consisting of hydrochloric acid (HC1), hydrobromic acid (HBr), hydroiodic acid (HI), perchloric acid (HCIO4), chloric acid (HCIO3), sulfuric acid (H2SO4), and nitric acid (HNO3). In some embodiments, the strong acid is HC1. In some embodiments, in (g2) the base is triethylamine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a MALDI-TOF spectrum of DODA-PG34.

FIG. IB depicts a MALDI-TOF spectrum of DODA-PG45.

FIG. 1C depicts a MALDI-TOF spectrum of DODA-PG58.

FIG. ID shows “Scheme 1” showing synthesis of DODA-PG41 and DODA-PG46.

FIG. IE shows “Scheme 2” showing synthesis of DODA-PG45 and DODA-PG58.

FIG. 2A shows the total flux measured by the total photon counts per the region of interest, z.e., the liver, measured in mice by In vivo Imaging System (IVIS) at Day 4 post-dosing for LNP formulations of the disclosure and a negative control (PBS).

FIG. 2B shows the total flux measured by the total photon counts per the region of interest, z.e., the liver, measured in mice by IVIS at Day 7 post-dosing for LNP formulations of the disclosure and a negative control (PBS).

FIG. 2C shows the total flux measured by the total photon counts per the region of interest, z.e., the liver, measured in mice by IVIS across two collection days (Day 4 and Day 7) post-dosing for LNP formulations of the disclosure and a negative control (PBS).

FIG. 2D shows percent change in body weight (BW) of mice at Day 1 post-dosing with LNP formulations of the disclosure.

FIG. 3 shows luciferase activity for LNP formulations of the disclosure containing different lipid-anchored polymers.

FIG. 4A is a schematic depicting the proposed mechanism of opsonization-driven cell uptake of LNPs.

FIG. 4B is a schematic depicting the assay used for evaluating opsonization-driven cell uptake of LNPs.

FIG. 4C shows DiD fluorescence area normalized to area of live nuclei measured for LNP formulations of the disclosure containing different lipid-anchored polymers.

FIG. 5 shows DiD fluorescence area normalized to area of live nuclei for LNP formulations of the disclosure containing different amounts of polyglycerol-conjugated lipids and a control.

FIG. 6 shows the amount of endosomal escape measured as the amount of luciferase expression normalized to DiD uptake in mouse hepatocytes treated with LNP formulations of the disclosure containing different amounts of polyglycerol-conjugated lipids and a control.

FIG. 7 shows the whole blood clearance of the Control LNP, and the different Lipid Z carrying LNPs of the disclosure.

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MEl\57916143.vl 131698-30520

FIG. 8A shows the total flux quantified by total photon counts per the region of interest, i. e. , the liver, measured in mice by IVIS at Day 7 post-dosing with LNP formulations of the disclosure and a negative control (DPBS).

FIG. 8B shows the percentage change in body weight at Day 1 of mice injected with LNP formulations of the disclosure.

FIGs. 9A-9D show the levels of various cytokines that are implicated in the regulation of innate immune response, z.e., IFN-alpha (FIG. 9A), IFN-gamma (FIG. 9B), IL-6 (FIG. 9C) and IL- 18 (FIG. 9D) measured in mice following administration LNP formulations of the disclosure.

FIG. 10 shows DiD fluorescence area normalized to area of live nuclei for LNP formulations of the disclosure containing different amounts of polyglycerol-conjugated lipids and a control, and formulated with DSPE-PEG5K-Ns using a mole percentage of 0.5%.

FIG. 11A shows the levels of mRNA cargo (pg/mL) measured by qPCR in the blood of mice (n = 3 per group) following a single dose or three doses of LNPs containing either 3% DSG-PEG2000 or 3% DODA-PG45.

FIG. 11B shows the levels of mRNA cargo (pg/mL) measured by qPCR in the blood of mice (n = 3 per group) following a single dose or three doses of LNPs containing either 3% DSG-PEG2000 or 3% DODG-PG45.

FIG. 12 is a graph that shows repeat dosing of LNPs containing 2% or 4% PG 34 does not induce an antibody response and maintains an extended blood circulation profile.

FIG. 13 is a panel of graphs that show repeat dosing of stealth LNPs containing PG50 maintained an extended blood circulation profile and are not rapidly cleared from the blood, regardless which ionizable lipid is used.

FIG. 14 shows a graph of in vivo luciferase mRNA cargo expression, as measured by the total photon counts per the region of interest, i. e. , the liver, measured in mice (n = 4 per group) by IVIS at 24 hours post-dosing of 0.05 mg/kg (on Day 0) LNP formulations containing mRNA cargo and 2%, 3%, or 4% DODA-PG45, DODA-PG34, with two different ionizable lipids, Lipid 87 and L319.

FIG. 15 is a graph of in vivo ssDNA luciferase cargo expression, as measured by the total photon counts per the region of interest, z.e., the liver, measured in mice (n = 5 per group) by IVIS at Day 7 post dose of 0.5 mg/kg (on Day 0) for LNP formulations containing ssDNA cargo, 3% PG50 or 3% PEG, and two different ionizable lipids, Lipid 87 or L-319.

FIG. 16 is a graph showing in vivo luciferase mRNA expression (as measured by IVIS) for a single dose of ctLNPs comprising only PEG- or only PG-conjugated lipids.

FIG. 17A shows the beta-2 -microglobulin (B2M) siRNA sequences and modification patterns.

FIG. 17B is a graph that shows ctLNP delivery increased in a dose dependent manner for both PEG-Mal and PG-Mal LNP formulations, but not for the control (y-axis, DiD labelling (gMFl); x-axis, siRNA dose (nM)).

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MEl\57916143.vl 131698-30520

FIG. 17C is a graph that shows there was dose dependent target knockdown in non-activated T cells in vitro for both PEG-Mal and PG-Mal LNP formulations, but not for the control(y-axis, foldreduction in hB2M from baseline; x-axis, siRNA dose (nM)).

FIG. 18A is a graph that shows that B2M siRNAs with specific modification patterns (siRNA216 and siRNA217; Mod pattern #1 and Mod pattern #2) were more potent and durable than negative control siRNA with a random modification (control mod) in humanized mice (NP= N/P ratio; PEG-Mal = PEG-maleimide; PG-Mal = Polyglycerol-maleimide).

FIG. 18B is a panel of five graphs that show percent knockdown of housekeeping gene beta- 2-microglobulin (B2M) at days 2 (D2), 6 (D6), 10 (DIO), 14 (D14) and 17 (D17).

FIG. 19 is a graph showing the concentration of mRNA cargo present in the circulation of mice treated with single or multiple doses of LNPs comprising only DODA-PG39, only DSG-PEG2k, DODA-PG39/DODA-PG68-Mal (0.2% or 0.5%), or DODA-PG39/PEG5K-Mal (0.5%).

FIG. 20 is a graph showing the percentage of mRNA cargo present in the circulation of mice 6 hours after treatment with 3 doses of LNPs comprising only 3% PG-conjugated lipids (DODA- PG34, DODA-PG39, DODA-PG45, or DODA-PG50), as compared to LNPs comprising 3% DSG- PEG2.

FIG. 21 shows “Scheme 3” showing synthesis of DODA-PG39.

FIG. 22A shows “Scheme 4A” showing synthesis of the intermediate DODA-1.

FIG. 22B shows “Scheme 4B” showing synthesis of DODA-PG-Mal.

FIG. 23 is a graph that shows percent (%) knockdown of beta 2 microglobulin (B2M) expression in T cells, NK cells and B cells at 48 hours after dosing NHPs with a ctLNP conjugated to a CD7 antibody as described in Example 24. The median fluorescence value of the antibody against B2M at 48 hours following dose was converted to a ratio relative the median fluorescence value for that animal pre-dose for each cell type. As shown in FIG 23, siRNA against B2M achieved more than 80% knock-down of B2M expression in T and NK cells when delivered using a ctLNP conjugated to a CD7 antibody.

FIG. 24 is a graph that shows on-target delivery of the ctLNP to CD7-expressing cells in a NHP study as described in Example 24. As shown in FIG. 24, the percentage of cells expressing CD7 at pre-dose were tightly correlated with the percentage of cells that were stained with the DiD dye delivered by the formulated LNP at 48 hours.

FIG. 25 is a graph that shows the percentage B2M expression post-dose relative to pre-dose baseline (baseline = 100%)at 48 hours post-dose in the NHP study of Example 24.

FIG. 26 is a graph that shows the percentage B2M expression relative to each animal’s predose B2M geometric mean (gMFI) in PBMC samples in the NHP study of Example 24. B2M expression in total T cells, CD4+ T cells, CD8+ T cells and NK cells showed average gMFI decrease to an average of 31%, 25%, 35% and 23% of pre-dose level through day 15 post-dose, respectively.

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FIG. 27 is a graph that shows the distribution of B2M gMFI reduction or knockdown relative to predose over time in PBMC samples in the NHP study of Example 24.

FIG. 28 is a graph that shows the ratio of post dose DiD+ or DiD- B2M gMFI to predose total B2M gMFI for each immune cell type in PBMC samples over four weeks post-dose in the NHP study of Example 24. The y-axis shows % of CD3+ T cells.

FIG. 29 depicts graphs demonstrating that CD7-ctLNP is well-tolerated in non-human primates (NHP) with no significant clinical pathology changes after multiple doses.

DETAILED DESCRIPTION

The present disclosure provides polymer-conjugated lipids, comprising, e.g., a polyglycerol (PG) conjugated to a lipid, and methods of their synthesis. The present disclosure also provides lipid nanoparticles (LNPs) comprising, inter alia, polymer-conjugated lipids of the disclosure, and methods of treatment of various disorders comprising administering to a subject in need thereof LNPs of the disclosure. The present disclosure further provides methods for treating diseases, disorders, or conditions comprising administering multiple doses of the LNPs described herein. It has been surprisingly discovered that LNPs comprising polymer-conjugated lipids of the present disclosure are characterized by low levels of undesirable opsonization-driven uptake of LNPs into non-target cells, and balanced with desirable levels of endosomal escape, thereby achieving advantageous stealth/endosomal escape tradeoff, as described herein. It has further been surprisingly discovered that the LNPs described herein can be administered in multiple doses without inducing antibody- mediated clearance of the LNPs from the blood. Moreover, it has been shown that repeat dosing of LNPs comprising a polyglycerol (PG) or a PG derivative conjugated lipid surprisingly maintained an extended blood circulation profile (e.g., increased blood ti/2) and were not rapidly cleared when compared to LNPs comprising a polyethylene glycol (PEG) conjugated lipid.

I. Definitions

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc. , described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, 6th Edition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), Knipe, D.M. and Howley, P.M. (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell

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MEl\57916143.vl 131698-30520

Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al. Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.

As used herein, the term “about,” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

As used herein, “comprise,” “comprising,” and “comprises” are synonymous with “include”, “including”, “includes” or “contain”, “containing”, “contains” and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

The term “consisting of’ refers to compositions, methods, processes, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure.

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MEl\57916143.vl 131698-30520

As used herein the terms, “administration,” “administering” and variants thereof refers to introducing a composition or agent (e.g., an LNP, TNA, ceDNA, ssDNA, mRNA, etc.) into a subject and includes concurrent and sequential introduction of one or more compositions or agents. “Administration” can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. “Administration” also encompasses in vitro and ex vivo treatments. The introduction of a composition or agent into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intratumorally, or topically. Administration includes self-administration and the administration by another. Administration can be carried out by any suitable route. A suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.

The term “immunogenicity of an LNP” or “immunogenicity of a composition comprising an LNP”, as used herein, refers to the ability of a composition comprising LNPs of the present disclosure to stimulate an undesired immune response in a subject after the LNPs of the disclosure or a composition comprising the LNPs of the disclosure are administered to the subject. In some embodiments, the immune response, e.g., before and after administration of a composition comprising LNPs of the present disclosure, may be measured by measuring levels of one or more pro- inflammatory cytokines. Exemplary pro-inflammatory cytokines that may be used to determine immunogenicity of LNPs of the present disclosure or a composition comprising LNPs of the present disclosure include, but are not limited to, granulocyte colony stimulating factor (G-CSF), interleukin 1 alpha (IL- la), interleukin 1 beta (IL-1 P), interleukin 6 (IL-6), interleukin 8 (IL-8 or CXCL8), interleukin 11 (IL- 11), interleukin 17 (IL- 17), interleukin 18 (IL- 18), interferon a (IFN-a), interferon (IFN-P), interferon y (IFN-y), C-X-C motif chemokine ligand 10 (CXCL10 or IP- 10), monocyte chemoattractant protein 1 (MCP-1), CD40L, CCL2, CCL3, CCL4, CCL5, CCL11, tumor necrosis factor a (TNF-a), and combinations thereof.

The term “off-target delivery”, as used herein, refers to delivery of LNPs of the disclosure to non-target cells. For example, an LNP of the disclosure comprising GalNAc targets delivery of the LNP to hepatocytes, and off-target delivery of the LNP refers to the delivery of the LNP to random, non-target cells that are not, for example, hepatocytes. In some embodiments, the non-target cell may be a blood cell, e.g. , a leukocyte, a neutrophil, an eosinophil, a basophil, a macrophage, or a monocyte. In some embodiments, the non-target cell may be an immune cell, such as a T-cell, B-cell or a macrophage. In some embodiments, the non-target cell may be a liver sinusoidal endothelial cell (LSEC cell), a spleen cell or a Kupffer cell.

After administration to a subject, an LNP may be delivered to a non-target cell, e.g., one or more of blood cells listed above, and may result in expression of a therapeutic nucleic acid (TNA) in the non-target cell, or may be degraded once engulfed by, e.g., a macrophage. In some embodiments,

35

MEl\57916143.vl 131698-30520 a reference LNP that does not contain a polygycerol-conjugated lipid, may be characterized by a higher rate of delivery to a non-target cell, e.g. , one or more of blood cells listed above, as compared to an LNP of the present disclosure. In some embodiments, an LNP of the present disclosure results in an uptake level of TNA (e.g., ceDNA, mRNA or siRNA) in a non-target cell, e.g., a blood cell, that is lower than that of a reference LNP. In some embodiments, the reference LNP is an LNP that does not comprise a polymer-conjugated lipid. In some embodiments, the blood cell is a cell selected from the group consisting of a red blood cell, a leukocyte, a neutrophil, a macrophage, a monocyte, a T- cell, a B-cell, a macrophage and a peripheral blood mononuclear cell.

As used herein, the term “aqueous solution” refers to a composition comprising in whole, or in part, water.

As used herein, the term “bases” includes purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.

As used herein, the terms “carrier” and “excipient” include any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically- acceptable” refers to molecular entities and compositions that do not produce a toxic, an allergic, or similar untoward reaction when administered to a host.

As used herein, the term “ceDNA” refers to capsid-free closed-ended linear double stranded (ds) duplex DNA for non-viral gene transfer, synthetic or otherwise. According to some embodiments, the ceDNA is a closed-ended linear duplex (CELiD) CELiD DNA. According to some embodiments, the ceDNA is a DNA-based minicircle. According to some embodiments, the ceDNA is a minimalistic immunological-defmed gene expression (MIDGE)-vector. According to some embodiments, the ceDNA is a ministering DNA. According to some embodiments, the ceDNA is a dumbbell shaped linear duplex closed-ended DNA comprising two hairpin structures of ITRs in the 5 ’ and 3’ ends of an expression cassette. According to some embodiments, the ceDNA is a doggybone™ DNA. Detailed description of ceDNA is described in International Patent Application No. PCT/US2017/020828, fded March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for the production of ceDNA comprising various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Patent Application Nos. PCT/US 18/49996, filed September 7, 2018, and PCT/US2018/064242, filed December 6, 2018 each of which is incorporated herein in its entirety by reference. Certain methods for the production of synthetic ceDNA vectors comprising various ITR

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MEl\57916143.vl 131698-30520 sequences and configurations are described, e.g., in International Application PCT/US2019/14122, filed on January 18, 2019, the entire content of which is incorporated herein by reference.

As used herein, the term “closed-ended DNA vector” refers to a capsid-free DNA vector with at least one covalently closed end and where at least part of the vector has an intramolecular duplex structure.

As used herein, the terms “ceDNA vector” and “ceDNA” are used interchangeably and refer to a closed-ended DNA vector comprising at least one terminal palindrome. In some embodiments, the ceDNA comprises two covalently-closed ends.

As used herein, the term “ceDNA-bacmid” refers to an infectious baculovirus genome comprising a ceDNA genome as an intermolecular duplex that is capable of propagating in E. coli as a plasmid, and so can operate as a shuttle vector for baculovirus.

As used herein, the term “ceDNA-baculovirus” refers to a baculovirus that comprises a ceDNA genome as an intermolecular duplex within the baculovirus genome.

As used herein, the terms “ceDNA-baculovirus infected insect cell” and “ceDNA-BIIC” are used interchangeably, and refer to an invertebrate host cell (including, but not limited to an insect cell (e.g., an Sf9 cell)) infected with a ceDNA-baculovirus.

As used herein, the term “ceDNA genome” refers to an expression cassette that further incorporates at least one inverted terminal repeat (ITR) region. A ceDNA genome may further comprise one or more spacer regions. In some embodiments the ceDNA genome is incorporated as an intermolecular duplex polynucleotide of DNA into a plasmid or viral genome.

As used herein, the terms “ssDNA”, “ssDNA construct” “ssDNA molecule”, and “ssDNA vector” refer to a linear, substantially single-stranded DNA molecule comprising a nucleic acid sequence of interest. In some embodiments, the nucleic acid sequence of interest comprises at least one promoter or promoter set (comprising, e.g., a promoter and additional expression control elements). In some embodiments, an “ssDNA”, “ssDNA construct”, “ssDNA molecule”, or “ssDNA vector” may include at least one stem -loop structure at its 3’ end comprising at least one stem and one loop, and/or at least one stem -loop structure comprising at least one stem and one loop at its 5’ end. In some embodiments, an “ssDNA”, “ssDNA construct”, “ssDNA molecule”, or “ssDNA vector” may comprise an extended double-stranded region adjacent to its 3’ and/or 5’ end, and this double -stranded region may comprise the promoter region, including the promoter itself, one or more enhancers, the transcription start site, and/or other regulatory elements. In some embodiments, an “ssDNA”, “ssDNA construct”, “ssDNA molecule”, or “ssDNA vector” may further comprise double -stranded regions comprising hybridized, noncovalently-bound oligonucleotides. Although the terms “ssDNA”, “ssDNA construct” “ssDNA molecule”, and “ssDNA vector” generally refer to a linear DNA molecule, it should also be understood that in some embodiments, the term “circular ssDNA” molecule may be used to refer to circular, partially single-stranded DNA molecules comprising one fully circular strand and at least one noncovalently-bound linear strand hybridized to the fully circular

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MEl\57916143.vl 131698-30520 strand to produce one or more double -stranded regions. Although lacking the 3’ and/or 5’ stem-loop structures described elsewhere herein for ssDNA molecules, circular ssDNA molecules are nevertheless useful for the same applications as any of the instantly described ssDNA molecules. Additional description of ssDNA molecules may be found, for example, in International Patent Application No. WO 2024/119017, incorporated herein in its entirety by this reference.

As used herein, the term “stem-loop structure” refers to a nucleic acid sequence located at the 5 ’ and/or 3 ’ terminus of the ssDNA vectors disclosed herein, which comprises at least one partial duplex (referred to herein as a “stem”) and one loop (comprising 3 or more unbound, single-stranded nucleotides). According to some embodiments, the stem -loop structure may be an artificial sequence (e.g. , contains no sequences derived from a virus), or it may be derived in part or entirely from a virus (e.g. an AAV such as AAV2). An ssDNA molecule may further comprise one stem-loop structure (e.g., a “hairpin”), or more than one stem-loop structure (e.g, 2, 3, 4, 5, or more stem-loop structures). For example, an ssDNA molecule may comprise two stem-loop structures (e.g., a “hammerhead”, “doggy-bone”, or “dumbbell”), three stem-loop structures (e.g., “cruciform”), or more complex structures. A stem -loop structure may further comprise an aptamer sequence or one or more chemical modifications.

In some embodiments, a stem -loop structure at the 3 ’ and/or 5 ’ ends of an ssDNA molecule may be referred to as an “inverted terminal repeat” or “ITR”. For the purposes of the disclosure herein, the terms “inverted terminal repeat” and “ITR” are not intended to represent only viral-derived sequences, but are intended to refer to any sequence, fully synthetic or viral -derived, that contain inverted, palindromic sequences that can self-hybridize to form a stem -loop structure.

As used herein, the terms “DNA regulatory sequences,” “control elements,” and “regulatory elements,” are used interchangeably herein, and refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., DNA-targeting RNA) or a coding sequence (e.g., site-directed modifying polypeptide, or Cas9/Csnl polypeptide) and/or regulate translation of an encoded polypeptide.

As used herein, an “ITR” or “stem-loop structure” can be artificially synthesized using a set of oligonucleotides comprising one or more desirable functional sequences (e.g., palindromic sequence, RBS). The ITR sequence can be an AAV ITR, an artificial non-AAV ITR, or an ITR physically derived from a viral AAV ITR (e.g., ITR fragments removed from a viral genome). For example, the ITR can be derived from the family Parvoviridae, which encompasses parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin that serves as the origin of SV40 replication can be used as an ITR, which can further be modified by truncation, substitution, deletion, insertion and/or addition. Parvoviridcie family viruses consist of two subfamilies: Parvovirincie, which infect vertebrates, and Densovirinae , which infect invertebrates. Dependoparvoviruses include the viral

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MEl\57916143.vl 131698-30520 family of the adeno-associated viruses (AAV) which are capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine and ovine species. Typically, ITR sequences can be derived not only from AAV, but also from Parvovirus, lentivirus, goose virus, Bl 9, in the configurations of wildtype, “doggy bone” and “dumbbell shape”, symmetrical or even asymmetrical ITR orientation. Although the ITRs are typically present in both 5’ and 3’ ends of an AAV vector, an ITR can be present in only one of end of the linear vector. For example, the ITR can be present on the 5’ end only. In some other cases, the ITR can be present on the 3’ end only in synthetic AAV vector. For convenience herein, an ITR located 5 ’ to (“upstream of’) an expression cassette in a synthetic AAV vector (e.g., ceDNA or ssDNA) is referred to as a “5’ ITR” or a “left ITR”, and an ITR located 3’ to (“downstream of’) an expression cassette in a vector or synthetic AAV is referred to as a “3’ ITR” or a “right ITR”.

As used herein, a “wild-type ITR” or “WT-ITR” refers to the sequence of a naturally occurring ITR sequence in an AAV genome or other dependovirus that remains, e.g., Rep binding activity and Rep nicking ability. The nucleotide sequence of a WT-ITR from any AAV serotype may slightly vary from the canonical naturally occurring sequence due to degeneracy of the genetic code or drift, and therefore WT-ITR sequences encompasses for use herein include WT-ITR sequences as result of naturally occurring changes (e.g., a replication error).

As used herein, the term “substantially symmetrical WT-ITRs” or a “substantially symmetrical WT-ITR pair” refers to a pair of WT-ITRs within a synthetic AAV vector that are both wild type ITRs that have an inverse complement sequence across their entire length. For example, an ITR can be considered to be a wild-type sequence, even if it has one or more nucleotides that deviate from the canonical naturally occurring canonical sequence, so long as the changes do not affect the physical and functional properties and overall three-dimensional structure of the sequence (secondary and tertiary structures). In some aspects, the deviating nucleotides represent conservative sequence changes. As one non-limiting example, a sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (as measured, e.g., using BLAST at default settings), and also has a symmetrical three-dimensional spatial organization to the other WT-ITR such that their 3D structures are the same shape in geometrical space. The substantially symmetrical WT-ITR has the same A, C-C’ and B-B’ loops in 3D space. A substantially symmetrical WT-ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE’) and terminal resolution site (trs) that pairs with the appropriate Rep protein. One can optionally test other functions, including transgene expression under permissive conditions.

As used herein, the phrases of “modified ITR” or “mod-ITR” or “mutant ITR” are used interchangeably and refer to an ITR with a mutation in at least one or more nucleotides as compared to the WT-ITR from the same serotype. The mutation can result in a change in one or more of A, C, C’, B, B’ regions in the ITR, and can result in a change in the three-dimensional spatial organization

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MEl\57916143.vl 131698-30520

(i.e., its 3D structure in geometric space) as compared to the 3D spatial organization of a WT-ITR of the same serotype.

As used herein, the term “asymmetric ITRs” also referred to as “asymmetric ITR pairs” refers to a pair of ITRs within a single synthetic AAV genome that are not inverse complements across their full length. As one non-limiting example, an asymmetric ITR pair does not have a symmetrical three- dimensional spatial organization to their cognate ITR such that their 3D structures are different shapes in geometrical space. Stated differently, an asymmetrical ITR pair have the different overall geometric structure, i.e., they have different organization of their A, C-C’ and B-B’ loops in 3D space (e.g. , one ITR may have a short C-C’ arm and/or short B-B’ arm as compared to the cognate ITR). The difference in sequence between the two ITRs may be due to one or more nucleotide addition, deletion, truncation, or point mutation. In one embodiment, one ITR of the asymmetric ITR pair may be a wild-type AAV ITR sequence and the other ITR a modified ITR as defined herein (e.g., a nonwild-type or synthetic ITR sequence). In another embodiment, neither ITRs of the asymmetric ITR pair is a wild-type AAV sequence and the two ITRs are modified ITRs that have different shapes in geometrical space i.e., a different overall geometric structure). In some embodiments, one mod-ITRs of an asymmetric ITR pair can have a short C-C’ arm and the other ITR can have a different modification (e.g., a single arm, or a short B-B’ arm etc.) such that they have different three- dimensional spatial organization as compared to the cognate asymmetric mod-ITR.

As used herein, the term “symmetric ITRs” refers to a pair of ITRs within a single stranded AAV genome that are wild-type or mutated (e.g., modified relative to wild-type) dependoviral ITR sequences and are inverse complements across their full length. In one non-limiting example, both ITRs are wild type ITRs sequences from AAV2. In another example, neither ITRs are wild type ITR AAV2 sequences (i.e. , they are a modified ITR, also referred to as a mutant ITR), and can have a difference in sequence from the wild type ITR due to nucleotide addition, deletion, substitution, truncation, or point mutation. For convenience herein, an ITR located 5’ to (upstream of) an expression cassette in a synthetic AAV vector is referred to as a “5 ’ ITR” or a “left ITR”, and an ITR located 3 ’ to (downstream of) an expression cassette in a synthetic AAV vector is referred to as a “3 ’ ITR” or a “right ITR”.

As used herein, the terms “substantially symmetrical modified-ITRs” or a “substantially symmetrical mod-ITR pair” refers to a pair of modified-ITRs within a synthetic AAV that are both that have an inverse complement sequence across their entire length. For example, the modified ITR can be considered substantially symmetrical, even if it has some nucleotide sequences that deviate from the inverse complement sequence so long as the changes do not affect the properties and overall shape. As one non-limiting example, a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (as measured using BLAST at default settings), and also has a symmetrical three-dimensional spatial organization to their cognate modified ITR such that their 3D structures are the same shape in geometrical space. Stated differently, a substantially

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MEl\57916143.vl 131698-30520 symmetrical modified-ITR pair have the same A, C-C’ and B-B’ loops organized in 3D space. In some embodiments, the ITRs from a mod-ITR pair may have different reverse complement nucleotide sequences but still have the same symmetrical three-dimensional spatial organization - that is both ITRs have mutations that result in the same overall 3D shape. For example, one ITR (e.g., 5’ ITR) in a mod-ITR pair can be from one serotype, and the other ITR (e.g., 3’ ITR) can be from a different serotype, however, both can have the same corresponding mutation (e.g. , if the 5 ’ITR has a deletion in the C region, the cognate modified 3’ITR from a different serotype has a deletion at the corresponding position in the C’ region), such that the modified ITR pair has the same symmetrical three- dimensional spatial organization. In such embodiments, each ITR in a modified ITR pair can be from different serotypes (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) such as the combination of AAV2 and AAV6, with the modification in one ITR reflected in the corresponding position in the cognate ITR from a different serotype. In one embodiment, a substantially symmetrical modified ITR pair refers to a pair of modified ITRs (mod-ITRs) so long as the difference in nucleotide sequences between the ITRs does not affect the properties or overall shape and they have substantially the same shape in 3D space. As a non-limiting example, a mod-ITR that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the canonical mod-ITR as determined by standard means well known in the art such as BLAST (Basic Local Alignment Search Tool), or BLASTN at default settings, and also has a symmetrical three-dimensional spatial organization such that their 3D structure is the same shape in geometric space. A substantially symmetrical mod-ITR pair has the same A, C-C’ and B-B’ loops in 3D space, e.g., if a modified ITR in a substantially symmetrical mod-ITR pair has a deletion of a C-C’ arm, then the cognate mod-ITR has the corresponding deletion of the C-C’ loop and also has a similar 3D structure of the remaining A and B-B’ loops in the same shape in geometric space of its cognate mod-ITR.

As used herein, the phrase an “effective amount” or “therapeutically effective amount” of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce the desired effect, e.g., inhibition of expression of a target sequence in comparison to the expression level detected in the absence of a therapeutic nucleic acid. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

As used herein, the term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. As used herein, the phrase “expression products” include RNA transcribed from a gene (e.g., transgene), and polypeptides obtained by translation of mRNA transcribed from a gene.

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MEl\57916143.vl 131698-30520

As used herein, the term “expression vector” refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the host cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The expression vector may be a recombinant vector.

As used herein, the term “flanking” refers to a relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, B is flanked by A and C. The same is true for the arrangement AxBxC. Thus, a flanking sequence precedes or follows a flanked sequence but need not be contiguous with, or immediately adjacent to the flanked sequence.

As used herein, the term “spacer region” refers to an intervening sequence that separates functional elements in a vector or genome. In some embodiments, spacer regions keep two functional elements at a desired distance for optimal functionality. In some embodiments, the spacer regions provide or add to the genetic stability of the vector or genome. In some embodiments, spacer regions facilitate ready genetic manipulation of the genome by providing a convenient location for cloning sites and a gap of design number of nucleotide.

As used herein, the terms “expression cassette” and “expression unit” are used interchangeably, and refer to a heterologous DNA sequence that is operably linked to a promoter or other DNA regulatory sequence sufficient to direct transcription of a transgene of a DNA vector, e.g., synthetic AAV vector. Suitable promoters include, for example, tissue specific promoters. Promoters can also be of AAV origin.

As used herein, a “vector” or “expression vector” is a replicon, which can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral in origin in the final form. For the purpose of the present disclosure, a “vector” generally refers to synthetic, capsid-free AAV, for example a single-stranded (ss) synthetic vector or a nicked ceDNA vector. Accordingly, the term “vector” encompasses any genetic element that is capable of replication or expression when associated with the proper control elements and that can transfer gene sequences to cells. In some embodiments, a vector can be a recombinant vector or an expression vector. It is to be understood that the term “single -stranded (ss) synthetic vector” as used herein includes a single -stranded AAV-like vector that may not have any viral sequence(s).

As used herein, the term “genetic disease” refers to a disease, partially or completely, directly or indirectly, caused by one or more abnormalities in the genome, especially a condition that is present from birth and can be treated by a single -stranded (ssDNA) molecule as described herein. The abnormality may be a mutation, an insertion or a deletion. The abnormality may affect the coding sequence of the gene or its regulatory sequence. The genetic disease may be, but not limited to

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MEl\57916143.vl 131698-30520 phenylketonuria (PKU), melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis, Huntington’s disease (Huntington’s chorea), familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson’s disease, congenital hepatic porphyria, inherited disorders of hepatic metabolism, Lesch- Nyhan syndrome, sickle cell disease, thalassemias, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, and mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS Type I), Scheie syndrome (MPS Type I S), Hurler-Scheie syndrome (MPS Type I H-S), Hunter syndrome (MPS Type II), Sanfilippo Types A, B, C, and D (MPS Types III A, B, C, and D), Morquio Types A and B (MPS IVA and MPS IVB), Maroteaux- Lamy syndrome (MPS Type VI), Sly syndrome (MPS Type VII), hyaluronidase deficiency (MPS Type IX)), Niemann-Pick Disease Types A/B, Cl and C2, Fabry disease, Schindler disease, GM2- gangliosidosis Type II (Sandhoff Disease), Tay-Sachs disease, Metachromatic Leukodystrophy, Krabbe disease, Mucolipidosis Type I, II/III and IV, Sialidosis Types I and II, Glycogen Storage disease Types I and II (Pompe disease), Gaucher disease Types I, II and III, cystinosis, Batten disease, Aspartylglucosaminuria, Salla disease, Danon disease (LAMP -2 deficiency), Lysosomal Acid Lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipidoses, and galactosialidosis. Also included in genetic disorders are amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Alzheimer’s disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich’s ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophies (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber Congenital Amaurosis (LCA, e.g., LCA10 [CEP290]), Stargardt macular dystrophy (ABCA4), and Cathepsin A deficiency.

As used herein, the terms “autoimmune disease”, “autoimmune disorder”, and “autoimmune condition” refer to diseases, disorders, and/or conditions that result when a subject’s own immune system attacks the subject’s own tissues and/or cells, as if they were foreign (e.g., as if they were a foreign pathogen or infectious agent). Autoimmune diseases or disorders may include, but are not limited, to, rheumatoid arthritis, multiple sclerosis, type I diabetes, systemic lupus erythematosus, psoriasis, psoriatic arthritis, Sjogren’s syndrome, Crohn’s disease, Celiac disease, ulcerative colitis, Graves’ disease, Hashimoto’s thyroiditis, Addison’s disease, dermatomyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome, myasthenia gravis, autoimmune vasculitis, pernicious anemia, vitiligo, systemic sclerosis, scleroderma, diffuse scleroderma, limited scleroderma, linear scleroderma, localized scleroderma, hemolytic anemia, inflammatory bowel disease (IBD), achantholysis, acute disseminated encephalomyelitis, adult-onset Still disease, allergic glomerulonephritis, ANCA associated vasculitis, ankylosing spondylitis, anti -glomerular basement membrane disease, anti-N-methyl-D-aspartate receptor encephalitis, antiphospholipid syndrome, antisynthetase syndrome, autoimmune gastritis, autoimmune hypophysitis, autoimmune liver disease, autoimmune lung disease, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis,

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MEl\57916143.vl 131698-30520 autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, autoimmune skin disease, autoimmune thyroiditis, autoimmune uveitis, Behcet diseae, bullous pemphigoid, Churg Strauss syndrome, dermatitis herpetiformis, endocrine ophthalmopathy, erythematous pemphigus, Felty syndrome, Giant cell arteritis, IgA glomerulonephritis, membranous glomerulonephritis, Goodpasture syndrome, Granulomatosis with polyangiitis, Graves’ ophthalmopathy, Hailey Hailey syndrome, heparin induced thrombocytopenia, autoimmune hepatitis, idiopathic hypoparathryroidism, idiopathic thrombocytopenic purpura, IgA pemphigus, immune complex nephritis, immunoglobulin A nephropathy, juvenile rheumatoid arthritis, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, linear IgA bullous dermatosis, lupus nephritis, lupus vasculitis, microscopic polyangiitis, morphea, mucous membrane phemphigoid, myasthenia gravis, nonarticular rheumatism, sympathetic ophthalmia, opsoclonous myoclonus syndrome, paraneoplastic pemphigus, pemphigoid, pemphigoid gestationis, pemphigus, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, autoimmune polyendocrinopathies, polyradiculoneuropathy, postpartum thyroiditis, primary biliary cirrhosis, rheumatic disease, rheumatic heart disease, rheumatoid polymyalgia, rheumatoid nodule, rheumatoid vasculitis, stiff-person syndrome, CREST syndrome, systemic juvenile idiopathic arthritis, sarcoidosis, and uveomeningoencephalitic syndrome. Autoimmune diseases, disorders, and conditions may be idiopathic (that is, they occur without any perceived cause), or they may be induced, by, e.g., treatments (drugs or other types of therapy) or infectious agents (e.g., an immune response mounted against a virus or bacteria that then also attacks the subject’s own cells and/or tissues).

The phrase “disease, disorder or condition associated with an elevated expression of a tumor antigen” as used herein is meant to include, but is not limited to, a disease associated with expression of a tumor antigen as described herein or condition associated with cells which express a tumor antigen as described herein including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia; or a noncancer related indication associated with cells which express a tumor antigen as described herein. In some aspects, a cancer associated with expression of a tumor antigen as described herein is a hematological cancer. In some aspects, a cancer associated with expression of a tumor antigen as described herein is a solid cancer. Further diseases associated with expression of a tumor antigen described herein include, but not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of a tumor antigen as described herein. Non-cancer related indications associated with expression of a tumor antigen as described herein include, but are not limited to, e.g., autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation. In some embodiments, the tumor antigenexpressing cells express, or at any time expressed, mRNA encoding the tumor antigen. In an embodiment, the tumor antigen-expressing cells produce the tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal levels or reduced levels. In an

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MEl\57916143.vl 131698-30520 embodiment, the tumor antigen-expressing cells produced detectable levels of a tumor antigen protein at one point, and subsequently produced substantially no detectable tumor antigen protein.

The term “cancer” as used herein refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.

The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

According to some embodiments, a polypeptide of the disclosure is an ApoE or an ApoB polypeptide. According to some embodiments, the ApoE polypeptide is a functional fragment (or a functional portion) of the full length ApoE polypeptide. According to some embodiments, the ApoE polypeptide is a functional fragment (or a functional portion) of the full length ApoB polypeptide. According to some embodiments, the ApoE polypeptide is 30 amino acids in length or less. According to some embodiments, the ApoB polypeptide is 30 amino acids in length or less.

As used herein, the term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and P-acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipids described above can be mixed with other lipids including triglycerides and sterols.

As used herein, the term “polyglycerol” refers to an organic compound that is a polymeric condensation product of glycerol. Polyglycerols obtained from the dehydration of glycerol can have linear, branched, or cyclic structures. In some embodiments, the polyglycerol of the disclosure is linear or branched. In one embodiment, the polyglycerol is linear. In one embodiment, the polyglycerol is branched. In some embodiments, the term “polyglycerol” encompasses a population of polyglycerol molecules. A population of polyglycerol molecules may comprise a distribution of polyglycerol molecules of different lengths, z.e., a distribution of polyglycerol molecules comprising different numbers of monomeric units. Thus, the term “average molecular weight”, when used herein

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MEl\57916143.vl 131698-30520 in reference to polyglycerol, refers to an average molecular weight of a population of polyglycerol molecules. The average molecular weight of a polyglycerol may be determined by any method known in the art, e.g. , MALDI-MS or NMR. The term “average”, when used herein in reference to the number of monomeric units present in a polyglycerol, refers to an average number of monomeric units per polyglycerol molecule in a population of polyglycerol molecules. Thus, the language, e.g., “an average of 45 monomeric units”, when used herein in reference to the number of monomeric units present in a polyglycerol, refers to an average of 45 monomeric units per polyglycerol molecule in a population of polyglycerol molecules. The average number of monomeric units per polyglycerol molecule may be calculated based on an average molecular weight of a polyglycerol.

In some embodiments, a polyglycerol may comprise an average of 8 to 100 monomeric units, e.g., an average of 8 to 40 monomeric units, an average of 15-75 monomeric units, an average of 20 to 50 monomeric units, an average of 30 to 70 monomeric units, an average of 40 to 90 monomeric units or an average of 50 to 100 monomeric units. In some embodiments, the average number of monomeric units present in a polyglycerol molecule is at least about 20, at least about 25, at least about 27, at least about 30, at least about 34, at least about 35, at least about 39, at least about 40, at least about 45, at least about 46, at least about 50, at least about 55, at least about 58, at least about 60, at least about 65, at least about 68, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 monomeric units.

In some embodiments, the polyglycerol of the disclosure comprises an average of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 monomeric units.

As used herein, the term “linear”, as it refers to a polyglycerol or an aliphatic hydrocarbon chain, means that the chain is unbranched.

As used herein, the term “polyglycerol derivative” or a “PG derivative” refers to polyglycerol in which free alcohol groups have been modified. In some embodiments, the polyglycerol derivative of the disclosure is linear or branched. In one embodiment, a polyglycerol derivative is linear. In one embodiment, the polyglycerol derivative is branched.

In some embodiments, the term “polyglycerol derivative” encompasses a population of polyglycerol derivative molecules. A population of polyglycerol derivative molecules may comprise a distribution of polyglycerol derivative molecules of different lengths, z.e., distribution of polyglycerol derivative molecules comprising different numbers of monomeric units. Thus, the term “average molecular weight”, when used herein in reference to a polyglycerol derivative, refers to an average molecular weight of a population of polyglycerol derivative molecules. An average molecular weight of a polyglycerol derivative may be determined by any method known in the art, e.g., MALDI-MS or NMR. The term “average”, when used herein in reference to the number of

46

MEl\57916143.vl 131698-30520 monomeric units present in a polyglycerol derivative, refers to an average number of monomeric units per polyglycerol derivative molecule in a population of polyglycerol derivative molecules. Thus, the language, e.g, “an average of 8 monomeric units”, when used herein in reference to the number of monomeric units present in a polyglycerol derivative, refers to an average of 8 monomeric units per polyglycerol derivative molecule in a population of polyglycerol derivative molecules. An average number of monomeric units per polyglycerol derivative molecule may be calculated based on an average molecular weight of a polyglycerol derivative.

In some embodiments, a polyglycerol derivative of the disclosure may comprise an average of 8 to 100 monomeric units, e.g., an average of 8 to 40 monomeric units, an average of 15-75 monomeric units, an average of 20 to 50 monomeric units, an average of 30 to 70 monomeric units, an average of 40 to 90 monomeric units or an average of 50 to 100 monomeric units. In some embodiments, the polyglycerol of the disclosure comprises an average of 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,

41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

95, 96, 97, 98, 99 or 100 monomeric units.

In some embodiments, a polyglycerol derivative may be a carboxylated polyglycerol, i.e., a polyglycerol in which the free alcohol groups have been modified by converting them into a moiety comprising one or more carboxylate groups, e.g., 2 -carboxy cyclohexane-1 -carboxylated polyglycerol. In some embodiments, a polyglycerol derivative may be a glutarylated polyglycerol, i.e., a polyglycerol in which free alcohol groups have been modified by converting them into a glutarate or a glutarate derivative, e.g., 3-methyl glutarylated polyglycerol. In some embodiments, a polyglycerol derivative may be conjugated to a lipid moiety. In some embodiments, a polyglycerol derivative may be conjugated to a lipid moiety represented by Formula (I) as described herein. In some embodiments, a polyglycerol derivative that is conjugated to a lipid moiety is represented by the following structural formula: wherein: n is an integer ranging from 8 to 100; and

R is selected from the group consisting

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MEl\57916143.vl 131698-30520

As used herein, the term “hydrophobic tail” refers to a hydrocarbon chain, i.e., a chain containing carbon and hydrogen atoms, that can be saturated or unsaturated. In one embodiment, the hydrophobic tail may comprise 10-30 carbon atoms, e.g., 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In some embodiments, the hydrocarbon chain of a hydrophobic tail is unsaturated, z.e., does not comprise double or triple bonds. In other embodiments, the hydrocarbon chain of a hydrophobic tail is unsaturated, comprising one or more double bonds and/or one or more triple bonds. In some embodiments, the hydrocarbon chain may be a linear chain. In other embodiments, the hydrocarbon chain may be a branched chain. Non-limiting examples of backbone hydrophobic tail of the present disclosure include the hydrophobic tails present in lauric acid, myristic acid, myristoleic acid, octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, and a derivative thereof.

As used herein, the term “click chemistry reaction product” refers to a moiety formed by two click chemistry reagents of a “click pair”. In some embodiments, the click chemistry reaction product is a product of a reaction between: a) a tetrazine reagent (z. e. , a reagent comprising a tetrazine moiety) and a transcyclooctene reagent (z'.e., a reagent comprising a transcyclooctene moiety); b) a tetrazine reagent and a norbomene reagent (z'.e., a reagent comprising a norbomene moiety); or c) a an azide reagent (z'.e., a reagent comprising an azide moiety) and an alkyne reagent, e.g., a dibenzocyclooctyne (DBCO) reagent.

As used herein, the term “lipid-anchored polymer”, which may be used herein interchangeably with the term “lipid conjugate”, refers to a molecule comprising a lipid moiety covalently attached to a polymer, optionally via a linker. Without wishing to be bound by a specific theory, it is believed that a lipid-anchored polymer can inhibit aggregation of LNPs and provide steric stabilization and prolonged blood half-life (ti/2) in vivo. As used herein, the terms “lipid-anchored polymer” and “polymer-conjugated lipid” may be used interchangeably. Exemplary lipid-anchored polymers include, but are not limited to polymer-conjugated lipids as described herein, PEGylated lipids such as, e.g., PEG coupled to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to dimyristolglycerol (e.g., PEG-DMG), PEG coupled to distearoyl glycerol (e.g., PEG-DSG), PEG coupled to poly(2 -methacryloyloxyethyl phosphorylcholine) (e.g., PEG-PMPC), PEG coupled to l,2-distearoyl-sn-glycero-3- phosphoethanolamine (e.g., PEG-DSPE), polyglycerol (PG)-lipid conjugates such as DODA-PG, and mixtures thereof, comprising from about 5-100 monomeric units, including DODA-PG34, DODA- PG39, DODA-PG45, DODA-PG-46, DODA-PG50, DODA-PG58, and DODA-PG68, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides (see, e.g., U.S. Patent No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates; see, e.g., U.S. Provisional Application No. 61/294,828, fded Jan. 13, 2010,

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MEl\57916143.vl 131698-30520 and U.S. Provisional Application No. 61/295,140, filed Jan. 14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Additional examples of POZ -lipid conjugates are described in International Patent Application Publication No. WO 2010/006282. PEG, PG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG, PG or the POZ to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, non-ester containing linker moieties, such as amides or carbamates, are used. The disclosures of each of the above patent documents are herein incorporated by reference in their entirety for all purposes.

Exemplary linkers that may be used to conjugate a lipid moiety to the polymer in a lipid- anchored polymer of the present disclosure may be selected from the group consisting of an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker (e.g., a glutaryl linker, a succinyl linker), an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, a click reaction product, and any combination thereof. In some embodiments, the linker may be selected from the group consisting of -(CEEjn-, -C(O)(CH2)_n-, - C(O)O(CH₂)_n-, -OC(O)(CH2)_nC(O)O-, and -NH(CH2)_nC(O)O-, wherein n is an integer ranging from 1 to 20. In some embodiments, the linker is -C(O)(CH2)_n-, and wherein n is 2, 3, 4, 5, or 6. In one specific embodiment, n is 4.

As used herein, the term “lipid encapsulated” refers to a lipid particle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., a ceDNA or an siRNA), with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form a nucleic acid containing lipid particle).

As used herein, the terms “lipid particle” or “lipid nanoparticle” or “LNP” refers to a lipid formulation that can be used to deliver a therapeutic agent such as nucleic acid therapeutics to a target site of interest (e.g., cell, tissue, organ, and the like). In one embodiment, the lipid particle of the disclosure is a nucleic acid containing lipid particle, which is typically formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle. In other preferred embodiments, a therapeutic agent such as a therapeutic nucleic acid may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation. In one embodiment, the lipid particle comprises a nucleic acid (e.g., ceDNA, ssDNA, mRNA, siRNA etc.) and a lipid comprising one or more tertiary amino groups, one or more phenyl ester bonds and a disulfide bond.

According to some embodiments, lipid particles of the disclosure typically have a mean diameter of from about 20 nm to about 75 nm, about 20 nm to about 70 nm, about 25 nm to about 75 nm, about 25 nm to about 70 nm, from about 30 nm to about 75 nm, from about 30 nm to about 70 nm, from about 35 nm to about 75 nm, from about 35 nm to about 70 nm, from about 40 nm to about 75 nm, from about 40 nm to about 70 nm, from about 45 nm to about 75 nm, from about 50 nm to

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MEl\57916143.vl 131698-30520 about 75 nm, from about 50 nm to about 70 nm, from about 60 rim to about 75 nm, from about 60 nm to about 70 nm, from about 65 nm to about 75 nm, from about 65 nm to about 70 nm, or about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 51 nm, about 52 nm, about 53 nm, about 54 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, about 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, or about 75 nm (± 3 nm) in size.

Generally, the LNPs of the disclosure have a mean diameter selected to provide an intended therapeutic effect. For example, the LNPs of the disclosure have a mean diameter that is compatible with a target organ, such that the LNPs of the disclosure are able to diffuse through the fenestrations of atarget organ (e.g., liver) or a target cell subpopulation (e.g., hepatocytes).

According to some embodiments, the lipid particles of the disclosure typically have a mean diameter of less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, less than about 20 nm in size.

As used herein, the term “cationic lipid” refers to any lipid that is positively charged at physiological pH. The cationic lipid in the lipid particles may comprise, e.g., one or more cationic lipids such as l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N,N-dimethylaminopropane (y- DLenDMA), 2, 2-dilinoleyl-4-(2-dimethylaminoethyl)-[ 1,3] -dioxolane (DLin-K-C2-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), “SS-cleavable lipid”, or a mixture thereof. In some embodiments, a cationic lipid can also be an ionizable lipid, i.e., an ionizable cationic lipid, i.e. The term “cationic lipids” also encompasses lipids that are positively charged at any pH, e.g., lipids comprising quaternary amine groups, i.e., quarternary lipids. Any cationic lipid described herein comprising a primary, secondary or tertiary amine group may be converted to a corresponding quaternary lipid, for example, by treatment with chloromethane (CH3CI) in acetonitrile (CH3CN) and chloroform (CHCI3).

As used herein, the term “ionizable lipid” refers to a lipid, e.g., cationic lipid, having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipids be present in the charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. In some embodiments, ionizable lipid may include “cleavable lipid” or “SS- cleavable lipid”.

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MEl\57916143.vl 131698-30520

As used herein, the term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

As used herein, the term “non-cationic lipid” refers to any amphipathic helper lipid as well as any other neutral lipid or anionic lipid.

As used herein, the term “cleavable lipid” or “SS-cleavable lipid” refers to an ionizable lipid comprising a disulfide bond cleavable unit. Cleavable lipids may include cleavable disulfide bond (“ss”) containing lipid-like materials that comprise a pH-sensitive amine, e.g., a tertiary amine, and self-degradable phenyl ester. For example, a SS-cleavable lipid can be an ss-OP lipid (COATSOME® SS-OP), an ss-M lipid (COATSOME® SS-M), an ss-E lipid (COATSOME® SS-E), an ss-EC lipid (COATSOME® SS-EC), an ss-LC lipid (COATSOME® SS-LC), an ss-OC lipid (COATSOME® SS- OC), and an ss-PalmE lipid (see, for example, Formulae I-IV), or a lipid described by Togashi et al., (2018) Journal of Controlled Release “A hepatic pDNA delivery system based on an intracellular environment sensitive vitamin E -scaffold lipid-like material with the aid of an anti-inflammatory drug” 279:262-270. Additional examples of cleavable lipids are described in US Patent No. 9,708,628, and US Patent No. 10,385,030, the entire contents of which are incorporated herein by reference. In one embodiment, cleavable lipids comprise a tertiary amine, which responds to an acidic compartment, e.g., an endosome or lysosome for membrane destabilization and a disulfide bond that can be cleaved in a reducing environment, such as the cytoplasm. In one embodiment, a cleavable lipid is a cationic lipid. In one embodiment, a cleavable lipid is an ionizable cationic lipid. Cleavable lipids are described in more detail herein.

As used herein, the term “organic lipid solution” refers to a composition comprising in whole, or in part, an organic solvent having a lipid.

As used herein, the term “liposome” refers to lipid molecules assembled in a spherical configuration encapsulating an interior aqueous volume that is segregated from an aqueous exterior. Liposomes are vesicles that possess at least one lipid bilayer. Liposomes are typical used as carriers for drug/ therapeutic delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient. Liposome compositions for such delivery are typically composed of phospholipids, especially compounds having a phosphatidylcholine group, however these compositions may also include other lipids.

As used herein, the term “local delivery” refers to delivery of an active agent such as an interfering RNA (e.g., siRNA) directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site such as a tumor or other target site such as a site of inflammation or a target organ such as the liver, heart, pancreas, kidney, and the like.

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As used herein, the term “nucleic acid” refers to a polymer containing at least two nucleotides (z.e., deoxyribonucleotides or ribonucleotides) in either single- or double -stranded form and includes DNA, RNA, and hybrids thereof. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. DNA may be in the form of minicircle, plasmid, bacmid, minigene, ministring DNA (linear covalently closed DNA vector), closed-ended linear duplex DNA (CELiD or ceDNA), doggybone™ DNA, dumbbell shaped DNA, minimalistic immunological- defined gene expression (MIDGE) -vector, viral vector or nonviral vectors. RNA may be in the form of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), messenger RNA (mRNA), rRNA, tRNA, gRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs and/or modified residues include, without limitation, phosphorothioates, phosphorodiamidate morpholino oligomer (morpholino), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, locked nucleic acid (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.

As used herein, the phrases “nucleic acid therapeutic”, “therapeutic nucleic acid” and “TNA” are used interchangeably and refer to any modality of therapeutic using nucleic acids as an active component of therapeutic agent to treat a disease or disorder. As used herein, these phrases refer to RNA-based therapeutics and DNA-based therapeutics. Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNAs (RNAi), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA) or guide RNA (gRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (e.g., Lentiviral or AAV genome) or nonviral synthetic DNA vectors, closed-ended linear duplex DNA (ceDNA / CELiD), single stranded DNA (ssDNA) molecules, plasmids, bacmids, DOGGYBONE™ DNA vectors, minimalistic immunological-defmed gene expression (MIDGE)-vector, nonviral ministring DNA vector (linear- covalently closed DNA vector), or dumbbell-shaped DNA minimal vector (“dumbbell DNA”).

As used herein, “nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.

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As used herein, the terms “single-stranded DNA molecule”, “ssDNA molecule”, or “SSD molecule” refer to a deoxyribonucleic acid (DNA) molecule comprising at least one single-stranded nucleic acid sequence flanked by at least one stem -loop structure at the 3’ end. In some embodiments, the single -stranded DNA molecule further comprises at least one stem -loop structure at the 5’ end. As used herein, a single-stranded DNA molecule may comprise regions of double-stranded DNA (or partial duplexes), e.g., a stem -loop structure, e.g., an inverted terminal repeat or portion thereof, at the terminal end(s), e.g., the 3’ end and/or the 5’ end. In some embodiments, a ssDNA molecule is a synthetic ssDNA molecule. In some embodiments, a ssDNA molecule comprises at least one stemloop structure at the 5’ end and at least one stem -loop structure at the 3’ end.

As used herein, the term “single-stranded (ss) synthetic DNA molecules”, “single-stranded (ss) synthetic vectors”, “synthetic production of ss DNA molecules” and “synthetic production of ss vectors” refers to a single-stranded (ss) synthetic DNA molecule (ssDNA), a single-stranded vector and synthetic production methods thereof in an entirely cell-free environment. The production may involve one or more molecules in a manner that does not involve replication or other multiplication of the molecule by or inside of a cell or using a cellular extract. Synthetic production avoids contamination of the produced molecule with cellular contaminants, e.g. , cellular proteins or cellular nucleic acid, viral protein or DNA, insect protein or DNA and further minimizes unwanted cellular- specific modification of the molecule during the production process, e.g., methylation or glycosylation or other post-translational modification.

As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound.

As used herein, the term “gap” refers to a discontinued portion of synthetic DNA vector of the present disclosure, creating a stretch of single stranded DNA portion in otherwise double stranded ceDNA. The gap can be 1 nucleotide to 100 nucleotides in length in one strand of a duplex DNA. Typical gaps, designed and created by the methods described herein and synthetic vectors generated by the methods can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nt long in length. Exemplified gaps in the present disclosure can be 1 nt to 10 nt long, 1 to 20 nt long, 1 to 30 nt long in length.

As used herein, the term “nick” refers to a discontinuity in a double stranded DNA molecule where there is no phosphodiester bond between adjacent nucleotides of one strand typically through damage or enzyme action. It is understood that one or more nicks allow for the release of torsion in

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MEl\57916143.vl 131698-30520 the strand during DNA replication and that nicks are also thought to play a role in facilitating binding of transcriptional machinery.

As used herein, the term “ceDNA” refers to capsid-free closed-ended linear double-stranded (ds) duplex DNA for non-viral gene transfer, synthetic or otherwise. Detailed description of ceDNA is described in International Patent Application No. PCT/US2017/020828, fded March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for the production of ceDNA comprising various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International applications PCT/US 18/49996, filed September 7, 2018, and PCT/US2018/064242, filed December 6, 2018 each of which is incorporated herein in its entirety by reference. Certain methods for the production of synthetic ceDNA vectors comprising various ITR sequences and configurations are described, e.g., in International application PCT/US2019/14122, filed January 18, 2019, the entire content of which is incorporated herein by reference. As used herein, the terms “ceDNA vector” and “ceDNA” are used interchangeably. According to some embodiments, the ceDNA is a closed-ended linear duplex (CELiD) CELiD DNA. According to some embodiments, the ceDNA is a DNA-based minicircle. According to some embodiments, the ceDNA is a minimalistic immunological -defined gene expression (MIDGE) -vector. According to some embodiments, the ceDNA is a ministring DNA. According to some embodiments, the ceDNA is a doggybone™ DNA. According to some embodiments, the ceDNA comprises one or more phosphorothioate-modified nucleotides. According to some embodiments, the ceDNA comprises no phosphorothioate-modified nucleotides.

As used herein, the term “neDNA” or “nicked ceDNA” refers to a closed-ended DNA having a nick or a gap of 1-100 nucleotides in a stem region or spacer region upstream of an open reading frame (e.g., a promoter and transgene to be expressed).

As used herein, the terms “inverted terminal repeat” or “ITR” refer to a nucleic acid sequence located at the 5’ and/or 3’ terminus of the ssDNA molecules disclosed herein, which comprises at least one stem -loop structure comprising a partial duplex and at least one loop.

As used herein, the term “stem-loop structure” refers to a nucleic acid structure comprising at least one double-stranded region (referred to herein as a “stem”) and at least one single -stranded region (referred to herein as a “loop”). In some embodiments, a stem-lop structure is a hairpin structure. In some embodiments, a stem -loop structure comprises more than one stem and more than one loop. In some embodiments, a loop is located at the end of a stem (such that a single loop connects the two strands of a duplex stem, e.g., as in a hairpin structure). In some embodiments, a loop may be located between two stems (which may be referred to herein as a “bulge” or a “bubble”), such that the loop connects two strands of different stems. In some embodiments, as described in more detail herein, a stem-loop structure may comprise more complex secondary structures comprising multiple stems and multiple loops.

According to some embodiments, the 5’ and/or 3’ terminus of certain ssDNA molecules

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MEl\57916143.vl 131698-30520 comprise inverted terminal repeats (ITRs) of about 145 nucleotides at both ends, or fragments thereof. The terminal 125 nucleotides in each ITR form a palindromic double-stranded T-shaped hairpin structure, in which the A-A' palindrome forms the stem, and the two smaller palindromes, B-B' and the C-C, form the cross-arms of the T. The other 20 nucleotides in ITR remain single -stranded, and are called the D sequence. The D(-) sequence (also referred to herein as “the ssD(-) sequence”) is at the 3' end, and the complementary D(+) sequence (also referred to herein as “the ssD(+) sequence”) is at the 5' end. Second-strand DNA synthesis turns both ssD(-) and ssD(+) sequences into a doublestranded (ds) D(±) sequence, each of which comprises a D region and a D’ region. Ling et al. J Virol. 2015 Jan 15 ;89(2):952-61 , WO2016081927A2, incorporated by reference in its entirety herein, described ssD(+)-sequence-substituted ssAAV genomes. ssD(-) and ssD(+) have been reported to contain one or more transcription factor binding sites and to be required for packaging and replication (Ling et al. J Virol. 2015 Jan 15 ;89(2): 952-61 ; WO2016081927A2, incorporated by reference in its entirety herein).

According to some embodiments, the ITR may be a viral ITR (e.g., AAV or other dependo virus), a sequence derived or modified from a viral ITR (e.g., truncation, deletion, substitution, insertion and/or addition), or an entirely artificial sequence (e.g., the ITRs contain no sequences derived from a virus). The ITR may further comprise one stem-loop structure (e.g., a “hairpin”), or more than one stem-loop structure. For example, the ITR may comprise two stem-loop structures (e.g., a “hammerhead”, “doggy-bone”, or “dumbbell”), three stem-loop structures (e.g., “cruciform”), or more complex structures (e.g., quadruplex stem-loop structure). The ITR may comprise an aptamer sequence or one or more chemical modifications. The ITR can be made entirely out of an aptamer sequence having at least one stem region and at least one loop region.

As used herein, the term “substantially symmetrical WT-ITRs” or a “substantially symmetrical WT-ITR pair” refers to a pair of WT-ITRs within a single -stranded DNA (ssDNA) molecule that are both wild type ITRs that have an inverse complement sequence across their entire length. For example, an ITR can be considered to be a wild-type sequence, even if it has one or more nucleotides that deviate from the canonical naturally occurring canonical sequence, so long as the changes do not affect the physical and functional properties and overall three-dimensional structure of the sequence (secondary and tertiary structures). In some aspects, the deviating nucleotides represent conservative sequence changes. As one non-limiting example, a sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (as measured, e.g, using BLAST at default settings), and also has a symmetrical three-dimensional spatial organization to the other WT- ITR such that their 3D structures are the same shape in geometrical space. The substantially symmetrical WT-ITR has the same ssD(-)/ssD(+), A-A’, C-C’ and B-B’ loops in 3D space. A substantially symmetrical WT-ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE’) and terminal resolution site (TRS) that pairs with the appropriate Rep protein. One can optionally test other functions, including transgene expression under

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MEl\57916143.vl 131698-30520 permissive conditions.

As used herein, the phrases “modified ITR” or “mod-ITR” or “mutant ITR” are used interchangeably and refer to an ITR with a mutation in at least one or more nucleotides as compared to the WT-ITR from the same serotype. The mutation can result in a change in one or more of ssD(-) or ssD(+), A, A’, C, C’, B, B’ regions in the ITR, and can result in a change in the three-dimensional spatial organization (z.e., its 3D structure in geometric space) as compared to the 3D spatial organization of a WT-ITR of the same serotype.

The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes single, double, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer including purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. “Oligonucleotide” generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as “oligomers” or “oligos” and may be isolated from genes, or chemically synthesized by methods known in the art. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. According to some embodiments, the nucleic acid is a single -stranded DNA (ssDNA) molecule described by the present disclosure. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. DNA may be in the form of minicircle, plasmid, bacmid, minigene, ministring DNA (linear covalently closed DNA vector), closed-ended linear duplex DNA (CELiD or ceDNA), doggybone (dbDNA ™) DNA, dumbbell shaped DNA, minimalistic immunological-defined gene expression (MIDGE)-vector, viral vector or nonviral vectors. RNA may be in the form of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs and/or modified residues include, without limitation, phosphorothioates, phosphorodiamidate morpholino oligomer (morpholino), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, locked nucleic acid (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively

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MEl\57916143.vl 131698-30520 modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.

An “inhibitory polynucleotide” as used herein refers to a DNA or RNA molecule that reduces or prevents expression (transcription or translation) of a second (target) polynucleotide. Inhibitory polynucleotides include antisense polynucleotides, ribozymes, and external guide sequences. The term “inhibitory polynucleotide” further includes DNA and RNA molecules, e.g., RNAi that encode the actual inhibitory species, such as DNA molecules that encode ribozymes.

“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.

“Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.

By “receptor” is meant a polypeptide, or portion thereof, present on a cell membrane that selectively binds one or more ligands. The term “receptor” as used herein is intended to encompass the entire receptor or ligand-binding portions thereof. These portions of the receptor particularly include those regions sufficient for specific binding of the ligand to occur.

As used herein, the term “in vivo” refers to assays or processes that occur in or within an organism, such as a multicellular animal. In some of the aspects described herein, a method or use can be said to occur “in vivo” when a unicellular organism, such as a bacterium, is used. The term “ex vivo” refers to methods and uses that are performed using a living cell with an intact membrane that is outside of the body of a multicellular animal or plant, e.g., explants, cultured cells, including primary cells and cell lines, transformed cell lines, and extracted tissue or cells, including blood cells, among others. The term “in vitro” refers to assays and methods that do not require the presence of a cell with an intact membrane, such as cellular extracts, and can refer to the introducing of a programmable synthetic biological circuit in a non-cellular system, such as a medium not comprising cells or cellular systems, such as cellular extracts.

As used herein, the term “subject” refers to a human or animal, to whom treatment, including prophylactic treatment, with the therapeutic nucleic acid according to the present disclosure, is provided. Usually, the animal is a vertebrate such as, but not limited to a primate, rodent, domestic animal or game animal. Primates include but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate or a human. A subject can be male or female. Additionally, a subject can be an infant or a child. In some embodiments, the subject can

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MEl\57916143.vl 131698-30520 be a neonate or an unborn subject, e.g., the subject is in utero. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of diseases and disorders. In addition, the methods and compositions described herein can be used for domesticated animals and/or pets. A human subject can be of any age, gender, race or ethnic group, e.g., Caucasian (white), Asian, African, black, African American, African European, Hispanic, Mideastem, etc. In some embodiments, the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment. In some embodiments, the subject is an embryo, a fetus, neonate, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human neonate, human infant, human child, human adolescent, or human adult. In some embodiments, the subject is an animal embryo, or non-human embryo or non-human primate embryo. In some embodiments, the subject is a human embryo.

As used herein, the phrase “subject in need” refers to a subject that (i) will be administered a lipid nanoparticle (LNP) comprising a therapeutic nucleic acid (TNA) (or pharmaceutical composition comprising an LNP comprising a TNA) according to the described disclosure; (ii) is receiving an LNP comprising a TNA (or pharmaceutical composition comprising an LNP comprising a TNA) according to the described disclosure; or (iii) has received an LNP comprising a TNA (or pharmaceutical composition comprising an LNP comprising a TNA) according to the described disclosure, unless the context and usage of the phrase indicates otherwise.

As used herein, the term “suppress,” “decrease,” “interfere,” “inhibit” and/or “reduce” (and like terms) generally refers to the act of reducing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.

As used herein, the term “systemic delivery” refers to delivery of lipid particles that leads to a broad biodistribution of an active agent such as an interfering RNA (e.g., siRNA) within an organism. Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body. To obtain broad biodistribution generally requires a blood lifetime such that the agent is not rapidly degraded or cleared (such as by first pass organs (liver, lung, etc.) or by rapid, nonspecific cell binding) before reaching a disease site distal to the site of administration. Systemic delivery of LNPs can be by any means known in the art including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of LNPs is by intravenous delivery.

As used herein, the terms “effective amount”, which may be used interchangeably with the terms “therapeutic amount”, “therapeutically effective amount”, an “amount effective”, or “pharmaceutically effective amount” of an active agent (e.g., a ceDNA or an siRNA as described herein), refers to an amount that is sufficient to provide the intended benefit of treatment or effect,

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MEl\57916143.vl 131698-30520 e.g., expression or inhibition of expression of a target sequence in comparison to the expression level detected in the absence of a therapeutic nucleic acid. Suitable assays for measuring expression of a target gene or target sequence include, e.g. , examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art. Dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Additionally, the terms “effective amount”, “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the described invention. In prophylactic or preventative applications of the described invention, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment. The terms “dose” and “dosage” are used interchangeably herein. In one aspect of any of the aspects or embodiments herein, “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” refer to non-prophylactic or non-preventative applications.

As used herein the term “therapeutic effect” refers to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.

For any therapeutic agent described herein therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models. A therapeutically effective dose may also be determined from human data. The applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan. General principles for determining therapeutic effectiveness, which may be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.

Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations

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MEl\57916143.vl 131698-30520 where the drug's plasma concentration can be measured and related to therapeutic window, additional guidance for dosage modification can be obtained.

As used herein, the terms “treat,” “treating,” and/or “treatment” include abrogating, inhibiting, slowing or reversing the progression of a condition, ameliorating clinical symptoms of a condition, or preventing the appearance of clinical symptoms of a condition, obtaining beneficial or desired clinical results. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorders) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s). In one aspect of any of the aspects or embodiments herein, the terms “treat,” “treating,” and/or “treatment” include abrogating, inhibiting, slowing or reversing the progression of a condition, or ameliorating clinical symptoms of a condition.

Beneficial or desired clinical results, such as pharmacologic and/or physiologic effects include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (z.e., not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

As used herein, the term “combination therapy” refers to treatment regimens for a clinical indication that comprise two or more therapeutic agents. Thus, the term refers to a therapeutic regimen in which a first therapy comprising a first composition (e.g. , active ingredient) is administered in conjunction with a second therapy comprising a second composition (active ingredient) to a patient, intended to treat the same or overlapping disease or clinical condition. The first and second compositions may both act on the same cellular target, or discrete cellular targets. The phrase “in conjunction with,” in the context of combination therapies, means that therapeutic effects of a first therapy overlaps temporarily and/or spatially with therapeutic effects of a second therapy in the subject receiving the combination therapy. Thus, the combination therapies may be formulated as a single formulation for concurrent administration, or as separate formulations, for sequential administration of the therapies.

As used herein, the term “alkyl” refers to a saturated monovalent hydrocarbon radical of 1 to

20 carbon atoms (i.e. , C1-20 alkyl). “Monovalent” means that alkyl has one point of attachment to the remainder of the molecule. In one embodiment, the alkyl has 1 to 12 carbon atoms (i.e. , C1-12 alkyl) or 1 to 10 carbon atoms (i.e., Ci-w alkyl). In one embodiment, the alkyl has 1 to 8 carbon atoms (i.e., Ci-

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MEl\57916143.vl 131698-30520 s alkyl), 1 to 7 carbon atoms (z.e., C1-7 alkyl), 1 to 6 carbon atoms (z.e., C1-6 alkyl), 1 to 4 carbon atoms (z.e., C1-4 alkyl), or 1 to 3 carbon atoms (z.e., C1-3 alkyl). Examples include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2 -methyl- 1 -propyl, 2-butyl, 2 -methyl -2 -propyl, 1 -pentyl, 2- pentyl, 3 -pentyl, 2-methyl-2-butyl, 3 -methyl -2 -butyl, 3 -methyl- 1 -butyl, 2 -methyl- 1 -butyl, 1 -hexyl, 2- hexyl, 3 -hexyl, 2 -methyl -2 -pentyl, 3 -methyl -2 -pentyl, 4-methyl -2 -pentyl, 3 -methyl-3 -pentyl, 2- methyl-3 -pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. A linear or branched alkyl, such as a “linear or branched CM alkyl,” “linear or branched C1.4 alkyl,” or “linear or branched C1.3 alkyl” means that the saturated monovalent hydrocarbon radical is a linear or branched chain. As used herein, the term “linear” as referring to aliphatic hydrocarbon chains means that the chain is unbranched.

The term “alkylene” as used herein refers to a saturated divalent hydrocarbon radical of 1 to 20 carbon atoms (z.e., C1.20 alkylene), examples of which include, but are not limited to, those having the same core structures of the alkyl groups as exemplified above. “Divalent” means that the alkylene has two points of attachment to the remainder of the molecule. In one embodiment, the alkylene has 1 to 12 carbon atoms (z.e., CM 2 alkylene) or 1 to 10 carbon atoms (z.e., CMO alkylene). In one embodiment, the alkylene has 1 to 8 carbon atoms (z.e., Ci-s alkylene), 1 to 7 carbon atoms (z.e., C1-7 alkylene), 1 to 6 carbon atoms (z.e., C1-6 alkylene), 1 to 4 carbon atoms (z.e., C1.4 alkylene), 1 to 3 carbon atoms (z.e., C1-3 alkylene), ethylene, or methylene. A linear or branched alkylene, such as a “linear or branched C1-6 alkylene,” “linear or branched C1.4 alkylene,” or “linear or branched C1-3 alkylene” means that the saturated divalent hydrocarbon radical is a linear or branched chain.

The term “alkenyl” refers to straight or branched aliphatic hydrocarbon radical with one or more (e.g., one or two) carbon-carbon double bonds, wherein the alkenyl radical includes radicals having “cis” and “trans” orientations, or by an alternative nomenclature, “E” and “Z” orientations.

The term “alkenylene” refers to aliphatic divalent hydrocarbon radical of 2 to 20 carbon atoms (i. e. , C2-20 alkenylene) with one or two carbon-carbon double bonds, wherein the alkenylene radical includes radicals having “cA” and “trans” orientations, or by an alternative nomenclature, “E” and “Z” orientations. “Divalent” means that alkenylene has two points of attachment to the remainder of the molecule. In one embodiment, the alkenylene has 2 to 12 carbon atoms (z.e., C2-12 alkenylene), 2 to 10 carbon atoms (z.e., C2-10 alkenylene). In one embodiment, the alkenylene has 2 to four carbon atoms (C2-4). Examples include, but are not limited to, ethylenylene or vinylene (-CH=CH-), allyl (- CH2CH=CH-), and the like. A linear or branched alkenylene, such as a “linear or branched C2-6 alkenylene,” “linear or branched C2-4 alkenylene,” or “linear or branched C2-3 alkenylene” means that the unsaturated divalent hydrocarbon radical is a linear or branched chain.

The term “cycloalkylene”, as used herein refers to a divalent saturated carbocyclic ring radical having 3 to 12 carbon atoms as a monocyclic ring, or 7 to 12 carbon atoms as a bicyclic ring. “Divalent” means that the cycloalkylene has two points of attachment to the remainder of the molecule. In one embodiment, the cycloalkylene is a 3 - to 7-membered monocyclic or 3- to 6-

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MEl\57916143.vl 131698-30520 membered monocyclic. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, cyclodecylene, cycloundecylene, cyclododecylene, and the like. In one embodiment, the cycloalkylene is cyclopropylene.

The terms “heterocycle,” “heterocyclyl,” heterocyclic and “heterocyclic ring” are used interchangeably herein and refer to a cyclic group which contains at least one N atom has a heteroatom and optionally 1-3 additional heteroatoms selected from N and S, and are non-aromatic (z.e., partially or fully saturated). It can be monocyclic or bicyclic (bridged or fused). Examples of heterocyclic rings include, but are not limited to, aziridinyl, diaziridinyl, thiaziridinyl, azetidinyl, diazetidinyl, triazetidinyl, thiadiazetidinyl, thiazetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, isothiazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, azepanyl, azocanyl, and the like. The heterocycle contains 1 to 4 heteroatoms, which may be the same or different, selected from N and S. In one embodiment, the heterocycle contains 1 to 3 N atoms. In another embodiment, the heterocycle contains 1 or 2 N atoms. In another embodiment, the heterocycle contains 1 N atom. A “4- to 8 -membered heterocyclyl” means a radical having from 4 to 8 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. A “5- or 6-membered heterocyclyl” means a radical having from 5 or 6 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. The term “heterocycle” is intended to include all the possible isomeric forms. Heterocycles are described in Paquette, Leo A., Principles of Modern Heterocyclic Chemistry (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. The heterocyclyl groups may be carbon (carbon-linked) or nitrogen (nitrogen-linked) attached to the rest of the molecule where such is possible.

If a group is described as being “optionally substituted,” the group may be either (1) not substituted, or (2) substituted. If a carbon of a group is described as being optionally substituted with one or more of a list of substituents, one or more of the hydrogen atoms on the carbon (to the extent there are any) may separately and/or together be replaced with an independently selected optional substituent.

Suitable substituents for an alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl, are those which do not significantly adversely affect the biological activity of the molecule. Unless otherwise specified, exemplary substituents for these groups include linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, halogen, guanidinium [-NH(C=NH)NH2], -ORioo, NR101R102, -NO2, -NR101COR102, -SR100, a sulfoxide represented by -SOR101, a sulfone represented by -SO2R101, a sulfonate -SO3M, a sulfate -OSO3M, a sulfonamide represented by -SO2NR101R102, cyano, an azido, -COR101, -OCOR101, -OCONR101R102 and

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MEl\57916143.vl 131698-30520 a polyethylene glycol unit (-OCH2CH2)_nRioi wherein M is H or a cation (such as Na⁺ or K⁺); Rioi, R102 and R103 are each independently selected from H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH2CH2)_n-Rio4, wherein n is an integer from 1 to 24, an aryl having from 6 to 10 carbon atoms, a heterocyclic ring having from 3 to 10 carbon atoms and a heteroaryl having 5 to 10 carbon atoms; and R104 is H or a linear or branched alkyl having 1 to 4 carbon atoms, wherein the alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl in the groups represented by R100, R101, R102, R103 and R104 are optionally substituted with one or more (e.g., 2, 3, 4, 5, 6 or more) substituents independently selected from halogen, -OH, -CN, -NO2, and unsubstituted linear or branched alkyl having 1 to 4 carbon atoms. Preferably, the substituent for the optionally substituted alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl described above is selected from the group consisting of halogen, -CN, -NR101R102, -CF3, -OR100, aryl, heteroaryl, heterocyclyl, -SR101, -SOR101, -SO2R101, and -SO3M. Alternatively, the suitable substituent is selected from the group consisting of halogen, -OH, -NO2, -CN, C1.4 alkyl, -OR100, NR101R102, -NR101COR102, - SR100, -SO2R101, -SO2NR101 R102, -COR101, -OCOR101, and -OCONR101R102, wherein R100, R101, and R102 are each independently -H or C 1.4 alkyl.

The term “halogen”, as used herein, refers to F, Cl, Br or I. “Cyano” is -CN.

The terms “amine” or “amino” are used herein interchangeably and refer to a functional group that contains a basic nitrogen atom with a lone pair.

The term “pharmaceutically acceptable salt”, as used herein, refers to pharmaceutically acceptable organic or inorganic salts of an ionizable lipid of the disclosure. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate “mesylate,” ethanesulfonate, benzene sulfonate, p-toluenesulfonate, pamoate (z.e., l,l’-methylene-bis-(2 -hydroxy-3 -naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion. The counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion.

Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or

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MEl\57916143.vl 131698-30520 patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

In some embodiments, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

Other terms are defined herein within the description of the various aspects of the disclosure.

All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may

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MEl\57916143.vl 131698-30520 also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. It should be understood that this disclosure is not limited in any manner to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

II. Polymer-Conjugated Lipids

The present disclosure provides polymer-conjugated lipids (also referred to herein as “lipid- anchored polymers”), comprising, e.g., a polyglycerol (PG) conjugated to a lipid, methods of their synthesis, and methods of treatment of various disorders comprising administering to a subject in need thereof LNPs of the disclosure. In particular, the present disclosure provides novel LNPs comprising polymer-conjugated lipids that are surprisingly characterized by low levels of undesirable opsonization-driven uptake of LNPs into non-target cells, balanced with desirable levels of endosomal escape, thereby achieving advantageous stealth/endosomal escape tradeoff, as described herein. It has further been surprisingly discovered that the LNPs described herein can be administered in multiple doses without inducing antibody-mediated clearance of the LNPs from the blood, as well as well as physiological characteristics of prolonged blood circulation time (e.g., increased blood ti/2).

In some embodiments, the present disclosure provides a lipid nanoparticle (LNP) comprising a polymer-conjugated lipid, wherein the polymer-conjugated lipid comprises: (i) a polyglycerol (PG) or a PG derivative; (ii) a lipid moiety; and (iii) a linker conjugating the PG or the PG derivative to the lipid moiety.

In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 3 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 4 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 5 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 6 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 7 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 8 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 9 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 10 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 11 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 12 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 14 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 16 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 18 hours. In one

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MEl\57916143.vl 131698-30520 embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 20 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 22 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 24 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 28 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 32 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 36 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 40 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 44 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 48 hours. In one embodiment, the half-life (ti/2) of the ctLNP in blood in vivo is greater than 72 hours.

In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is less than 72 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is less than 96 hours.

In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 3 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 4 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 5 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 6 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 7 hours and about 48 hours. In one embodiment, the halflife (ti/2) of the LNP in blood in vivo is between about 8 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 9 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 10 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 11 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 12 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 16 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 20 hours and about 48 hours. In one embodiment, the halflife (ti/2) of the LNP in blood in vivo is between about 24 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 36 hours and about 48 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 8 hours and about 36 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 12 hours and about 36 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is between about 24 hours and about 36 hours.

Polyglycerol and Polyglycerol Derivative

The PG or the PG derivative comprised in the polymer-conjugated lipid of the disclosure may be linear or branched. In one specific embodiment, the PG or the PG derivative is linear. In another embodiment, the PG or the PG derivative is branched.

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The polymer-conjugated lipid of the disclosure may comprise PG or a PG derivative comprising an average of 5 to 100 monomeric units, e.g., an average of 10 to 100 monomeric units, e.g., an average of 10 to 40 monomeric units, an average of 15-75 monomeric units, an average of 20 to 50 monomeric units, an average of 30 to 70 monomeric units, an average of 40 to 90 monomeric units or an average of 50 to 100 monomeric units. In some embodiments, the first PG or PG derivative may comprise an average of about 5-100, about 10-100, about 15-100, about 20-100, about 25-100, about 27-100, about 30-100, about 34-100, about 35-100, about 39-100, about 40-100, about 45-100, about 46-100, about 50-100, about 55-100, about 58-100, about 60-100, about 65-100, about 68-100, about 70-100, about 75-100, about 80-100, about 85-100, about 90-100, or about 95-100 monomeric units.

In some embodiments, the PG or PG derivative may comprise at least about 20, at least about 25, at least about 27, at least about 30, at least about 34, at least about 35, at least about 39, at least about 40, at least about 45, at least about 46, at least about 50, at least about 55, at least about 58, at least about 60, at least about 65, at least about 68, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 monomeric units.

91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 monomeric units.

In one embodiment, PG or the PG derivative of the disclosure comprises an average of 8 monomeric units.

In one embodiment, the PG or the PG derivative of the disclosure comprises an average of 34 monomeric units.

In one embodiment, the PG or the PG derivative of the disclosure comprises an average of 39 monomeric units.

In one embodiment, the PG or the PG derivative of the disclosure comprises an average of 45 monomeric units.

In one embodiment, the PG or the PG derivative of the disclosure comprises an average of 46 monomeric units.

In one embodiment, the PG or the PG derivative of the disclosure comprises an average of 50 monomeric units.

In one embodiment, the PG or the PG derivative of the disclosure comprises an average of 58 monomeric units.

In one embodiment, the PG or the PG derivative of the disclosure comprises an average of 68 monomeric units.

In one embodiment, the polymer-conjugated lipid of the disclosure comprises PG.

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In one embodiment, the polymer-conjugated lipid of the disclosure comprises PG derivative.

The PG derivative comprised in the polymer-conjugated lipid of the disclosure may be a carboxylated PG, e.g., 2-carboxycyclohexane-l-carboxylated polyglycerol.

The PG derivative comprised in the polymer-conjugated lipid of the disclosure may also be a glutarylated PG, e.g., 3 -methyl glutarylated PG.

In some embodiments, the PG derivative comprised in the polymer-conjugated lipid of the disclosure is represented by the following structural formula: wherein: n is an integer ranging from 8 to 100; and

R is selected from the group consisting

Lipid Moiety

In some embodiments, a polyglycerol derivative may be conjugated to a lipid moiety.

In some embodiments, the lipid moiety comprised in the polymer-conjugated lipid of the disclosure is represented by Formula (I) or a pharmaceutically acceptable salt thereof, wherein:

R¹ is absent, hydrogen, Ci-Ce alkyl, or a hydrophobic tail comprising 10-30 carbon atoms;

R² is absent, hydrogen, Ci-Ce alkyl, or a hydrophobic tail comprising 10-30 carbon atoms; wherein, when R¹ and R² are each hydrogen, Ci-Ce alkyl, or a hydrophobic tail comprising 10-30 carbon atoms, N is positively charged; and

R³ is a hydrophobic tail comprising 10-30 carbon atoms; wherein i_n Formula (I) is a bond conjugating the lipid moiety and the linker.

In some embodiments, R¹ is absent, and R² and R³ are each independently a hydrophobic tail comprising 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In some embodiments, R² and R³ are each independently a hydrophobic tail comprising 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In one

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MEl\57916143.vl 131698-30520 specific embodiment, R² and R³ are each independently a hydrophobic tail comprising 18 carbon atoms, and the lipid moiety is dioctadecylamine (DODA).

Linker

The linker conjugating the polyglycerol or the polyglycerol derivative to the lipid moiety in the polymer-conjugated lipid of the present disclosure may be an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker (e.g., a glutaryl linker, a succinyl linker, etc.), an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, or any combination thereof.

In some embodiments, the linker may be selected from the group consisting of -(CH₂)_n-, - C(O)(CH₂)_n-, -C(O)O(CH₂)_n, -OC(O)(CH₂)_nC(O)O-, and -NH(CH₂)_nC(O)O-, wherein n is an integer ranging from 1 to 20. In some embodiments, the linker is -C(O)(CH₂)_n-, and n is 2, 3, 4, 5, or 6. In one embodiment, n is 4.

In some embodiments, the polymer-conjugated lipid of the disclosure is DODA-PG34 represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

In some embodiments, the polymer-conjugated lipid of the disclosure is DODA-PG45 represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

In some embodiments, the polymer-conjugated lipid of the disclosure is DODA-PG46 represented by the following structure:

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In some embodiments, the polymer-conjugated lipid of the disclosure is DODA-PG58 represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

In some embodiments, the polymer-conjugated lipid of the disclosure may be represented by the following structure or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

In some embodiments, the polymer-conjugated lipid of the disclosure is DODA-PG39 or DODA-PG68, or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

The polymer-conjugated lipid of the present disclosure may also comprise a targeting moiety. The various targeting moieties that may be comprised in the polymer-conjugated lipid of the disclosure are described herein in the section “Targeting Moiety”.

The polymer-conjugated lipid of the present disclosure may also comprise a reactive species conjugated to the PG or the PG derivative. The terms “reactive species” and “reactive moiety” are used interchangeably herein. The reactive species present in the polymer-conjugated lipid of the present disclosure may be used for conjugation, e.g., to a targeting moiety which has been functionalized with a complementary reactive species, i. e. , a reactive species capable of reacting with the reactive species comprised in the polymer-conjugated lipid of the present disclosure. In some embodiments, the reactive species conjugated to the polymer-conjugated lipid of the present disclosure may be a thiol reagent, a maleimide reagent, or click chemistry reagent, e.g., a reagent

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MEl\57916143.vl 131698-30520 selected from the group consisting of an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent and an azide (AZ) reagent.

In some embodiments, the polymer-conjugated lipid of the present disclosure may comprise a targeting moiety that has been conjugated to the polymer-conjugated lipid via the reactive species. For example, the polymer-conjugated lipid of the present disclosure comprising an azide reagent as the reactive species may be reacted with a targeting moiety functionalized with a DBCO reagent as a complementary reactive species to produce a polymer-conjugated lipid comprising a targeting moiety. In another example, the polymer-conjugated lipid of the present disclosure comprising a thiol reagent may be reacted with a targeting moiety functionalized with a maleimide reagent to produce a polymer-conjugated lipid comprising a targeting moiety. Any targeting moiety described herein may be conjugated to a polymer-conjugated lipid of the present disclosure.

Synthesis of Polymer-Conjugated Lipid

A polymer-conjugated lipid of the present disclosure, wherein the polymer is PG, may be synthesized by a method comprising:

(a) reacting a lipid moiety which is conjugated to a linker with 2,3 -epoxy- 1-(1- ethoxyethoxyjpropane (EEGE) in the presence of a base, or in the presence of an organocatalyst, under argon atmosphere to produce a lipid moiety conjugated to a linker and polymerized EEGE; and

(b) subjecting the lipid moiety conjugated to a linker and polymerized EEGE to acidic conditions to produce the polymer-conjugated lipid.

In some embodiments, molar ratio of the lipid moiety conjugated to a linker to EEGE may be varied from about 1 :20 to about 1 : 100. For example, in step (a), the molar ratio of the lipid moiety conjugated to a linker to EEGE may be about 1:20 to about 1:40, about 1:25 to about 1:50, about 1:30 to about 1:60, about 1:50 to about 1:75 or about 1:60 to about 1: 100, e.g., about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:55, about 1:60, about 1:65, about 1:70, about 1:75, about 1:80, about 1:85, about 1:90 or about 1: 100. In one embodiment, the ratio of a lipid moiety conjugated to a linker to EEGE may be about 1:50. In another embodiment, the ratio of a lipid moiety conjugated to a linker to EEGE may be about 1:60.

In some embodiments, the ratio of the lipid moiety conjugated to a linker to EEGE in step (a) determines the average number of monomeric units present in the PG portion of the polymer- conjugated lipid in the final product.

The base useful for carrying out step (a) of the method may be a phosphazene base, such as P4-t-Bu.

In some embodiments, the organocatalyst useful for carrying out step (a) of the method may be an N-heterocyclic carbene (NHC) or an N-heterocyclic olefin (NHO).

In some embodiments, step (a) may be carried out overnight.

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The acidic conditions useful for carrying out step (b) of the method comprise a strong acid, such as hydrochloric acid (HC1). Other strong acids that may be used in this step comprise hydrobromic acid (HBr), hydroiodic acid (HI), perchloric acid (HCIO4), chloric acid (HCIO3), sulfuric acid (H2SO4), and nitric acid (HNO3).

The lipid moiety may be any lipid suitable for use in the methods described herein.

In some embodiments, the lipid moiety is DODA and the lipid moiety conjugated to a linker is represented by the following structure:

In some embodiments, the polymer-conjugated lipid is DODA-PG, wherein the PG comprises an average of 5-100 monomeric units. In some embodiments, the polymer-conjugated lipid is DODA-PG34, DODA-PG39, DODA-PG45, DODA-PG46, DODA-PG58, DODA-PG50, or DODA- PG68.

III. Lipid Nanoparticles (LNPs)

The present disclosure also provides lipid nanoparticles (LNPs) comprising: (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid; (iii) a sterol; and (iv) a first lipid-anchored polymer, and optionally further comprising a helper lipid, wherein the first lipid-anchored polymer comprises the polymer-conjugated lipid of the present disclosure. Also provided herein are LNPs consisting essentially of: (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid; (iii) a sterol; and (iv) a first lipid-anchored polymer, and optionally further consisting essentially of a helper lipid, wherein the first lipid-anchored polymer comprises the polymer-conjugated lipid of the present disclosure. Also provided herein are LNPs consisting of: (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid; (iii) a sterol; and (iv) a first lipid-anchored polymer, and optionally further consisting of a helper lipid, wherein the first lipid-anchored polymer comprises the polymer-conjugated lipid of the present disclosure.

A. Ionizable Lipids

In some embodiments, the ionizable lipid is present in the LNP provided by the present disclosure in an amount of about 20 mol% to about 60 mol%, about 35 mol% to about 50 mol%, of the total lipid present in the LNP.

In some embodiments, the LNPs provided by the present disclosure comprise an ionizable lipid. Exemplary ionizable lipids in the LNPs of the present disclosure are described in International Patent Application Publication Nos. W02015/095340, WO2015/199952, W02018/011633, WO2017/049245, WO2015/061467, WO2012/040184, W02012/000104, W02015/074085,

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WO2016/081029, WO2017/004143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, WO2011/090965, W02013/016058, W02012/162210, W02008/042973, W02010/129709, W02010/144740, WO2012/099755, WO2013/049328, WO2013/086322, WO2013/086373, WO2011/071860, W02009/132131, W02010/048536, W02010/088537, WO2010/054401, WO2010/054406, WO2010/054405, W02010/054384, W02012/016184, W02009/086558, WO2010/042877, WO2011/000106, WO2011/000107, W02005/120152, WO2011/141705, WO2013/126803, W02006/007712, WO2011/038160, WO2005/121348, WO2011/066651, W02009/127060, WO2011/141704, W02006/069782, WO2012/031043, W02013/006825, WO2013/033563, W02013/089151, WO2017/099823, WO2015/095346, WO2013/086354, and W02021/102411, and US Patent Application Publication Nos. US2016/0311759, US2015/0376115, US2016/0151284, US2017/0210697, US2015/0140070, US2013/0178541, US2013/0303587, US2015/0141678, US2015/0239926, US2016/0376224, US2017/0119904, US2012/0149894, US2015/0057373, US2013/0090372, US2013/0274523, US2013/0274504, US2013/0274504, US2009/0023673, US2012/0128760, US2010/0324120, US2014/0200257, US2015/0203446, US2018/0005363, US2014/0308304, US2013/0338210, US2012/0101148, US2012/0027796, US2012/0058144, US2013/0323269, US2011/0117125, US2011/0256175, US2012/0202871, US2011/0076335, US2006/0083780, US2013/0123338, US2015/0064242, US2006/0051405, US2013/0065939, US2006/0008910, US2003/0022649, US2010/0130588, US2013/0116307, US2010/0062967, US2013/0202684, US2014/0141070, US2014/0255472, US2014/0039032, US2018/0028664, US2016/0317458, and US2013/0195920, the contents of all of which are incorporated herein by reference in their entirety.

In some embodiments, the ionizable lipid in the LNPs of the present disclosure is MC3 (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3- DMA or MC3) represented by the following structural formula:

The lipid DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34): 8529-8533, content of which is incorporated herein by reference in its entirety.

In some embodiments, the ionizable lipid is selected from the group consisting of N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N-[l-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTAP); l,2-dioleoyl-sn-glycero-3 -ethylphosphocholine

MEl\57916143.vl 131698-30520

(DOEPC); l,2-dilauroyl-sn-glycero-3 -ethylphosphocholine (DLEPC); l,2-dimyristoyl-sn-glycero-3- ethylphosphocholine (DMEPC); l,2-dimyristoleoyl-sn-glycero-3 -ethylphosphocholine ( 14: 1), N1 -[2- (( 1 S)- 1 -[(3 -aminopropyl)amino] -4 - [di (3 -amino-propyl) aminolbutylcarboxamidoiethy 1 ] -3 ,4- di[oleyloxy]-benzamide(MVL5); Dioctadecylamido-glycylspermine (DOGS); 3b-[N-(N’,N’- dimethylaminoethyl)carbamoyl] cholesterol (DC-Chol); Dioctadecyldimethylammonium Bromide (DDAB); a Saint lipid (e.g., SAINT-2, N-methyl-4-(dioleyl)methylpyridinium); 1,2- dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); l,2-dioleoyl-3 -dimethyl - hydroxyethyl ammonium bromide (DORIE); l,2-dioleoyloxypropyl-3 -dimethylhydroxyethyl ammonium chloride (DORI); Di-alkylated Amino Acid (DILA2) (e.g., C18: l-norArg-C16);

Dioleyldimethylammonium chloride (DODAC); 1 -palmitoyl -2 -oleoyl-sn-glycero-3- ethylphosphocholine (POEPC); and l,2-dimyristoleoyl-sn-glycero-3 -ethylphosphocholine (MOEPC). In some variations, the condensing agent, e.g. a cationic lipid, is a lipid such as, e.g., Dioctadecyldimethylammonium bromide (DDAB), l,2-dilinoleyloxy-3 -dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,3 l-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2- Dioleoyloxy-3-dimethylaminopropane (DODAP), l,2-Dioleyloxy-3 -dimethylaminopropane (DODMA), Morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-l,2-diyl dioleate hydrochloride (DODAPen-Cl), (R)-5-guanidinopentane-l,2-diyl dioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan- 1 -aminium chloride(DOTAPen) .

Formula (A)

In some embodiments, the ionizable lipid in the LNPs of the present disclosure is represented by Formula (A): or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R¹ are each independently C1-3 alkylene;

R² and R² are each independently linear or branched Ci-e alkylene, or C3-6 cycloalkylene;

R³ and R³ are each independently optionally substituted Ci-e alkyl or optionally substituted C3-6 cycloalkyl;

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MEl\57916143.vl 131698-30520 or alternatively, when R²is branched Ci-6 alkylene and when R³ is Ci-6 alkyl, R² and R³, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R² is branched Ci-6 alkylene and when R³ is Ci-6 alkyl, R² and R³, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl;

R⁴ and R⁴ are each independently -CH, -CH2CH, or -(Q^CH;

R⁵ and R⁵ are each independently hydrogen, C1-20 alkylene or C2-20 alkenylene;

R⁶ and R⁶ , for each occurrence, are independently C1-20 alkylene, C3-20 cycloalkylene, or C2-20 alkenylene; and m and n are each independently an integer selected from 1, 2, 3, 4, and 5.

In some embodiments, R² and R² are each independently C1-3 alkylene.

In some embodiments, the linear or branched C1-3 alkylene represented by R¹ or R¹ , the linear or branched Ci-e alkylene represented by R² or R² , and the optionally substituted linear or branched Ci-6 alkyl are each optionally substituted with one or more halo and cyano groups.

In some embodiments, R¹ and R² taken together are C1-3 alkylene and R¹ and R² taken together are C1-3 alkylene, e.g., ethylene.

In some embodiments, R³ and R³ are each independently optionally substituted C1-3 alkyl, e.g., methyl.

In some embodiments, R⁴ and R⁴ are each -CH.

In some embodiments, R² is optionally substituted branched C1-6 alkylene; and R² and R³, taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl. In some embodiments, R² is optionally substituted branched Ci-e alkylene; and R² and R³ , taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl, such as pyrrolidinyl or piperidinyl.

In some embodiments, R⁴is -C(R^a)2CR^a, or -[C(R^a)2hCR^a and R^a is C1-3 alkyl; and R³ and R⁴, taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl. In some embodiments, R⁴ is -C(R^a)2CR^a, or [ C( R )z | zC R and R^a is C1-3 alkyl; and R³ and R⁴ , taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl, such as pyrrolidinyl or piperidinyl.

In some embodiments, R⁵ and R⁵ are each independently C1-10 alkylene or C2-10 alkenylene. In one embodiment, R⁵ and R⁵ are each independently Cns alkylene or C1-6 alkylene.

In some embodiments, R⁶ and R⁶ , for each occurrence, are independently C1-10 alkylene, C3-10 cycloalkylene, or C2-10 alkenylene. In one embodiment, C1-6 alkylene, C3-6 cycloalkylene, or C2-6 alkenylene. In one embodiment the C3-10 cycloalkylene or the C3-6 cycloalkylene is cyclopropylene. In some embodiments, m and n are each 3.

In some embodiments, the ionizable lipid in the LNPs of the present disclosure may be selected from any one of the lipids listed in Table 1 below, or a pharmaceutically acceptable salt thereof.

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Table 1

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Formula (B)

In some embodiments, the ionizable lipid in the LNPs of the present disclosure is represented by Formula (B): or a pharmaceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20 (e.g., a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); b is an integer ranging from 2 to 10 (e.g., b is 2, 3, 4, 5, 6, 7, 8, 9, or 10); R¹ is absent or is selected from (C2-C2o)alkenyl, -C(0)0(C2-C2o)alkyl, and cyclopropyl substituted with (C2-C2o)alkyl; and

R² is (C₂-C2o)alkyl.

In a second embodiment of Formula (B), the ionizable lipid of Formula (B) is represented by Formula (B-l):

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MEl\57916143.vl 131698-30520 or a pharmaceutically acceptable salt thereof, wherein c and d are each independently integers ranging from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and wherein the remaining variables are as described for Formula (B).

In a third embodiment of Formula (B), c and d in Formula (B- 1) are each independently integers ranging from 2 to 8, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 8, 4 to 7, 4 to 6, 5 to 8, 5 to 7, or 6 to 8, wherein the remaining variables are as described for Formula (B-l).

In a fourth embodiment of Formula (B), c in Formula (B-l) is 2, 3, 4, 5, 6, 7, or 8, wherein the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B). Alternatively, c and d in Formula (B-l) are each independently 1, 3, 5, or 7, wherein the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B).

In a fifth embodiment of Formula (B), d in the cationic lipid of Formula (B-l) is 2, 3, 4, 5, 6, 7, or 8, wherein the remaining variables are as described for Formula (B), or the second, third or fourth embodiments of Formula (B). Alternatively, at least one of c and d in Formula (B-l) is 7, wherein the remaining variables are as described for Formula (B), or the second, third or fourth embodiments of Formula (B).

In a sixth embodiment of Formula (B), the ionizable lipid of Formula (B) or Formula (B-l) is represented by Formula (B-2): or a pharmaceutically acceptable salt thereof, wherein the remaining variables are as described for Formula (B) or Formula (B-l).

In a seventh embodiment of Formula (B), b in Formula (B), (B-l), or (B-2) is an integer ranging from 3 to 9, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth or sixth embodiments of Formula (B). Alternatively, b in Formula (B), (B-l), or (B-2) is an integer ranging from 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 9, 5 to 8, 5 to 7, 6 to 9, 6 to 8, or 7 to 9, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth or sixth embodiments of Formula (B). Alternatively, b in Formula (B),

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(B-l), or (B-2) is 3, 4, 5, 6, 7, 8, or 9, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth or sixth embodiments of Formula (B).

In an eighth embodiment of Formula (B), a in Formula (B), (B-l), or (B-2) is an integer ranging from 2 to 18, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B). Alternatively, a in Formula (B), (B- 1), or (B-2) is an integer ranging from 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 25 to 8, 5 to 7, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 18, 8 to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, 11 to 18, 11 to 17, 11 to 16, 11 to 15, 11 to 14, 11 to 13, 12 to 18, 12 to 17, 12 to 16, 12 to 15, 12 to 14, 13 to 18, 13 to 17, 13 to 16, 13 to 15, 14 to 18, 14 to 17, 14 to 16, 15 to 18, 15 to 17, or 16 to 18, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B). Alternatively, a in Formula (B), (B-l), or (B-2) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B).

In a ninth embodiment of Formula (B), R¹ in Formula (B), Formula (B-l), or Formula (B-2) is absent or is selected from (C5-Ci5)alkenyl, -C(O)O(C4-Cis)alkyl, and cyclopropyl substituted with (C4-Cie)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). Alternatively, R¹ in Formula (B), Formula (B-l), or Formula (B-2) is absent or is selected from (C5-Ci5)alkenyl, -C(O)O(C4-Cie)alkyl, and cyclopropyl substituted with (C4-Cie)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). Alternatively, R¹ in Formula (B), Formula (B-l), or Formula (B-2) is absent or is selected from (C5- Ci2)alkenyl, -C(O)O(C4-Ci2)alkyl, and cyclopropyl substituted with (C4-Ci2)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). In another alternative, R¹ in the cationic lipid of Formula (B), Formula (B-l), or Formula (B-2) is absent or is selected from (C5-Cio)alkenyl, -C(0)0(C4-Cio)alkyl, and cyclopropyl substituted with (C4-Cio)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B).

In a tenth embodiment of Formula (B), R¹ is C10 alkenyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B).

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In an eleventh embodiment of Formula (B), the alkyl in C(0)0(C2-C2o)alkyl, -C(O)O(C4- Cis)alkyl, -C(O)O(C4-Ci2)alkyl, or -C(0)0(C4-Cio)alkyl of R¹ in Formula (B), Formula (B-l), or Formula (B-2) is an unbranched alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiments of Formula (B). In one embodiment, R¹ is -C(O)O(Cg alkyl). Alternatively, the alkyl in -C(O)O(C4-Cis)alkyl, - C(O)O(C4-Ci2)alkyl, or -C(0)0(C4-Cio)alkyl of R¹ in Formula (B), Formula (B-l), or Formula (B-2) is a branched alkyl, wherein the remaining variables are as described for Formula (B), Formula (B-l), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiments of Formula (B). In one embodiment, R¹ is -C(O)O(Ci? alkyl), wherein the remaining variables are as described for Formula (B), Formula (B-l), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiments of Formula (B).

In a twelfth embodiment of Formula (B), R¹ in Formula (B), Formula (B-l), or Formula (B-2) is selected from any group listed in Table 2 below, wherein the wavy bond in each of the groups indicates the point of attachment of the group to the rest of the ionizable lipid molecule, and wherein the remaining variables are as described for Formula (B), Formula (B-l), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). The present disclosure further contemplates the combination of any one of the R¹ groups in Table 2 with any one of the R² groups in Table 3 in Formula (B), wherein the remaining variables are as described for Formula (B), Formula (B-l), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B).

Table 2

In a thirteenth embodiment, R² in Formula (B) or a pharmaceutically acceptable salt thereof is selected from any group listed in Table 3 below, wherein the wavy bond in each of the groups indicates the point of attachment of the group to the rest of the ionizable lipid molecule, and wherein the remaining variables are as described for Formula (B), Formula (B-l), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh or eighth, ninth, tenth, eleventh or twelfth embodiments of Formula (B).

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Table 4 below provides specific examples of ionizable lipids of Formula (B).

Pharmaceutically acceptable salts as well as ionized and neutral forms are also included. Table 4

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89

MEl\57916143.vl 131698-30520

Formula (C)

In some embodiments, the ionizable lipid in the LNPs of the present disclosure are represented by Formula (C): or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R¹ are each independently (Ci-Ce)alkylene optionally substituted with one or more groups selected from R^a;

R² and R² are each independently (Ci-C2)alkylene; R³ and R³ are each independently (Ci-Ce)alkyl optionally substituted with one or more groups selected from R^b;

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MEl\57916143.vl 131698-30520 or alternatively, R² and R³ and/or R² and R³ are taken together with their intervening N atom to form a 4- to 7-membered heterocyclyl;

R⁴ and R⁴’ are each a (C2-Ce)alkylene interrupted by -C(O)O-;

R⁵ and R⁵’ are each independently a (CS-C’s-Oalkyl or (C2-C3o)alkenyl, each of which are optionally interrupted with -C(O)O- or (C3-Ce)cycloalkyl; and

R^a and R^b are each halo or cyano.

In a second embodiment of Formula (C), R¹ and R¹ are each independently (Ci-Ce)alkylene, wherein the remaining variables are as described above for Formula (C). Alternatively, R¹ and R¹ are each independently (Ci-C3)alkylene, wherein the remaining variables are as described above for Formula (C).

In a third embodiment of Formula (C), the ionizable lipid of the Formula (C) is represented by Formula (C-l): or a pharmaceutically acceptable salt thereof, wherein R² and R² , R³ and R³ , R⁴ and R⁴’ and R⁵ and

R⁵’ are as described above for Formula (C) or the second embodiment of Formula (C).

In a fourth embodiment, the ionizable lipid of Formula (C) is represented by Formula (C-2) or Formula or a pharmaceutically acceptable salt thereof, wherein R⁴ and R⁴’ and R⁵ and R⁵’ are as described above for Formula (C).

In a fifth embodiment of Formula (C), the ionizable lipid of Formula (C) is represented by Formula (C-4) or (C-5):

91

MEl\57916143.vl 131698-30520 or a pharmaceutically acceptable salt thereof, wherein R⁵ and R⁵’ are as described above for Formula (C).

In a sixth embodiment of Formula (C), the ionizable lipid of Formula (C) is represented by Formula ( or a pharmaceutically acceptable salt thereof, wherein R⁵ and R⁵’ are as described above for Formula (XV).

In a seventh embodiment of Formula (C), at least one of R⁵ and R⁵ in Formula (C), (C-l), (C- 2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, one of R⁵ and R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).

In an eighth embodiment of Formula (C), R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (Ce-C26)alkyl or (Ce-C26)alkenyl, each of which are optionally interrupted with -C(O)O- or (C3-Ce)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C- 2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C₆-C₂6)alkyl or (C₆-C₂6)alkenyl, each of which are optionally interrupted with -C(O)O- or (C3-C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C₇-C₂6)alkyl or (C₇- C2e)alkenyl, each of which are optionally interrupted with -C(O)O- or (C3-C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9)

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MEl\57916143.vl 131698-30520 is a (Cs-C26)alkyl or (C₈-C2e)alkenyl, each of which are optionally interrupted with -C(O)O- or (C3- C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C- 6), (C-7), (C-8), or (C-9) is a (Ce-C24)alkyl or (Ce-C24)alkenyl, each of which are optionally interrupted with -C(O)O- or cyclopropyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C- 2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C₈-C24)alkyl or (C₈-C24)alkenyl, wherein said (Cs-C24)alkyl is optionally interrupted with -C(O)O- or cyclopropyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C₈-Cio)alkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (Ci4-Cie)alkyl interrupted with cyclopropyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (Cio-C₂4)alkyl interrupted with -C(O)O-, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (Cie-Ci₈)alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is -(CH₂)3C(O)O(CH₂)₈CH3, -(CH₂)5C(O)O(CH₂)₈CH3, - (CH₂)₇C(O)O(CH₂)₈CH3, -(CH₂)₇C(O)OCH[(CH₂)₇CH3]2, -(CH₂)₇-C3H₆-(CH₂)₇CH3, -(CH₂)₇CH₃, - (CH₂)9CH3, -(CH₂)16CH3, -(CH₂)₇CH=CH(CH₂)₇CH₃, or -(CH2)₇CH=CHCH₂CH=CH(CH₂)4CH3, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).

In a ninth embodiment, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C- 8), or (C-9) is a (Ci5-C2₈)alkyl interrupted with -C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (Ci₇-C₂₈)alkyl interrupted with -C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (Ci₉-C₂₈)alkyl interrupted with -C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (Ci₇-C2e)alkyl interrupted with -C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (Ci₉-C₂6)alkyl interrupted with -C(O)O-, and the remaining variables are as described above for Formula (C) or the

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MEl\57916143.vl 131698-30520 second or eighth embodiments of Formula (C). Alternatively, R⁵ in Formula (C), (C-l), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C₂o-C₂6)alkyl interrupted with -C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R⁵ is a (C₂₂-C₂4)alkyl interrupted with -C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R⁵ is -(CH₂)₅C(O)OCH[(CH₂)₇CH₃]₂, -(CH₂)₇C(O)OCH[(CH₂)₇CH₃]₂, - (CH₂)₅C(O)OCH(CH₂)₂[(CH₂)₇CH₃]₂, or -(CH₂)₇C(O)OCH(CH₂)₂[(CH₂)₇CH₃]₂, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). In some embodiments, the ionizable lipid of Formula (C), (C-l), (C-3), (C-3), (C-4), (C-5),

(C-7), (C-8), or (C-9) may be selected from any of the lipids listed in Table 5 below, or pharmaceutically acceptable salts thereof.

Table 5

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Formula (D)

In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (D): or a pharmaceutically acceptable salt thereof, wherein:

R’ is absent, hydrogen, or Ci-Ce alkyl; provided that when R’ is hydrogen or Ci-Ce alkyl, the nitrogen atom to which R’, R¹, and R² are all attached is positively charged;

R¹ and R² are each independently hydrogen, Ci-Ce alkyl, or C2-C6 alkenyl;

R³ is Ci -Ci 2 alkylene or C2-C 12 alkenylene;

R⁴ is Ci-Cis unbranched alkyl, C2-C18 unbranched alkenyl, or ; wherein:

R^4a and R^4b are each independently Ci-Cie unbranched alkyl or C2-C16 unbranched alkenyl;

R⁵ is absent, Ci-Cs alkylene, or C2-C8 alkenylene;

R^6a and R^6b are each independently C7-C16 alkyl or C7-C16 alkenyl; provided that the total number of carbon atoms in R^6a and R^6b as combined is greater than 15;

X¹ and X² are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(R^a)=N-, -N=C(R^a)-, -C(R^a)=NO-, -O-N=C(R^a)-, -C(=O)NR^a-, -NR^aC(=O)-, -NR^aC(=O)NR^a-, -OC(=O)O-, -OSi(R^a)₂O-, -C(=O)(CR^a ₂)C(=O)O-, or OC(=O)(CR^a ₂)C(=O)-; wherein:

R^a, for each occurrence, is independently hydrogen or Ci-Ce alkyl; and

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MEl\57916143.vl 131698-30520 n is an integer selected from 1, 2, 3, 4, 5, and 6.

In a second embodiment of Formula (D), X¹ and X² are the same; and all other remaining variables are as described for Formula (C).

In a third embodiment of Formula (D), X¹ and X² are each independently -OC(=O)-, - SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -S-S-; or X¹ and X² are each independently -C(=O)O-, - C(=O)S-, or -S-S-; or X¹ and X² are each independently -C(=O)O- or -S-S-; and all other remaining variables are as described for Formula (D) or the second embodiment of Formula (D).

In a fourth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure, is represented by Formula (D-l):

(D-l) or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, 3, and 4; and all other remaining variables are as described for Formula (D) or the second or third embodiments of Formula (D).

In a fifth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure, is represented by Formula (D-2):

(D-2) or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, and 3; and all other remaining variables are as described for Formula (D) or the second or third embodiments of Formula (D).

In a sixth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosu

MEl\57916143.vl 131698-30520 or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (D) or the second or third embodiments of Formula (D).

In a seventh embodiment of Formula (D), in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), or the second or third embodiments of Formula (D), R¹ and R² are each independently hydrogen, Ci-Ce alkyl or C2-C6 alkenyl, or C1-C5 alkyl or C2-C5 alkenyl, or C1-C4 alkyl or C2-C4 alkenyl, or Ce alkyl, or C5 alkyl, or C4 alkyl, or C3 alkyl, or C2 alkyl, or Ci alkyl, or Ce alkenyl, or C5 alkenyl, or C4 alkenyl, or C3 alkenyl, or C2 alkenyl; and all other remaining variables are as described for Formula (D), Formula (D-l), Formula (D-2), Formula (D-3) or the second or third embodiments of Formula (D).

In an eighth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclo

(D-4) or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (D), Formula (D-l), Formula (D-2), Formula (D-3) or the second, third or seventh embodiments of Formula (D).

In a ninth embodiment of Formula (D), in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third or seventh embodiments of Formula (D), R³ is C1-C9 alkylene or C2-C9 alkenylene, C1-C7 alkylene or C2- C7 alkenylene, C1-C5 alkylene or C2-C5 alkenylene, or C2-C8 alkylene or C2-C8 alkenylene, or C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene; or R³ is C12 alkylene, Cn alkylene, C10 alkylene, C9 alkylene, or Cs alkylene, or C7 alkylene, or Ce alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or Ci alkylene, or C12 alkenylene, Cn alkenylene, C10 alkenylene, C9 alkenylene, or Cs alkenylene, or C7 alkenylene, or Ce alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene; and all other remaining variables are as described for Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third or seventh embodiments of Formula (D).

In a tenth embodiment of Formula (D), in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third or seventh embodiments of Formula (D), R⁵ is absent, Ci-Ce alkylene, or C2-C6 alkenylene; or R⁵ is absent, C1-C4 alkylene, or C2-C4 alkenylene; or R⁵ is absent; or R⁵ is Cs alkylene, C7 alkylene, Ce alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, Ci alkylene, Cs alkenylene, C7 alkenylene, Ce alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene, or C2 alkenylene; and all other remaining

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MEl\57916143.vl 131698-30520 variables are as described for Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third, seventh, or ninth embodiments of Formula (D).

In an eleventh embodiment of Formula (D), in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third, seventh, ninth or tenth embodiments of Formula (D), R⁴is Ci-Ci 4 unbranched alkyl, C2-

C14 unbranched alkenyl, or , wherein R^4a and R^4b are each independently C1-C12 unbranched alkyl or C2-C12 unbranched alkenyl; or R⁴is C2-C 12 unbranched alkyl or C2-C12 unbranched alkenyl; or R⁴ is C5-C7 unbranched alkyl or C5-C7 unbranched alkenyl; or R⁴is Cie unbranched alkyl, C15 unbranched alkyl, C14 unbranched alkyl, C13 unbranched alkyl, C12 unbranched alkyl, Cn unbranched alkyl, Ciounbranched alkyl, C9 unbranched alkyl, Cs unbranched alkyl, C7 unbranched alkyl, Ce unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, Ci unbranched alkyl, Cie unbranched alkenyl, C15 unbranched alkenyl, C14 unbranched alkenyl, C13 unbranched alkenyl, C12 unbranched alkenyl, Cn unbranched alkenyl, C10 unbranched alkenyl, C9 unbranched alkenyl, Cs unbranched alkenyl, C7 unbranched alkenyl, Ce unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl; or R⁴ is , wherein R^4a and R^4b are each independently C2-C10 unbranched alkyl or

C2-C10 unbranched alkenyl; or R⁴ is , wherein R^4a and R^4b are each independently Cie unbranched alkyl, C15 unbranched alkyl, C14 unbranched alkyl, C13 unbranched alkyl, C12 unbranched alkyl, Cn unbranched alkyl, Ciounbranched alkyl, C9 unbranched alkyl, Cs unbranched alkyl, C7 unbranched alkyl, Ce unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, Ci alkyl, Ciounbranched alkenyl, C15 unbranched alkenyl, C14 unbranched alkenyl, C13 unbranched alkenyl, C12 unbranched alkenyl, Cn unbranched alkenyl, Ciounbranched alkenyl, C9 unbranched alkenyl, Cs unbranched alkenyl, C7 unbranched alkenyl, Co unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl; and all other remaining variables are as described for Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third, seventh, ninth or tenth embodiments of Formula (D).

In a twelfth embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth or eleventh embodiments of Formula (D), R^6aand R^6b are each independently Ce-Ci4 alkyl or Ce- C14 alkenyl; or R^6a and R^6b are each independently Cs-Ci2 alkyl or Cs-Ci2 alkenyl; or R^6a and R^6b are

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MEl\57916143.vl 131698-30520 each independently Cie alkyl, C15 alkyl, C14 alkyl, C13 alkyl, C12 alkyl, Cn alkyl, C10 alkyl, C , alkyl. Cs alkyl, C7 alkyl, Cie alkenyl, C15 alkenyl, C14 alkenyl, C13 alkenyl, C12 alkenyl, Cn alkenyl, C10 alkenyl, Cg alkenyl, Cs alkenyl, or C7 alkenyl; provided that the total number of carbon atoms in R^6a and R^6b as combined is greater than 15; and all other remaining variables are as described for Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth or eleventh embodiments of Formula (D).

In a thirteenth embodiment of Formula (D), in the ionizable lipid, e.g. , cationic lipid, according to Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh or twelfth embodiments of Formula (D), or a pharmaceutically acceptable salt thereof, R^6a and R^6b contain an equal number of carbon atoms with each other; or R^6a and R^6b are the same; or R^6a and R^6b are both Cie alkyl, C15 alkyl, C14 alkyl, C13 alkyl, C12 alkyl, Cn alkyl, C 10 alkyl, Cg alkyl, Cs alkyl, C7 alkyl, Cie alkenyl, C15 alkenyl, C14 alkenyl, C13 alkenyl, C12 alkenyl, Cn alkenyl, C10 alkenyl, Cg alkenyl, Cs alkenyl, or C7 alkenyl; provided that the total number of carbon atoms in R^6a and R^6b as combined is greater than 15; and all other remaining variables are as described for Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third, seventh, ninth, tenth, eleventh or twelfth embodiments of Formula (D).

In a fourteenth embodiment of Formula (D), in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiments of Formula (D), R^6a and R^6b as defined in any one of the preceding embodiments each contain a different number of carbon atoms with each other; or the number of carbon atoms R^6a and R^6b differs by one or two carbon atoms; or the number of carbon atoms R^6a and R^6b differs by one carbon atom; or R^6a is C7 alkyl and R^6a is Cs alkyl, R^6a is Cs alkyl and R^6a is C7 alkyl, R^6a is Cs alkyl and R^6a is Cg alkyl, R^6a is Cg alkyl and R^6a is Cs alkyl, R^6a is Cg alkyl and R^6a is C10 alkyl, R^6a is C10 alkyl and R^6a is Cg alkyl, R^6a is C10 alkyl and R^6a is Cn alkyl, R^6a is Cn alkyl and R^6a is C10 alkyl, R^6a is Cn alkyl and R^6a is C12 alkyl, R^6a is C12 alkyl and R^6a is Cn alkyl, R^6a is C7 alkyl and R^6a is Cg alkyl, R^6a is Cg alkyl and R^6a is C7 alkyl, R^6a is Cs alkyl and R^6a is C10 alkyl, R^6a is C10 alkyl and R^6a is Cs alkyl, R^6a is Cg alkyl and R^6a is Cn alkyl, R^6a is C alkyl and R^6a is Cg alkyl, R^6a is C10 alkyl and R^6a is C12 alkyl, R^6a is C12 alkyl and R^6a is C10 alkyl, R^6a is Cn alkyl and R^6a is C13 alkyl, or R^6a is C13 alkyl and R^6a is Cn alkyl, etc.; and all other remaining variables are as described for Formula I, Formula II, Formula III, Formula IV, Formula V, or the second, third, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiments of Formula (D).

In a fifteenth embodiment of Formula (D), R⁴ is C1-C16 unbranched alkyl, C2-C16 unbranched alkenyl, , wherein R^4a and R^4b are as described above for the second, third, fourth,

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MEl\57916143.vl 131698-30520 fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenth embodiments of Formula (D).

In one embodiment, the ionizable lipid, e.g. , cationic lipid, of the present disclosure or the ionizable lipid of Formula (D), Formula (D-l), Formula (D-2), Formula (D-3), or Formula (D-4) is any one lipid selected from the lipids listed in Table 6 below, or a pharmaceutically acceptable salt thereof:

Table 6

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In one embodiment, the ionizable lipid in the LNPs of the present disclosure is Ionizable

Lipid 87: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

Formula (E)

In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E): or a pharmaceutically acceptable salt thereof, wherein:

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R’ is absent, hydrogen, or C1-C3 alkyl; provided that when R’ is hydrogen or C1-C3 alkyl, the nitrogen atom to which R’, R¹, and R² are all attached is positively charged;

R¹ and R² are each independently hydrogen or C1-C3 alkyl;

R³ is C3-C10 alkylene or C3-C10 alkenylene; R₄b

R⁴ is Ci-Cie unbranched alkyl, C2-C16 unbranched alkenyl, or D ^K4a ; wherein:

R⁵ is absent, Ci-Ce alkylene, or C2-C6 alkenylene;

R^6a and R^6b are each independently C7-C14 alkyl or C7-C14 alkenyl;

X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(R^a)=N-, -N=C(R^a)-, -C(R^a)=NO-, -O-N=C(R^a)-, -C(=O)NR^a-, -NR^aC(=O)-, -NR^aC(=O)NR^a-, -OC(=O)O-, -OSi(R^a)₂O-, -C(=O)(CR^a ₂)C(=O)O-, or OC(=O)(CR^a ₂)C(=O)-; wherein:

R^a, for each occurrence, is independently hydrogen or Ci-Ce alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6.

In a second embodiment of Formula (E), in the ionizable lipid, e.g., cationic lipid, according to the first embodiment, or a pharmaceutically acceptable salt thereof, X is -OC(=O)-, -SC(=O)-, - OC(=S)-, -C(=O)O-, -C(=O)S-, or -S-S-; and all other remaining variables are as described for Formula I or the first embodiment.

In a third embodiment of Formula (E), the ionizable lipid, e.g. , cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-l):

(E-l) or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, 3, and 4; and all other remaining variables are as described for Formula (E) or the second embodiment of Formula (E). Alternatively, n is an integer selected from 1, 2, and 3; and all other remaining variables are as described for Formula (E) or the second embodiment of Formula (E).

In a fourth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-2):

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MEl\57916143.vl 131698-30520 or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-l) or the second embodiment of Formula (E).

In a fifth embodiment of Formula (E), in the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure, R¹ and R² are each independently hydrogen or C1-C2 alkyl, or C2-C3 alkenyl; or R’, R¹, and R² are each independently hydrogen, C1-C2 alkyl; and all other remaining variables are as described for Formula (E), Formula (E-l) or the second embodiment of Formula (E).

In a sixth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-3):

(E-3) or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-l), Formula (E-2) or the second or fifth embodiments of Formula (E).

In a seventh embodiment of Formula (E), in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-l), Formula (E-2), Formula (E-3) or the second or firth embodiments of Formula (E), R⁵ is absent or Ci-Cs alkylene; or R⁵ is absent, Ci-Ce alkylene, or C2-C6 alkenylene; or R⁵ is absent, C1-C4 alkylene, or C2-C4 alkenylene; or R⁵ is absent; or R⁵ is Cs alkylene, C7 alkylene, Ce alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, Ci alkylene, Cs alkenylene, C7 alkenylene, Ce alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene, or C2 alkenylene; and all other remaining variables are as described for Formula (E), Formula (E-l), Formula (E-2), Formula (E-3) or the second or fifth embodiments of Formula (E).

In an eighth embodiment of Formula (E), he ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-4):

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(E-4) or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-l), Formula (E-2), Formula (E-3) or the second, fifth or seventh embodiments of Formula (E).

In a ninth embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-l), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth or seventh embodiments of Formula (E), or a pharmaceutically acceptable salt thereof, R⁴ is C1-C14 unbranched alkyl, C2-C14 unbranched alkenyl, or , wherein R^4a and R^4b are each independently C1-C12 unbranched alkyl or C2-C12 unbranched alkenyl; or R⁴is C2-C 12 unbranched alkyl or C2-C12 unbranched alkenyl; or R⁴is C5-C12 unbranched alkyl or C5-C12 unbranched alkenyl; or R⁴is Cie unbranched alkyl, C15 unbranched alkyl, C14 unbranched alkyl, C13 unbranched alkyl, C12 unbranched alkyl, Cn unbranched alkyl, Ciounbranched alkyl, C9 unbranched alkyl, Cs unbranched alkyl, C7 unbranched alkyl, Ce unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, Ci unbranched alkyl, Cie unbranched alkenyl, C15 unbranched alkenyl, C14 unbranched alkenyl, C13 unbranched alkenyl, C12 unbranched alkenyl, Cn unbranched alkenyl, C10 unbranched alkenyl, C9 unbranched alkenyl, Cs unbranched alkenyl, C7 unbranched alkenyl, Ce unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl; or R⁴ is , wherein R^4a and R^4b are each independently C2-C10 unbranched alkyl or y^{5 R4b}

C2-C10 unbranched alkenyl; or R⁴ is p ^K4a , wherein R^4a and R^4b are each independently Cie unbranched alkyl, C15 unbranched alkyl, C14 unbranched alkyl, C13 unbranched alkyl, C12 unbranched alkyl, Cn unbranched alkyl, Ciounbranched alkyl, C9 unbranched alkyl, Cs unbranched alkyl, C7 unbranched alkyl, Ce unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, Ci alkyl, C io unbranched alkenyl, C15 unbranched alkenyl, C14 unbranched alkenyl, C13 unbranched alkenyl, C12 unbranched alkenyl, Cn unbranched alkenyl, Ciounbranched alkenyl, C9

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MEl\57916143.vl 131698-30520 unbranched alkenyl, Cs unbranched alkenyl, C7 unbranched alkenyl, Ce unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl; and all other remaining variables are as described for Formula (E), Formula (E- 1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth or seventh embodiments of Formula (E).

In a tenth embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-l), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh or ninth embodiments of Formula (E), R³ is Cs-Cs alkylene or Cs-Cs alkenylene, C3-C7 alkylene or C3-C7 alkenylene, or C3-C5 alkylene or C3-C5 alkenylene,; or R³ is Cs alkylene, or C7 alkylene, or Ce alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or Ci alkylene, or Cs alkenylene, or C7 alkenylene, or Ce alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene; and all other remaining variables are as described for Formula (E), Formula (E-l), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh or ninth embodiments of Formula (E).

In an eleventh embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-l), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth or tenth embodiments of Formula (E), R^6aand R^6b are each independently C7-C12 alkyl or C7-C12 alkenyl; or R^6a and R^6b are each independently Cs-Cio alkyl or Cs-Cio alkenyl; or R^6a and R^6b are each independently Cs alkyl, Cn alkyl, Cio alkyl, C9 alkyl, Cs alkyl, C? alkyl. Cn alkenyl, Cn alkenyl, C10 alkenyl, C9 alkenyl, Cs alkenyl, or C7 alkenyl; and all other remaining variables are as described for Formula (E), Formula (E-l), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth or tenth embodiments of Formula (E).

In a twelfth embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-l), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth or eleventh embodiments of Formula (E), R^6a and R^6b contain an equal number of carbon atoms with each other; or R^6a and R^6b are the same; or R^6a and R^6b are both C12 alkyl, Ci 1 alkyl, C10 alkyl, C9 alkyl, Cs alkyl, C₇ alkyl, C 12 alkenyl, Cn alkenyl, C10 alkenyl, C9 alkenyl, Cs alkenyl, or C7 alkenyl; and all other remaining variables are as described for Formula (E), Formula (E-l), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth or eleventh embodiments of Formula (E).

In a thirteenth embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-l), Formula (E-2), Formula (E-3), Formula (E-4), R^6aand R^6b as defined in any one of the preceding embodiments each contain a different number of carbon atoms with each other; or the number of carbon atoms R^6a and R^6b differs by one or two carbon atoms; or the number of carbon atoms R^6a and R^6b differs by one carbon atom; or R^6a is C7 alkyl and R^6a is Cs alkyl, R^6a is Cs alkyl and R^6a is C7 alkyl, R^6a is Cs alkyl and R^6a is C9 alkyl, R^6a is C9 alkyl and R^6a is Cs alkyl, R^6a is C9 alkyl and R^6a is Cio alkyl, R^6a is Cioalkyl and R^6a is C9 alkyl, R^6a is Cio alkyl and R^6a is Cn alkyl, R^6a is Cn alkyl and R^6a is C10 alkyl, R^6a is Cn alkyl and R^6a is C12 alkyl, R^6a is C12 alkyl and R^6a is Cn alkyl, R^6a is C7 alkyl and R^6a is C9 alkyl, R^6a is C9 alkyl and R^6a is C7 alkyl, R^6a is Cs alkyl and R^6a is C10 alkyl, R^6a is

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Cio alkyl and R^6a is Cs alkyl, R^6a is Cg alkyl and R^6a is Cn alkyl, R^6a is Cn alkyl and R^6a is Cg alkyl, R^6a is Cio alkyl and R^6a is C12 alkyl, R^6a is C12 alkyl and R^6a is Cio alkyl, etc.; and all other remaining variables are as described for Formula (E), Formula (E- 1), Formula (E-2), Formula (E-3), Formula (E- 4) or the second, fifth, seventh, ninth, tenth, eleventh or twelfth embodiments of Formula (E). In a fourteenth embodiment, in the ionizable lipid, e.g. , cationic lipid, according to Formula

(E), Formula (E- 1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiments of Formula (E), R’ is absent; and all other remaining variables are as described for Formula (E), Formula (E-l), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiments of Formula (E).

In one embodiment, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure or the cationic lipid of Formula (E), Formula (E-l), Formula (E-2), Formula (E-3), Formula (E-4) is any one lipid selected from the lipids in Table 7 or a pharmaceutically acceptable salt thereof:

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Specific examples are provided in the exemplification section below and are included as part of the cationic or ionizable lipids described herein. Pharmaceutically acceptable salts as well as neutral forms are also included.

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Cleavable Lipids

In some embodiments, the LNPs provided by the present disclosure comprise an ionizable lipid that is also a cleavable lipid. As used herein, the term “cleavable lipid”, which may be used interchangeably with the term “SS-cleavable lipid” refers to an ionizable lipid comprising a disulfide bond (“SS”). The SS in the cleavable lipid is a cleavable unit. In one embodiment, a cleavable lipid comprises an amine, e.g., a tertiary amine, and a disulfide bond. In this cleavable lipid, an amine can become protonated in an acidic compartment (e.g., in an endosome or a lysosome), leading to LNP destabilization, and the cleavable lipid can become cleaved in a reductive environment (e.g., in the cytoplasm). Cleavable lipids also include pH-activated lipid-like materials, such as ss-OP lipids, ssPalm lipids, ss-M lipids, ss-E lipids, ss-EC lipids, ss-LC lipids and ss-OC lipids, etc.

According to some embodiments, SS-cleavable lipids are described in International Patent Application Publication No. WO2019188867, incorporated by reference in its entirety herein.

In one embodiment, a cleavable lipid may comprise three components: an amine head group, a linker group, and a hydrophobic tail(s). In one embodiment, the cleavable lipid comprises one or more phenyl ester bonds, one of more tertiary amino groups, and a disulfide bond. The tertiary amine groups provide pH responsiveness and induce endosomal escape, the phenyl ester bonds enhance the degradability of the structure (self- degradability) and the disulfide bond becomes cleaved in a reductive environment.

In one embodiment, the cleavable lipid is an ss-OP lipid. In one embodiment, an ss-OP lipid comprises the structure of Lipid A shown below:

Lipid A

In one embodiment, the SS-cleavable lipid is an SS-cleavable and pH-activated lipid-like material (ssPalm). ssPalm lipids are well known in the art. For example, see Togashi et al., Journal of Controlled Release, 279 (2018) 262-270, the entire contents of which are incorporated herein by reference. In one embodiment, the ssPalm is an ssPalmM lipid comprising the structure of Lipid B shown below:

Lipid B

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In one embodiment, the ssPalmE lipid is a ssPalmE-P4-C2 lipid comprising the structure of

Lipid C below:

Lipid C

In one embodiment, the ssPalmE lipid is a ssPalmE-Paz4-C2 lipid, comprising the structure of

Lipid D below:

Lipid D

In one embodiment, the cleavable lipid is an ss-M lipid, In one embodiment, an ss-M lipid comprises the structure shown in Lipid E below:

Lipid E

In one embodiment, the cleavable lipid is an ss-E lipid. In one embodiment, an ss-E lipid comprises the structure shown in Lipid F below:

Lipid F

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In one embodiment, the cleavable lipid is an ss-EC lipid. In one embodiment, an ss-EC lipid comprises the structure shown for Lipid G below:

Lipid G

In one embodiment, the cleavable lipid is an ss-LC lipid. In one embodiment, an ss-LC lipid comprises the structure shown for Lipid H below:

Lipid H

In one embodiment, the cleavable lipid is an ss-OC lipid. In one embodiment, an ss-OC lipid comprises the structure shown for Lipid J below:

Lipid J

Other Lipids

In some embodiments, the ionizable lipid in the LNPs of the present disclosure is selected from the group consisting of N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP); 1,2- dioleoyl-sn-glycero-3 -ethylphosphocholine (DOEPC); l,2-dilauroyl-sn-glycero-3- ethylphosphocholine (DLEPC); l,2-dimyristoyl-sn-glycero-3 -ethylphosphocholine (DMEPC); 1,2- dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14: 1), Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4- [ di (3 -amino-propyl) aminolbutylcarboxamidoiethyl] -3 ,4 -di [oleyloxy] -benzamide (MVL5 ) ; Dioctadecylamido-glycylspermine (DOGS); 3b-[N-(N’,N’-dimethylaminoethyl)carbamoyl]

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MEl\57916143.vl 131698-30520 cholesterol (DC-Chol); Dioctadecyldimethylammonium Bromide (DDAB); a Saint lipid (e.g., SAINT -2, N -methyl -4-(dioleyl)methylpyridinium) ; 1 ,2-dimyristyloxypropyl-3 - dimethylhydroxyethylammonium bromide (DMRIE); l,2-dioleoyl-3 -dimethyl -hydroxyethyl ammonium bromide (DORIE); 1,2-dioleoyloxypropyl -3 -dimethylhydroxyethyl ammonium chloride (DORI); Di-alkylated Amino Acid (DILA2) (e.g., C18:l-norArg -C16); Dioleyldimethylammonium chloride (DODAC); 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -ethylphosphocholine (POEPC); and 1,2- dimyristoleoyl-sn-glycero-3 -ethylphosphocholine (MOEPC). In some variations, the condensing agent, e.g. a cationic lipid, is a lipid such as, e.g., Dioctadecyldimethylammonium bromide (DDAB),

1.2-dilinoleyloxy-3 -dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)- [1,3] -dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,3 l-tetraen-19-yl-4- (dimethylamino)butanoate (DLin-MC3-DMA), l,2-Dioleoyloxy-3 -dimethylaminopropane (DODAP),

1.2-Dioleyloxy-3 -dimethylaminopropane (DODMA), Morpholinocholesterol (Mo-CHOL), (R)-5- (dimethylamino)pentane-l,2-diyl dioleate hydrochloride (DODAPen-Cl), (R)-5-guanidinopentane-

1.2-diyl dioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-l- aminium chloride(DOTAPen).

In some embodiments, the ionizable lipid in the LNP of the present disclosure is represented by the following structure:

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B. Structural Lipids

In some embodiments, the LNPs provided by the present disclosure comprise a structural lipid. Without wishing to be bound by a specific theory, it is believed that a structural lipid, when present in an LNP, contributes to membrane integrity and stability of the LNP.

In some embodiments, the structural lipid is a sterol, e.g, cholesterol, or a derivative thereof. In one embodiment, the structural lipid is cholesterol. In another embodiment, the structural lipid is a derivative of cholesterol. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 5p-coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4’- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4’-hydroxy)- butyl ether. In some embodiments, cholesterol derivative is cholestryl hemisuccinate (CHEMS).

Exemplary cholesterol derivatives are described in International Patent Application Publication No. W02009/127060 and U.S. Patent Application Publication No. US2010/0130588, contents of both of which are incorporated herein by reference in their entirety.

In some embodiments, the sterol in the LNPs of the present disclosure is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and derivatives thereof, and any combination thereof. In one embodiment, the sterol is cholesterol. In another embodiment, the sterol is beta-sitosterol.

In some embodiments, the structural lipid, e.g., a sterol, constitutes about 20 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid, e.g., a sterol, constitutes about 30 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid, e.g., cholesterol, constitutes about 30 mol% of the total lipid present in the LNP.

In some embodiments, the structural lipid is dexamethasone or dexamethasone -palmitate.

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C. Helper Lipids

Ceramide helper lipids

In some embodiments, the LNPs provided by the present disclosure comprise a helper lipid. In some embodiments, the helper lipid is a ceramide. The ceramides in the LNPs of the present disclosure are not conjugated to a polymer, such as polyethylene glycol or PEG. Ceramides are sphingolipids which is a class of cell membrane lipids. Ceramides contain an A'-acctylsphingosinc (z.e., (£)-JV-(l,3-dihydroxyoctadec-4-en-2-yl)acetamide) backbone and a fatty acid linked to the amide group. In some embodiments, the LNPs provided by the present disclosure comprise a ceramide, whereby the fatty acid portion of the ceramide is of a certain length or is a fatty acid having a certain number of carbon atoms as described below. As used herein, the term “helper lipid” refers to an amphiphilic lipid comprising at least one non-polar chain and at least one polar moiety. Without wishing to be bound by a specific theory, it is believed that a helper lipid functions to evade off- targeting of the LNP to the blood compartment, to increase the fusogenicity of the lipid bilayer of the LNP and to facilitate endosomal escape.

In some embodiments, the LNP of the present disclosure comprises ceramide as a helper lipid. In some embodiments, the helper lipid is represented by Formula (II):

Formula (II) or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, wherein:

'' is a single bond or a double bond;

R¹ is C1-C17 alkyl or C2-C17 alkenyl;

R² is C1-C22 alkyl or C2-C22 alkenyl;

R³ is hydrogen or C1-C2 alkyl; and

R⁴ is hydrogen or C1-C2 alkyl.

In some embodiments of Formula (II), '' is a single bond. In some embodiments of

Formula (II), ''' is a double bond.

In some embodiments of Formula (II), R¹ is C10-C20 alkenyl, R² is C10-C20 aklyl and R³ is hydrogen.

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In some embodiments of Formula (II), R¹ is Ci-Cw alkyl or C2-C10 alkenyl.

In some embodiments of Formula (II),

R¹ is C1-C10 alkyl or C2-C10 alkenyl;

R² is C1-C22 alkyl or C2-C22 alkenyl;

R³ is hydrogen or C1-C2 alkyl; and

R⁴ is hydrogen or C1-C2 alkyl.

In some embodiments of Formula (II), R³ and R⁴ are both hydrogen. In some embodiments of Formula (II), R³ and R⁴ are independently hydrogen or Ci alkyl.

In some embodiments of Formula (II), R¹ is C1-C7 alkyl or C2-C7 alkenyl. In one embodiment, R¹ is C1-C7 alkyl. In one embodiment, R¹ is Ci alkyl.

The term “salt” when used to refer to a helper lipid represented by Formula (II) means a pharmaceutically acceptable salt of a helper lipid represented by Formula (II), including both acid and base addition salts. A salt of a helper lipid represented by Formula (II) retains the biological effectiveness and properties of the free acid forms or free base forms of the helper lipid represented by Formula (II), which are not biologically or otherwise undesirable, and which are formed with inorganic acids or organic acids, or inorganic bases or organic bases. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; and examples of organic acids include, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzene sulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methane sulfonic acid, mucic acid, naphthalene- 1,5 -disulfonic acid, naphthalene-2- sulfonic acid, 1 -hydroxy-2 -naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2 -dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine,

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MEl\57916143.vl 131698-30520 glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

The term “ester”, when used to refer to a helper lipid represented by Formula (II), means an ester of a helper lipid represented by Formula (II). As a non-limiting example, a hydroxyl group of the helper lipid represented by Formula (II) may be linked to an organic acid such as phosphoric acid or carboxylic acid via the process of esterification to form an ester (e.g. , a carboxylate or a phosphate) of a helper lipid represented by Formula (II).

The term “deuterated analogue”, when used to refer to a helper lipid represented by Formula (II), means an analogue of a helper lipid represented by Formula (II), whereby any one or more hydrogen atoms of the helper lipid are substituted with deuterium, which is an isotope of hydrogen.

In one embodiment of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, ''' is a double bond; R¹, R², R³ and R⁴ are as defined above. In an alternative embodiment of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, ''' is a single bond; R¹, R², R³ and R⁴ are as defined above.

In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R¹ is C1-C15 alkyl or C2-C15 alkenyl.

In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing:

R¹ is C1-C15 alkyl or C2-C15 alkenyl;

R² is C1-C22 alkyl or C2-C22 alkenyl;

R³ is hydrogen or C1-C2 alkyl; and

R⁴ is hydrogen or C1-C2 alkyl.

In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R¹ is C1-C10 alkyl or C2-C10 alkenyl.

In some embodiments of Formula (II), or a salt or an ester thereof:

R¹ is C1-C10 alkyl or C2-C10 alkenyl;

R² is C1-C22 alkyl or C2-C22 alkenyl;

R³ is hydrogen or C1-C2 alkyl; and

R⁴ is hydrogen or C1-C2 alkyl.

In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R¹ is Ci-Cs alkyl or C2-C8 alkenyl. In one embodiment, R¹ is Ci-Cs alkyl. In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R¹ is C1-C7 alkyl or C2-C7 alkenyl. In one embodiment, R¹ is C1-C7 alkyl. In one

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MEl\57916143.vl 131698-30520 embodiment of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing:

R¹ is Ci-C₇ alkyl;

R² is C1-C22 alkyl or C2-C22 alkenyl;

R³ is hydrogen or C1-C2 alkyl; and

R⁴ is hydrogen or C1-C2 alkyl.

In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R¹ is Ci alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, Ce alkyl, or C₇ alkyl. In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R¹ is Ci alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, Ce alkyl, or C₇ alkyl. In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R¹ is Ci alkyl, C3 alkyl, C5 alkyl, or C₇ alkyl. In one embodiment of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R¹ is Ci alkyl. In one embodiment of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R¹ is C3 alkyl. In one embodiment of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R¹ is C5 alkyl. In one embodiment of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R¹ is C₇ alkyl.

In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R² is C3-C15 alkyl or C3-C15 alkenyl; and R¹, R³ and R⁴ are as defined above. In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R² is C5-C15 alkyl or C3-C15 alkenyl; and R¹, R³ and R⁴ are as defined above. In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R² is C₇-Cis alkyl or C3-C15 alkenyl; and R¹, R³ and R⁴ are as defined above. In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R² is C9-C15 alkyl or C9-C15 alkenyl; and R¹, R³ and R⁴ are as defined above. In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R² is C9 alkyl, C10 alkyl, Cn alkyl, C12 alkyl, C13 alkyl, C14 alkyl, or C15 alkyl; and R¹, R³ and R⁴ are as defined above. In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R² is C9 alkyl; and R¹, R³ and R⁴ are as defined above. In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R² is Cn alkyl; and R¹, R³ and R⁴ are as defined above. In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R² is C13 alkyl; and R¹, R³ and R⁴ are as defined above.

In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue thereof, R³ is hydrogen or Ci alkyl; and R¹, R² and R⁴ are as defined above. In one embodiment of Formula (II), or a salt or an ester thereof, or a deuterated analogue thereof, R³ is hydrogen; and R¹, R²

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MEl\57916143.vl 131698-30520 and R⁴ are as defined above. In one embodiment of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R³ is Ci alkyl; and R¹, R² and R⁴ are as defined above.

In some embodiments of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R⁴ is hydrogen or Ci alkyl; and R¹, R² and R³ are as defined above. In one embodiment of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R⁴ is hydrogen; and R¹, R² and R³ are as defined above. In one embodiment of Formula (II), or a salt or an ester thereof, or a deuterated analogue of any of the foregoing, R⁴ is Ci alkyl; and R¹, R² and R³ are as defined above.

In some embodiments of Formula (II), R¹ is C1-C7 alkyl or C2-C7 alkenyl.

In some embodiments, R¹ is Ci alkyl, C3 alkyl, C5 alkyl, or C7 alkyl. In some embodiments, R¹ is Ci alkyl.

In some embodiments of Formula (II), R² is C3-C15 alkyl or C3-C15 alkenyl. In some embodiments, R² is C10 alkyl, Cn alkyl, C12 alkyl, C13 alkyl, C14 alkyl, or C15 alkyl. In some embodiments, R² is C12 alkyl, C13 alkyl, or C14 alkyl. In some embodiments, R² is C13 alkyl. In some embodiments, R² is C12 alkyl. In some embodiments, R² is Cn alkyl.

In some embodiments of Formula (II), both R¹ and R² are hydrogen; and '' is a double bond.

In some embodiments of Formula (II), both R¹ and R² are hydrogen and ''' is a double bond; and R¹ is Ci alkyl, C3 alkyl, C5 alkyl or C7 alkyl. In one embodiment, R¹ is Ci alkyl. In another embodiment, R¹ is C3 alkyl. In yet another embodiment, R¹ is C5 alkyl. In yet another embodiment, R¹ is C7 alkyl.

In some embodiments of Formula (II), both R¹ and R² are hydrogen and '' is a double bond; R¹ is Ci alkyl, C3 alkyl, C5 alkyl or C7 alkyl and R² is C9 alkyl, Cn, or C 13 alkyl. In one embodiment, R² is C9 alkyl. In one embodiment, R² is Cn alkyl. In another embodiment, R² is C13 alkyl.

In some embodiments of Formula (II), R³ is hydrogen. In some embodiments of Formula (II), R³ is Ci alkyl.

In some embodiments of Formula (II), R⁴ is hydrogen. In some embodiments of Formula (II), R⁴ is Ci alkyl.

Other helper lipids

In some embodiments, a helper lipid comprised in an LNP of the present disclosure is a phospholipid, a phosphatidylcholine, or a derivative thereof. In some embodiments, the helper lipid comprised in an LNP of the present disclosure is selected from the group consisting of 1,2-distearoyl- sn-glycero-3 -phosphocholine (DSPC), hydrogenated soybean PC (HSPC), phosphatidylserine (PS), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine

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(DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), l,2-dilauroyl-sn-glycero-3- phosphocholine (DLPC), 1 -margaroyl -2 -oleoyl-sn-glycero-3 -phosphocholine (MOPC), 1-palmitoyl- 2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), 1 -stearoyl -2 -myristoyl-sn-glycero-3- phosphocholine (SMPC), l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), 1,2-dihexanoyl-sn- glycero-3 -phosphocholine (DHPC), l,2-dierucoyl-sn-glycero-3 -phosphocholine (DEPC), 1-palmitoyl- 2-oleoyl-glycero-3-phosphocholine (POPC), and l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE). In one embodiment, the helper lipid comprised in an LNP of the present disclosure is 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC).

In some embodiments, the helper lipid constitutes about 1 mol% to about 40 mol% of the total lipid present in the LNP, or about 5 mol% to about 15 mol%. In some embodiments, the helper lipid constitutes about 10% mol to about 20 mol% of the total lipid present in the LNP and such LNP having about 10% mol to about 20 mol% of the total lipid present in the LNP demonstrate overall increased tolerability (e.g., as demonstrated in body weight loss profdes in a subject and reduced cytokine response), as compared to the LNP comprising less than 10% of the same helper lipid.

D. Lipid-Anchored Polymers

In some embodiments, the LNPs provided by the present disclosure comprise at least one type of lipid-anchored polymer, e.g., a first lipid-anchored polymer and/or a second lipid-anchored polymer. As used herein, the term “lipid-anchored polymer” refers to a molecule comprising a lipid moiety covalently attached to a polymer, e.g., via a linker, e.g., a lipid-linker. Without wishing to be bound by a specific theory, it is believed that a lipid-anchored polymer can inhibit aggregation of LNPs and provide steric stabilization. In some embodiments, the LNPs provided by the present disclosure comprise two lipid-anchored polymers, i.e., a first lipid-anchored polymer and a second lipid-anchored polymer.

In some embodiments, the first lipid-anchored polymer comprised in an LNP of the present disclosure is the polymer-conjugated lipid of the present disclosure, e.g., DODA-PG34, DODA-PG39, DODA-PG45, DODA-PG46, DODA-PG58, or DODA-PG68. In some embodiments, the first lipid- anchored polymer comprises 1,2-O-dioctadecyl-sn-glycerol (DODG), e.g, DODG-PG34, DODG- PG39, DODG-PG45, DODG-PG46, DODG-PG58, or DODG-PG68.

In some embodiments, an LNP of the present disclosure comprises two types of a lipid- anchored polymer: a) the polymer-conjugated lipid of the present disclosure, e.g., DODA-PG34, , DODA-PG39, DODA-PG45, DODA-PG46, DODA-PG58, or DODA-PG68, as the first lipid- anchored polymer, and b) a second lipid-anchored polymer.

In some embodiments, a lipid-anchored polymer, e.g., a second lipid-anchored polymer in accordance with the present disclosure comprises:

(i) a lipid moiety comprising at least one hydrophobic tail; and

(ii) a hydrophilic polymer conjugated to the lipid moiety, optionally via a linker.

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(i) a lipid moiety comprising at least one hydrophobic tail;

(ii) a hydrophilic polymer;

(iii) a linker, wherein the polymer is conjugated to the lipid moiety via the linker; and

(iv) a targeting moiety conjugated to the polymer.

(i) a lipid moiety comprising at least one hydrophobic tail;

(ii) a hydrophilic polymer;

(iv) a reactive species conjugated to the polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety.

In some embodiments, the (ii) lipid moiety and the (iii) linker are collectively referred to as a “lipid-linker.” The terms “lipid-linker” or “lipid-linker moiety,” as used herein, refer to a lipid moiety comprising at least two hydrophobic tails, e.g., two hydrophobic tails, covalently attached to a linker.

In some embodiments, the lipid-anchored polymer comprises (i) a hydrophilic polymer, and (ii) a lipid-linker.

In one embodiment, the at least one (e.g., single or two) hydrophobic tail of the lipid moiety or of the lipid-linker moiety is a fatty acid (which may be linear or branched). Non-limiting examples of the at least one (e.g. , single or two) hydrophobic tail comprising 12 to 22 carbon atoms in a single aliphatic chain backbone include lauric acid, myristic acid, myristoleic acid, octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, and a derivative thereof.

The term “derivative,” when used herein in reference to hydrophobic tails in a lipid-anchored polymer, refers to a hydrophobic tail that has been modified as compared to the original or native hydrophobic tail. In some embodiments, the derivative contains one or more of the following modifications as compared to the original or native hydrophobic tail: a) carboxylate group has been replaced with an amine group, an amide group, an ether group, or a carbonate group; b) one or more points of saturation, e.g., double bonds, have been introduced into (e.g., via dehydrogenation) the hydrophobic tail; c) one or more points of saturation, e.g., double bonds, have been removed from (e.g., via hydrogenation) the hydrophobic tail; and d) configuration of one or more double bonds, if present, has been changed, e.g., from a cis configuration to a trans configuration, or from a trans

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MEl\57916143.vl 131698-30520 configuration to a cis configuration. The derivative contains the same number of carbon atoms as its original or native hydrophobic tail.

As used herein the term “a single aliphatic chain backbone” when referring to a hydrophobic tail in a lipid-anchored polymer refers the main linear aliphatic chain or carbon chain, z.e., the longest continuous linear aliphatic chain or carbon chain. For example, the alkyl chain below that has several branchings contains 18 carbon atoms in a single aliphatic chain backbone, z.e., the longest continuous linear alkyl chain contains 18 carbon atoms. Note that the one or two carbon atoms (all indicated with *) in the several branchings are not included in the carbon atom count in the single aliphatic chain backbone.

In one embodiment, a second lipid-anchored polymer in accordance with the present disclosure comprises a lipid moiety comprising at least one hydrophobic tail; and a polymer conjugated to the lipid moiety, optionally via a linker, wherein the lipid moiety of the second lipid- anchored polymer comprises a lipid-linker moiety selected from the group consisting of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 -palmitoyl -2 -oleoyl -glycero-3 -phosphocholine (POPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), 1 -palmitoyl -2 -oleoyl-sn- glycero-3-phospho-(l'-rac-glycerol) (POPG), l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), l,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn- phosphatidylethanolamine (DEPE), 1 -stearoyl -2 -oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE), l,2-dioleoyl-sn-glycero-3 -phosphoglycerol (DOPG), l,2-dipalmitoyl-sn-glycero-3 -phosphoglycerol (DPPG), 18-1-trans PE, l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn- glycero-3 -phosphoethanolamine (DPHyPE); and dioctadecylamine (DODA), dihexadecylamine, distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-O-dioctadecyl-sn-glycerol (DODG), and derivatives thereof. In some embodiments, the lipid moiety of the second lipid-anchored polymer comprises a lipid-linker moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, DODG, and a derivative thereof. In one specific embodiment, the lipid moiety of the second lipid-anchored polymer comprises DSPE. In one embodiment, the second lipid-anchored polymer further comprises a targeting moiety.

A lipid-anchored polymer of the present disclosure may also comprise a reactive species. In some embodiments, the reactive species is conjugated to the polymer in the lipid-anchored polymer. The reactive species present in a lipid-anchored polymer of the present disclosure may be used for conjugation, e.g., to a targeting moiety which has been functionalized with a complementary reactive

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MEl\57916143.vl 131698-30520 species, i.e., a reactive species capable of reacting with the reactive species comprised in the lipid- anchored polymer of the present disclosure. In some embodiments, the reactive species conjugated to the lipid-anchored polymer of the present disclosure may be a thiol reagent, a maleimide reagent, or click chemistry reagent, e.g., a reagent selected from the group consisting of an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent and an azide (AZ) reagent.

Linkers in lipid-anchored polymers

In some embodiments, in a lipid-anchored polymer of the present disclosure, a lipid moiety is covalently attached to a polymer via a linker. In some embodiments, the linker in the lipid-anchored polymer of the present disclosure is an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker (e.g., a glutary linker, a succinyl linker, etc. , an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, or any combination thereof. In some embodiments, the linker in the lipid-anchored polymer in the LNPs of the present disclosure is selected from the group consisting of -(CH2)_n-, -C(O)(CH2)_n-, -C(O)O(CH2)_n, -C(O)O(CH₂)_n-, -OC(O)(CH2)_nC(O)O-, and -NH(CH2)_nC(O)O-, wherein n is an number integer ranging from 1 to 20. Accordingly, in some embodiments, the linker is -C(O)(CH2)_n-, and in some embodiments, n is 2, 3, 4, 5, or 6.

In some embodiments, the linker of the second-lipid anchored polymer is a glycerol linker, a phosphate linker, an ether linker, an amide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, or -(CH2)_n-, -C(O)(CH2)_n-, or -C(O)O(CH₂)_n, wherein n is an integer ranging from 1 to 20, or any combination thereof.

The term “linker-lipid moiety”, as used herein, refers to a lipid moiety comprising at least one hydrophobic tail that is covalently attached to a linker. In some embodiments, the linker-lipid moiety may be a part of a lipid-anchored polymer.

As used herein, the term “derivative” when used in reference to a linker-lipid moiety means a linker-lipid moiety containing one or more of the following modifications: a) a phosphatidylethanolamine (PE) head group, if present, is modified to convert an amino group into a methylamino group or a dimethylamino group; b) the modified linker-lipid moiety comprises one or more additional functional groups or moieties, such as -OH, -OCH3, -NH2, a maleimide, an azide or a cyclooctyne such as dibonzeocyclooctyne (DBCO).

Polymers in lipid-anchored polymers

In some embodiments, the polymer comprised in the lipid-anchored polymer is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG),

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MEl\57916143.vl 131698-30520 polyglycerol (PG), polyvinyl alcohol (PVOH), polysarcosine (pSar), and a combination thereof. In one embodiment, the polymer is selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polysarcosine (pSar), poly(2 -methacryloyloxyethyl phosphorylcholine) (PMPC), and a combination thereof. In one embodiment, the polymer is polyethyelene glycol (PEG).

In some embodiments, the polymer comprised in the lipid-anchored polymer, e.g., the second lipid-anchored polymer, is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG), polyvinyl alcohol (PVOH), polysarcosine (pSar), polyglycerol (PG), and a derivative of any of the foregoing.

In some embodiments, the polymer comprised in the lipid-anchored polymer of the present disclosure, e.g., the first lipid-anchored polymer and/or the second lipid-anchored polymer, is polyglycerol (PG) or a PG derivative. The PG or the PG derivative may be linear or branched. The PG derivative may be a carboxylated PG, e.g., a glutarylated PG, such as 3-methyl glutarylated PG, or 2-carboxycyclohexane-l-carboxylated PG. In some embodiments, the PG or the PG derivative may comprise an average of 5-100 monomeric units. In some embodiments, the PG or the PG derivative may comprise an average of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,

53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,

80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, or about 34, 39, 45,

46, 58, or 68 monomeric units. In one embodiment, the PG or the PG derivative comprises an average of 34, 39, 45, 46, 50, 58, 58 ,or 68 monomeric units.

In some embodiments, the polymer in the lipid-anchored polymer has a molecular weight of between about 500 Da and about 5000 Da, e.g., between about 1500 Da and about 5000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 2000 Da.

Targeting moiety

In some embodiments, an LNP of the present disclosure further comprises one or more targeting moieties. The targeting moiety targets the LNP for delivery to a specific site or a tissue in a subject, e.g., liver. In some embodiments, the targeting moiety is capable of binding to specific liver cells, such as hepatocytes. The targeting moiety may be conjugated to a first lipid-anchored polymer, e.g., a polymer-conjugated lipid of the disclosure, or a second lipid-anchored polymer, as described herein.

In one embodiment, the targeting moiety is capable of binding to the asialoglycoprotein receptor (ASGPR), i.e., hepatocyte-specific ASGPR. In one embodiment, the targeting moiety comprises an '-acctylgalactosaminc molecule (GalNAc) or a GalNAc derivative thereof. As used herein, a “GalNAc derivative” refers to a modified GalNAc molecule or a conjugate of one or more GalNAc molecules (modified or unmodified) covalently linked to, for example, a lipid-anchored

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MEl\57916143.vl 131698-30520 polymer as defined herein. In one embodiment, the targeting moiety is a tri-antennary or tri-valent GalNAc conjugate (z.e., GalNAc3) which is a ligand conjugate having three GalNAc molecules or three GalNAc derivatives. In one embodiment, the targeting moiety is a tri-antennary GalNAc represented by the following structural formula:

In one embodiment, the targeting moiety is a tetra-antennary GalNAc conjugate. In one embodiment, the targeting moiety is a tetra-antennary or tetra- valent GalNAc conjugate (z.e., GalNAc4) which is a ligand having four GalNAc molecules or four GalNAc derivatives.

In one embodiment, the targeting moiety is capable of binding to low-density lipoprotein receptors (LDLRs), e.g., hepatocyte-specific LDLRs. In one embodiment, the targeting moiety comprises an apoliprotein E (ApoE) protein, an ApoE polypeptide (or peptide), an apoliprotein B (ApoB) protein, an ApoB polypeptide (or peptide), a fragment of any of the foregoing, or a derivative of any of the foregoing. In one embodiment, the ApoE polypeptide, ApoB polypeptide, or a fragment thereof is a ApoE polypeptide, ApoB polypeptide, or a fragment thereof as disclosed in International Patent Application Publication No. WO2022/261101, which is incorporated herein by reference in its entirety. In one embodiment, the ApoE protein is a modified ApoE protein and the ApoB protein is a modified ApoB protein.

In one embodiment, the ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence identity to the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYR GEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRER LGPLVEQGRVR (SEQ ID NO: 1). In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the ApoE protein consists essentially of the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO: 1.

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In one embodiment, the ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence identity to the following amino acid sequence:

MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYR GEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRER LGPLVEQGRVRHHHHHH (SEQ ID NO: 2). In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the ApoE protein consists essentially of the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO: 2.

MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYR GEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRER LGPLVEQGRVR (SEQ ID NO: 3). In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the ApoE protein consists essentially of the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO: 3.

MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYR GEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRER LGPLVEQGRVRHHHHHHGGSSGSGC (SEQ ID NO: 4). In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO: 4. In one embodiment, the ApoE protein

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MEl\57916143.vl 131698-30520 consists essentially of the amino acid sequence set forth in SEQ ID NO: 4. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO: 4.

As used herein, the term “sequence identity”, when used in reference to a polypeptide or a protein, refers to the ratio of the number of identical amino acids between the 2 aligned sequences over the aligned length, expressed as a percentage. In some embodiments, the 2 aligned sequences are identical in length, z.e., have the same number of amino acids.

In one embodiment, the targeting moiety in an LNP of the present disclosure is an ApoE protein conjugate in an ApoB protein conjugate, which is a conjugate of one or more ApoE and/or ApoB protein molecules (native or modified) or a fragment thereof covalently linked to, for example, a lipid-anchored polymer as defined herein. In one embodiment, the targeting moiety in an LNP of the present disclosure is an ApoE polypeptide conjugate in an ApoB polypeptide conjugate, which is a conjugate of one or more ApoE and/or ApoB polypeptide molecules or a fragment thereof covalently linked to, for example, a lipid-anchored polymer as defined herein.

In one embodiment the targeting moiety is an antibody or an antibody fragment, e.g. , an antibody or an antibody fragment that is capable of specifically binding to an antigen present on the surface of a cell. In one embodiment the antibody or an antibody fragment is a monoclonal antibody (mAb), a single chain variable fragment (scFv), a heavy chain antibody (hcAb), a nanobody (Nb), a heavy-chain-only immunoglobulin (HCIg), an immunoglobulin new antigen receptor (IgNAR), variable domain of immunoglobulin new antigen receptor (VNAR), a single-domain antibody, or a variable heavy chain-only antibody (VHH).

In some embodiments, an LNP of the present disclosure comprises a polymer-conjugated lipid of the present disclosure, and the targeting moiety as defined herein (and including GalNAc, ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, an antibody or an antibody fragment) is conjugated to the polymer-conjugated lipid of the present disclosure.

In some embodiments, an LNP of the present disclosure may comprise a polymer-conjugated lipid of the present disclosure as a first lipid-anchored polymer, and a targeting moiety as described herein conjugated to the polymer-conjugated lipid. In some embodiments the polymer in the polymer-conjugated lipid, e.g. , a PG or a PG derivative, is conjugated to a targeting moiety.

In some embodiments, the targeting moiety may be conjugated to the polymer-conjugated lipid via a reactive species. In some embodiments, the reactive species may be a thiol reagent, a maleimide reagent, or click chemistry reagent, e.g., a reagent selected from the group consisting of an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent and an azide (AZ) reagent. Accordingly, in an exemplary embodiment, the polymer-conjugated lipid of the present disclosure comprising an azide reagent as the reactive species may be reacted with a targeting moiety functionalized with a DBCO reagent as a complementary reactive species to produce a polymer-conjugated lipid conjugated to a targeting moiety via the reactive species. In another exemplary embodiment, the polymer-conjugated lipid of the present

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MEl\57916143.vl 131698-30520 disclosure comprising a thiol reagent may be reacted with a targeting moiety functionalized with a maleimide reagent to produce a polymer-conjugated lipid conjugated to a targeting moiety via the reactive species.

In some embodiments, an LNP of the present disclosure may comprise a polymer-conjugated lipid of the present disclosure as a first lipid-anchored polymer, a second lipid-anchored polymer and a targeting moiety as described herein conjugated to the second lipid-anchored polymer.

In some embodiments, the targeting moiety may be conjugated to the second lipid-anchored polymer via a reactive species. In some embodiments, the reactive species may be a thiol reagent, a maleimide reagent, or click chemistry reagent, e.g., a reagent selected from the group consisting of an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent and an azide (AZ) reagent. Accordingly, in an exemplary embodiment, the second lipid-anchored polymer of the present disclosure comprising an azide reagent as the reactive species may be reacted with a targeting moiety functionalized with a DBCO reagent as a complementary reactive species to produce a second lipid-anchored moiety conjugated to the targeting moiety via a reactive species. In another exemplary embodiment, the polymer-conjugated lipid of the present disclosure comprising a thiol reagent may be reacted with a targeting moiety functionalized with a maleimide reagent to produce a polymer-conjugated lipid comprising a targeting moiety.

Accordingly, in one embodiment of an LNP of the present disclosure, the LNP comprises a second lipid-anchored polymer and the targeting moiety as defined herein (and including GalNAc, ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, an antibody or an antibody fragment) is conjugated to the second lipid-anchored polymer. The second lipid-anchored polymer contains a lipid moiety conjugated to a polymer, optionally via a linker. In one embodiment, the second lipid-anchored polymer comprises a moiety selected from the group consisting of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 -palmitoyl -2 -oleoyl -glycero-3 -phosphocholine (POPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), 1 -palmitoyl -2 -oleoyl-sn- glycero-3-phospho-(l'-rac-glycerol) (POPG), l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), l,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn- phosphatidylethanolamine (DEPE), 1 -stearoyl -2 -oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE), l,2-dioleoyl-sn-glycero-3 -phosphoglycerol (DOPG), l,2-dipalmitoyl-sn-glycero-3 -phosphoglycerol (DPPG), 18-1-trans PE, l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn- glycero-3 -phosphoethanolamine (DPHyPE); and dioctadecylamine (DODA), dihexadecylamine, distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), and a derivative thereof. In one embodiment, the second lipid- anchored polymer comprises a linker-lipid moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, and a derivative thereof. In another embodiment the lipid moiety of the second lipid-anchored polymer comprises DSPE.

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In one embodiment, the ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, an antibody, or a fragment thereof, is covalently linked to a lipid-anchored polymer (e.g., first lipid anchored polymer or second lipid-anchored polymer) or to an LNP of the present disclosure via strain promoted alkyne-azide cycloaddition (SPAAC) chemistry, such as via an azide-modified lipid- anchored polymer (e.g., DSG-PEG2000-azide, DSPE-PEG2000-azide, DSG-PEG3400-azide, DSPE- PEG3400-azide, DSG-PEG5000-azide, DSPE-PEG5000-azide) and a dibenzocyclooctyne (DBCO)- functionalized ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, an antibody or a fragment thereof.

In an exemplary embodiment, the second lipid-anchored polymer conjugated to a targeting moiety is represented by the following structure:

In another exemplary embodiment, the second lipid-anchored polymer conjugated to a targeting moiety is represented by the following structure:

In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer. For example, the LNPs of the present disclosure may comprise a first lipid-anchored polymer that does not comprise a targeting moiety, and a second type of lipid-anchored polymer that comprises a targeting moiety, such as GalNAc or an antibody such as an scFv or VHH. For example, the LNPs of the present disclosure may comprise DSG-PEG2000 modified to comprise an additional OCHs group (DSG-PEG2000-OMe) as a first lipid-anchored polymer and DSPE-PEG2000-GalNAc3 as a second lipid-anchored polymer. In another example, the LNPs of the present disclosure may comprise a polymer-conjugated lipid as a first lipid-anchored polymer of the present disclosure and a second-anchored polymer, e.g, a second-anchored polymer conjugated to a targeting moiety. For example, the LNPs of the present disclosure may comprise DODA-PG45 as a first lipid-anchored polymer and DSPE-PEG2000-GH as the second lipid-anchored polymer.

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In one specific embodiment, the first lipid-anchored polymer is the polymer-conjugated lipid of the present disclosure, e.g., DODA-PG34, DODA-PG39, DODA-PG45, DODA-PG46, DODA- PG50, DODA-PG58, or DODA-PG68. For example, the LNPs of the present disclosure may comprise DODA-PG45 as a first lipid-anchored polymer and DODA-PG68-scFv as the second lipid- anchored polymer. Alternatively, the LNPs of the present disclosure may comprise DODA-PG39 or DODA-PG45 as a first lipid-anchored polymer and DODA-PG68-VHH as the second lipid-anchored polymer. The LNPs of the present disclosure may comprise DODA-PG45 as a first lipid-anchored polymer and DSPE-PEG2000-scFv as the second lipid-anchored polymer. The LNPs of the present disclosure may also comprise DODA-PG39 or DODA-PG45 as a first lipid-anchored polymer and DSPE-PEG2000-VHH as the second lipid-anchored polymer.

In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety. In some embodiments, the second lipid-anchored polymer comprises a lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, DODG, and a derivative of thereof. In some embodiments, the first lipid-anchored polymer is any lipid-anchored polymer as described hereinabove. In one specific embodiment, the first lipid-anchored polymer is the polymer-conjugated lipid of the present disclosure, e.g., DODA-PG34, DODA-PG39, DODA- PG45, DODA-PG46, DODA-PG58, or DODA-PG68. For example, the LNPs of the present disclosure may comprise DODA-PG45 as a first lipid-anchored polymer and DSPE-PEG2000- GalNAc3 as the second lipid-anchored polymer. In other embodiments, the first lipid-anchored polymer is DODG-PG34, , DODA-PG39, DODG-PG45, DODG-PG46, DODG-PG58, or DODA- PG68.

In some embodiments, the targeting moiety is conjugated to a DSPE-anchored polymer. In some embodiments, the DSPE-anchored polymer is DSPE-PEG or a derivative thereof.

In some embodiments, the targeting moiety is conjugated to a DSG-anchored polymer. In some embodiments, the DSG-anchored polymer is DSG-PEG or a derivative thereof.

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA); an ionizable lipid; a sterol; a helper lipid, and a first lipid-anchored polymer, wherein the first lipid-anchored polymer comprises the polymer-conjugated lipid.

In some embodiments, the ctLNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DSPE-PEG-IgG. In some embodiments, the ctLNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DSPE-PEG-IgG. In some embodiments, the ctLNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DSPE-PEG-IgG.

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In some embodiments, the ctLNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DSPE-PEG-VHH. In some embodiments, the ctLNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DSPE-PEG-VHH. In some embodiments, the ctLNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DSPE-PEG-VHH.

In some embodiments, the ctLNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DODA-PG-scFv. In some embodiments, the ctLNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DODA-PG-scFv. In some embodiments, the ctLNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DODA-PG-scFv.

In some embodiments, the ctLNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DODA-PG-VHH. In some embodiments, the ctLNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; and DODA-PG; and DODA-PG-VHH. In some embodiments, the ctLNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DODA-PG-VHH.

In some embodiments, the ctLNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; and DODA-PG46 (z. e. , polyglycerol having an average of 46 glycerol repeating units). In some embodiments, the ctLNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; and bis-DODA-PG46 (e.g., dl 8: 1/2:0 or dl4: 1/2:0). In some embodiments, the ctLNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; and DODA-PG46.

In some embodiments, the ctLNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; and DODA-PG34 (z. e. , polyglycerol having an average of 34 glycerol units). In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic

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MEl\57916143.vl 131698-30520 nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; and DODA-PG34. In some embodiments, the ctLNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; and DODA-PG34.

In some embodiments, the ctLNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA, e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG46; and DODA-PG46-VHH. In some embodiments, the ctLNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; DODA-PG46; and DODA-PG46-VHH. In some embodiments, the ctLNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG46; and DODA-PG46-VHH.

In some embodiments, thect LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA, e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG39; and DODA-PG68-VHH. In some embodiments, the ctLNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; DODA-PG39; and DODA-PG68-VHH. In some embodiments, the ctLNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG39; and DODA-PG68-VHH.

In some embodiments, the ctLNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA, e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG39; and DODA-PG58-VHH. In some embodiments, the ctLNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; DODA-PG39; and DODA-PG58-VHH. In some embodiments, the ctLNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG39; and DODA-PG58-VHH.

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; DODA-PG46; and DODA-PG46-scFv. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; DODA-PG46; and DODA-PG46-scFv. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG46; and DODA-PG46-scFv.

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In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; DODA-PG39; and DODA-PG68-scFv. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; DODA-PG39; and DODA-PG68-scFv. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG39; and DODA-PG68-scFv.

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OMe; and DODA-PG-VHH. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OMe; and DODA-PG-VHH. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OMe; and DODA- PG-VHH.

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OH; and DODA-PG-VHH. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OH; and DODA-PG-VHH. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OH; and DODA-PG-VHH.

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DODA-PG-scFv. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DODA-PG-scFv. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DODA-PG-scFv. In one embodiment, DODA-PG is DODA-PG39. In one embodiment, DODA-PG-scFv is DODA-PG68-scFv.

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DODA-PG-maleimide (Mai). In some embodiments, the

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LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DODA-PG-maleimide (Mai). In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG; and DODA-PG- maleimide (Mai). In one embodiment, DODA-PG is DODA-PG39. In one embodiment, DODA-PG- maleimide is DODA-PG58-maleimide (Mai). In one embodiment, DODA-PG-maleimide is DODA- PG68-maleimide (Mai).

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide); cholesterol; and DSG-PEG2000-OMe. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; and DSG-PEG2000- OMe. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OMe; and DSPE-PEG2000-VHH.

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; and DSG-PEG2000-OH. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OH; and DSPE-PEG2000-VHH. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g. , an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g. , DSPC, DOPE, ceramide) cholesterol; DSG-PEG2000-OH; and DSPE-PEG2000-VHH.

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OMe and DSPE-PEG2000-scFv. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OMe and DSPE-PEG2000-scFv. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OMe and DSPE- PEG2000-scFv.

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OH and DSPE-PEG2000-scFv. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g.,

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MEl\57916143.vl 131698-30520 an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OH and DSPE-PEG2000-scFv. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DSG-PEG2000-OH and DSPE- PEG2000-scFv.

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g., an siRNA); an ionizable lipid; helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; bis-DSG-PEG2000 and DSPE-PEG2000-scFv. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g. , an siRNA); an ionizable lipid; helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; bis-DSG- PEG2000 and DSPE-PEG2000-scFv. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA); an ionizable lipid; helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; bis-DSG-PEG2000 and DSPE-PEG2000-scFv.

In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG and DSPE-PEG-scFv. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG45 and DSPE-PEG2000-scFv. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA; e.g., an siRNA or mRNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; DODA-PG45 and DSPE-PEG2000-scFv.

In some embodiments, the lipid-anchored polymers (first and second lipid-anchored polymers in combination) constitute about 0.1 mol% to about 20 mol% of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 0.5 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 1 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute more than about 2 mol% (e.g., 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, 3.0 mol%) to about 10 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 3 mol% to about 8 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 3 mol% to about 7 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 3 mol% to about 5 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2 mol% to about 4 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2% to about 3% present in the LNP. In some embodiments, the lipid- anchored polymers constitute about 2 mol% present in the LNP. In some embodiments, the lipid- anchored polymers constitute about 2.5 mol% present in the LNP. In some embodiments, the lipid-

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MEl\57916143.vl 131698-30520 anchored polymers constitute about 3 mol% present in the LNP. In some embodiments, the lipid- anchored polymers constitute about 3.5 mol% present in the LNP. In some embodiments, the lipid- anchored polymers constitute about 4 mol% present in the LNP. In some embodiments, the lipid- anchored polymers constitute about 5 mol% present in the LNP. In some embodiments, the lipid- anchored polymers constitute about 6 mol% present in the LNP. In some embodiments, the lipid- anchored polymers constitute about 7 mol% present in the LNP. In some embodiments, the lipid- anchored polymers constitute about 8 mol% present in the LNP. In some embodiments, the lipid- anchored polymers constitute about 9 mol% present in the LNP. In some embodiments, the lipid- anchored polymers constitute about 10 mol% present in the LNP.

In some embodiments, the first lipid-anchored polymer is present at about 0. 1 mol% to about 10 mol% of the total lipid present in the LNP, or about 0.2 mol% to about 8 mol%, or about 0.2 mol% to about 7 mol%, or about 0.2% mol% to about 5 mol%, or about 0.3 mol to about 4 mol%, or about 0.4 mol% to about 4 mol%, or about 0.5 mol% to about 5 mol%, or about 0.5 mol% to about 4 mol%, or about 0.5 mol% to about 3.5 mol%, or about 0.5 mol% to about 3 mol%, or about 0.7 mol% to about 5 mol%, or about 0.7 mol% to about 4 mol%, or about 0.7 mol% to about 3.5 mol%, or about 0.7 mol% to about 3 mol%, or about 1 mol% to about 5 mol%, or about 1 mol% to about 4 mol%, or about 1 mol% to about 3.5 mol%, or about 1 mol% to about 3 mol%, or about 1.5 mol% to about 5 mol%, or about 1.5 mol% to about 4 mol%, or about 1.5 mol% to about 3.5 mol%, or about 1.5 mol% to about 3 mol%, or about 2 mol% to about 5 mol%, or about 2 mol% to about 4 mol%, or about 2 mol% to about 3.5 mol%, or about 2 mol% to about 3 mol%, or about 2.5 mol% to about 5 mol%, or about 2.5 mol% to about 4 mol%, or about 2.5 mol% to about 3.5 mol%, or about 2.5 mol% to about 3 mol%, or about 3 mol% to about 5 mol%, or about 3 mol% to about 4.5 mol% or about 3 mol% to about 4 mol%, or about 3 mol% to about 3.5 mol%, or about 3.5 mol% to about 5 mol%, or about 3.5 mol% to about 4.5 mol% or about 3.5 mol% to about 4 mol% or about 3 mol% to about 7 mol%. In some embodiments, the first lipid-anchored polymer is present at about 2.0 mol% to about 3.0 mol% of the total lipid present in the LNP.

In some embodiments, the second lipid-anchored polymer, if present, is present at about 0.01 mol% to about 5 mol% of the total lipid present in the LNP. In some embodiments, the second lipid- anchored polymer, if present, is present at about 0.01 mol% to about 4 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer, if present, is present at about 0.01 mol% to about 3 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer, if present, is present at about 0.01 mol% to about 2.5 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer, if present, is present at about 0.01 mol% to about 2 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer, if present, is present at about 0.01 mol% to about 1 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer, if present, is present at about 0.01 mol% to about 0.5 mol% of the total lipid present in the LNP. In some

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MEl\57916143.vl 131698-30520 embodiments, the second lipid-anchored polymer, if present, is present at about 0.01 mol% to about 0.25 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer, if present, is present at about 0.01 mol% to about 0.2 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer, if present, is present at about 0.01 mol% to about 0. 1 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer, if present, is present at about 0.05 mol% to about 0. 15 mol% of the total lipid present in the LNP.

In some embodiments, the second lipid-anchored polymer, if present, is present at about 0.005 mol% to about 5 mol% of the total lipid present in the LNP, or about 0.005 mol% to about 3 mol%, or about 0.005 mol% to about 2 mol%, or about 0.005 mol% to about 1 mol%, or about 0.005 mol% to about 0.5 mol%, or about 0.01 mol% to about 3 mol%, or about 0.01 mol% to about 2 mol%, or about 0.01 mol% to about 1 mol%, or about 0.01 mol% to about 0.5 mol%, or about 0.025 mol% to about 3 mol%, or about 0.025 mol% to about 2 mol%, or about 0.025 mol% to about 1 mol%, or about 0.025 mol% to about 0.5 mol%, or about 0.05 mol% to about 3 mol%, or about 0.05 mol% to about 2 mol%, or about 0.05 mol% to about 1 mol%, or about 0.05 mol% to about 0.5 mol%, or about 0.01 mol% to about 0.4 mol%, or about 0.01 mol% to about 0.3 mol%, or about 0.01 mol% to about 0.25 mol%, or about 0.01 mol% to about 0.2 mol%, or about 0.01 mol% to about 0. 1 mol%, or about 0.025 mol% to about 0.4 mol%, or about 0.025 mol% to about 0.3 mol%, or about 0.025 mol% to about 0.25 mol%, or about 0.025 mol% to about 0.2 mol%, or about 0.025 mol% to about 0. 1 mol%, or about 0.05 mol% to about 0.4 mol%, or about 0.05 mol% to about 0.3 mol%, or about 0.05 mol% to about 0.25 mol%, or about 0.05 mol% to about 0.2 mol%, or about 0.05 mol% to about 0.1 mol%.

In some embodiments, the second lipid-anchored polymer is present at about 0.01 mol%. In some embodiments, the second lipid-anchored polymer is present at about 0.05 mol%. In some embodiments, the second lipid-anchored polymer is present at about 0.06 mol%. In some embodiments, the second lipid-anchored polymer is present at about 0.08 mol%. In some embodiments, the second lipid-anchored polymer is present at about 0.5 mol%. In some embodiments, the second lipid-anchored polymer is present at about 0.1 mol%. In some embodiments, the second lipid-anchored polymer is present at about 0.2 mol%. In some embodiments, the second lipid-anchored polymer is present at about 0.3 mol%. In some embodiments, the second lipid-anchored polymer is present at about 0.4 mol%. In some embodiments, the second lipid-anchored polymer is present at about 0.1 mol% to about 0.4 mol%. In some embodiments, the second lipid-anchored polymer is present at about 0.1 mol% to about 0.3 mol%. In some embodiments, the second lipid-anchored polymer is present at about 0. 15 mol% to about 0.25 mol%.

In some embodiments, the ionizable lipid constitutes about 20 mol% to about 60 mol% of the total lipid present in the LNP. In some embodiments, the ionizable lipid constitutes about 35 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the sterol constitutes

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MEl\57916143.vl 131698-30520 about 20 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the sterol constitutes about 30 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid constitutes about 1 mol% to about 40 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid constitutes about 5 mol% to about 15 mol% of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer constitutes about 0.5 mol% to about 5 mol% of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer constitutes about 1.5 mol% to about 3 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer constitutes about 0.05 mol% to about 5 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer constitutes about 0.1 mol% to about 1 mol% of the total lipid present in the LNP.

Lipid nanoparticles (LNPs) comprising ceDNA are disclosed in International Patent Application No. PCT/US2018/050042, filed on September 7, 2018, which is incorporated herein in its entirety and envisioned for use in the methods and compositions as disclosed therein.

The size of LNPs can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK). In some embodiments, LNPs of the present disclosure have a mean diameter as determined by light scattering of less than about 90 nm, e.g., less than about 80 nm or less than about 75 nm. According to some embodiments, LNPs of the present disclosure have a mean diameter as determined by light scattering of between about 50 nm and about 75 nm or between about 50 nm and about 70 nm.

The pKa of formulated cationic lipids can be correlated with the effectiveness of the LNPs for delivery of nucleic acids (see Jayaraman et al., Angewandte Chemie. International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), both of which are incorporated by reference in their entireties). In one embodiment, the pKa of each cationic lipid is determined in lipid nanoparticles using an assay based on fluorescence of 2-(p-toluidino)-6- napthalene sulfonic acid (TNS). LNPs in PBS at a concentration of 0.4 mM total lipid can be prepared using the in-line process as described herein and elsewhere. TNS can be prepared as a 100 mM stock solution in distilled water. Vesicles can be diluted to 24 mM lipid in 2 mL of buffered solutions containing, 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH ranges from 2.5 to 11. An aliquot of the TNS solution can be added to give a final concentration of 1 mM and following vortex mixing fluorescence intensity is measured at room temperature in an SLM Aminco Series 2 Luminescence Spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best fit analysis can be applied to the fluorescence data and the pKa is measured as the pH giving rise to half-maximal fluorescence intensity.

In one embodiment, relative activity can be determined by measuring luciferase expression in the liver 4 hours following administration via tail vein injection. The activity is compared at a dose of

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0.3 and 1.0 mg ceDNA/kg and expressed as ng luciferase/g liver measured 4 hours after administration.

Without limitations, LNP of the present disclosure includes a lipid formulation that can be used to deliver a capsid-free, non-viral DNA vector to a target site of interest (e.g., cell, tissue, organ, and the like). Generally, the LNP comprises capsid-free, non-viral DNA vector and a cationic lipid or a salt thereof.

Further exemplary lipid-anchored polymers (e.g., second lipid-anchored polymer) include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, PG-lipid conjugates, polyamide -lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the second lipid-anchored polymer is a PEGylated lipid, for example, a (methoxy polyethylene glycol)-conjugated lipid. PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2, 3 -dimyristoylglycerol (PEG-DMG)), PEG- dialkyloxypropyl (DAA), PEG-phospholipid, a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2’,3’-di(tetradecanoyloxy)propyl-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl- methoxypoly ethylene glycol 2000)-l,2-distearoyl-sn-glycero-3 -phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in International Patent Application Publication Nos. WO 1996/010392, WO1998/051278, W02002/087541, W02005/026372, WO2008/147438, W02009/086558, W02012/000104, WO2017/117528, WO2017/099823, WO2015/199952, W02017/004143, WO2015/095346, WO2012/000104, WO2012/000104, and WO2010/006282, U.S. Patent Application Publication Nos. US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2013/0303587, US2018/0028664, US2015/0376115, US2016/0376224, US2016/0317458, US2013/0303587, US2013/0303587, and US20110123453, and U.S. Patent Nos. US5,885,613, US6,287,591, US6,320,017, and US6,586,559, the contents of all of which are incorporated herein by reference in their entireties.

Additional examples of PEG-DAA PEGylated lipids include, for example, PEG- dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG- disterylglycamide, PEG-cholesterol (l-[8’-(Cholest-5-en-3[beta]- oxy)carboxamido-3’,6’-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecoxylbenzyl- [omega]- methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]. In one embodiment, the PEG-lipid can be selected from the group consisting of PEG-DMG, l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000],

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Yet further exemplary lipid-anchored polymers (e.g., second lipid-anchored polymer) include N-(carbonyl-methoxyPEG_n)-l,2-dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE-PEG_n, where n is 350, 500, 750, 1000 or 2000), N-(carbonyl-methoxyPEG_n)-l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE-PEG_n, where n is 350, 500, 750, 1000 or 2000), DSPE-polyglycelin- cyclohexyl -carboxylic acid, DSPE-polyglycelin-2-methylglutar-carboxylic acid, 1,2-distearoyl-sn- glycero-3 -phosphoethanolamine (DSPE) conjugated polyethylene glycol (DSPE-PEG-OH), polyethylene glycol-dimyristolglycerol (PEG-DMG), or polyethylene glycol-distearoyl glycerol (PEG-DSG). In some examples of DMPE-PEG„, where n is 350, 500, 750, 1000 or 2000, the PEG- lipid is N-(carbonyl-methoxypolyethyleneglycol 2000)-l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE-PEG 2,000). In some examples of DSPE-PEG„. where n is 350, 500, 750, 1000 or 2000, the PEG-lipid is N-(carbonyl-methoxyPEG 2000)-l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE-PEG 2,000). In some embodiments, the PEG-lipid is DSPE-PEG-OH. In some embodiments, the PEG-lipid is PEG-DMG having two C14 hydrophobic tails and PEG2000.

E. Density of targeting moieties conjugated to the lipid-anchored polymer

In another embodiment of the present disclosure, the stealth T cell-targeted LNP (ctLNP) displays approximately 5, 10, 15, 20, 25, 30, 35, 40, 42, 45, 50, 52, 55, 60, 65, 70, 75, 80, 85, 90, 95,

100, 105, 110, 115, 120, 125, 126, 130, 135, 140, 145, 150, 155, 160, 165, 168, 170, 175, 180, 185,

190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285,

290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 340, 345, 350, 355, 360, 365, 370, 375,

380, 385, 390, 390, 395, or 400 targeting moieties per LNP.

In another embodiment, the stealth LNP displays at least 5 targeting moieties, at least 10 targeting moieties, at least 15 targeting moieties, at least 20 targeting moieties, at least 25 targeting

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MEl\57916143.vl 131698-30520 moieties, at least 30 targeting moieties, at least 35 targeting moieties, at least 40 targeting moieties, at least 45 targeting moieties, at least 50 targeting moieties, at least 55 targeting moieties, at least 60 targeting moieties, at least 65 targeting moieties, at least 70 targeting moieties, at least 75 targeting moieties, at least 80 targeting moieties, at least 85 targeting moieties, at least 90 targeting moieties, at least 95 targeting moieties, at least 100 targeting moieties, at least 110 targeting moieties, at least 120 targeting moieties, at least 130 targeting moieties, at least 140 targeting moieties, at least 150 targeting moieties, at least 160 targeting moieties, at least 170 targeting moieties, at least 180 targeting moieties, at least 190 targeting moieties, at least 200 targeting moieties, at least 210 targeting moieties, at least 220 targeting moieties, at least 230 targeting moieties, at least 240 targeting moieties, at least 250 targeting moieties per LNP, at least 260 targeting moieties, at least 270 targeting moieties, at least 280 targeting moieties, at least 290 targeting moieties, at least 300 targeting moieties per LNP, at least 310 targeting moieties per LNP, at least 320 targeting moieties per LNP, at least 330 targeting moieties per LNP, at least 340 targeting moieties per LNP, at least 350 targeting moieties per LNP, at least 360 targeting moieties per LNP, at least 370 targeting moieties per LNP, at least 380 targeting moieties per LNP, at least 390 targeting moieties per LNP, or at least 400 targeting moieties per LNP.

In another embodiment, the stealth LNP displays fewer than 400 targeting moieties, fewer than 390 targeting moieties, fewer than 380 targeting moieties, fewer than 370 targeting moieties, fewer than 360 targeting moieties, fewer than 350 targeting moieties, fewer than 340 targeting moieties, fewer than 330 targeting moieties, fewer than 320 targeting moieties, fewer than 310 targeting moieties, fewer than 300 targeting moieties, fewer than 290 targeting moieties, fewer than 280 targeting moieties, fewer than 270 targeting moieties, fewer than 260 targeting moieties, fewer than 250 targeting moieties, fewer than 240 targeting moieties, fewer than 230 targeting moieties, fewer than 220 targeting moieties, fewer than 210 targeting moieties, fewer than 200 targeting moieties, fewer than 190 targeting moieties, fewer than 180 targeting moieties, fewer than 170 targeting moieties, fewer than 160 targeting moieties, fewer than 150 targeting moieties, fewer than 140 targeting moieties, fewer than 130 targeting moieties, fewer than 120 targeting moieties, fewer than 110 targeting moieties, fewer than 100 targeting moieties, fewer than 95 targeting moieties, fewer than 90 targeting moieties, fewer than 85 targeting moieties, fewer than 80 targeting moieties, fewer than 75 targeting moieties, fewer than 70 targeting moieties, fewer than 65 targeting moieties, fewer than 60 targeting moieties, fewer than 55 targeting moieties, fewer than 50 targeting moieties, fewer than 45 targeting moieties, fewer than 40 targeting moieties, fewer than 35 targeting moieties, fewer than 30 targeting moieties, fewer than 25 targeting moieties, fewer than 20 targeting moieties, fewer than 15 targeting moieties, or fewer than 10 targeting moieties per LNP.

In another embodiment, the stealth LNP displays about 5-400 targeting moieties, about 10- 390 targeting moieties, about 20-380 targeting moieties, about 30-370 targeting moieties, about 40- 360 targeting moieties, about 50-350 targeting moieties, about 60-340 targeting moieties, about 70-

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330 targeting moieties, about 80-320 targeting moieties, about 90-310 targeting moieties, about 100- 300 targeting moieties, about 110-290 targeting moieties, about 120-280 targeting moieties, about 130-270 targeting moieties, about 140-260 targeting moieties, about 150-250 targeting moieties, about 160-249 targeting moieties, about 170-230 targeting moieties, about 180-220 targeting moieties, about 195-215 targeting moieties, or about 200-210 targeting moieties per LNP.

In another embodiment, the stealth LNP displays about 5-50 targeting moieties, about 50-100 targeting moieties, about 100-150 targeting moieties, about 150-200 targeting moieties, about 200-250 targeting moieties, about 250-300 targeting moieties, about 300-350 targeting moieties, or about 350- 400 targeting moieties.

In another embodiment, the stealth LNP displays about 5-100 targeting moieties, about 100- 200 targeting moieties, about 200-300 targeting moieties, or about 300-400 targeting moieties per LNP.

In another embodiment, the stealth LNP displays about 5-20 targeting moieties, about 20-40 targeting moieties, about 40-60 targeting moieties, about 60-80 targeting moieties, about 80-100 targeting moieties, about 100-120 targeting moieties, about 120-140 targeting moieties, about 140-160 targeting moieties, about 160-180 targeting moieties, about 180-200 targeting moieties, about 200-220 targeting moieties, about 220-240 targeting moieties, about 240-260 targeting moieties, about 260-280 targeting moieties, about 280-300 targeting moieties, about 200-320 targeting moieties, about 320-340 targeting moieties, about 340-360 targeting moieties, about 360-380 targeting moieties, or about 380- 400 targeting moieties per LNP.

In another embodiment, the stealth LNP displays about 5-10 targeting moieties, about 10-20 targeting moieties, about 20-30 targeting moieties, about 30-40 targeting moieties, about 40-50 targeting moieties, about 50-60 targeting moieties, about 60-70 targeting moieties, about 70-80 targeting moieties, about 80-90 targeting moieties, about 90-100 targeting moieties, about 100-110 targeting moieties, about 110-120 targeting moieties, about 120-130 targeting moieties, about 130-140 targeting moieties, about 140-150 targeting moieties, about 150-160 targeting moieties, about 160-170 targeting moieties, about 170-180 targeting moieties, about 180-190 targeting moieties, about 190-200 targeting moieties, about 210-220 targeting moieties, about 220-230 targeting moieties, about 230-240 targeting moieties, about 240-250 targeting moieties, about 250-260 targeting moieties, about 260-270 targeting moieties, about 270-280 targeting moieties, about 280-290 targeting moieties, about 290-300 targeting moieties, about 300-310 targeting moieties, about 310-320 targeting moieties, about 320-330 targeting moieties, about 330-340 targeting moieties, about 340-350 targeting moieties, about 350-360 targeting moieties, about 360-370 targeting moieties, about 370-380 targeting moieties, about 380-390 targeting moieties, or about 390-400 targeting moieties per LNP.

In another embodiment, the stealth LNP displays about 60-250 scFv targeting moieties, about 70-200 scFv targeting moieties, about 80-150 scFv targeting moieties, about 84-125 scFv targeting

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MEl\57916143.vl 131698-30520 moieties per LNP. In another embodiment, the stealth LNP displays about 84 or about 125 scFv targeting moieties per LNP.

In another embodiment, the stealth LNP displays about 20-400 VHH targeting moieties, about 30-350 VHH targeting moieties, about 40-300 VHH targeting moieties, about 50-250 VHH targeting moieties, about 52-210 VHH targeting moieties per LNP. In another embodiment, the stealth LNP displays about 52, 104 or 210 VHH targeting moieties per LNP.

F. Therapeutic Nucleic Acid

The LNPs provided by the present disclosure also comprise a therapeutic nucleic acid (TNA). According to embodiments, also provided are pharmaceutical compositions comprising the LNPs of the disclosure.

Illustrative therapeutic nucleic acids in the LNPs of the present disclosure can include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, deoxyribozymes, closed ended double stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA (“ministring”), doggybone™, protelomere closed ended DNA, or dumbbell linear DNA), dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), mRNA, tRNA, rRNA, gRNA, and DNA viral vectors, viral RNA vectors, non-viral vectors, and any combination thereof.

In any of the aspects and embodiments provided herein, the therapeutic nucleic acid can be a therapeutic DNA. Said therapeutic DNA can be ceDNA, ssDNA, CELiD, linear covalently closed DNA (“ministring” or otherwise), doggybone™, protelomere closed ended DNA, dumbbell linear DNA, minigenes, plasmids, or mini circles.

In one embodiment, the therapeutic nucleic acid can be a circular single -stranded polynucleotide comprised of at least three sections, two of which have sufficient complementarity to form a duplex, and an intervening sequence containing the single-stranded nucleic acid to be delivered, as described in described in WO2021/058984, the content of which is incorporated herein by reference in its entirety. siRNA or miRNA that can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi) are also contemplated by the present disclosure to be nucleic acid therapeutics. After siRNA or miRNA is introduced into the cytoplasm of a host cell, these double-stranded RNA constructs can bind to a protein called RISC. The sense strand of the siRNA or miRNA is removed by the RISC complex. The RISC complex, when combined with the complementary mRNA, cleaves the mRNA and release the cut strands. RNAi is by inducing specific destruction of mRNA that results in downregulation of a corresponding protein.

Antisense oligonucleotides (ASO) and ribozymes that inhibit mRNA translation into protein can be nucleic acid therapeutics. For antisense constructs, these single stranded deoxy nucleic acids

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MEl\57916143.vl 131698-30520 have a complementary sequence to the sequence of the target protein mRNA, and Watson - capable of binding to the mRNA by Crick base pairing. This binding prevents translation of a target mRNA, and/or triggers RNaseH degradation of the mRNA transcript. As a result, the antisense oligonucleotide has increased specificity of action (z.e., down-regulation of a specific disease-related protein).

In any of the aspects and embodiments provided herein, the therapeutic nucleic acid can be a therapeutic RNA. Said therapeutic RNA can be messenger RNA (mRNA) encoding a protein or peptide, an inhibitor of mRNA translation, agent of RNA interference (RNAi), catalytically active RNA molecule (ribozyme), transfer RNA (tRNA), an RNA that binds an mRNA transcript (ASO), protein or other molecular ligand (aptamer), or a guide RNA (gRNA). In any of the methods provided herein, the agent of RNAi can be a double -stranded RNA, single -stranded RNA, microRNA, short interfering RNA, short hairpin RNA, or a triplex-forming oligonucleotide.

Closed-ended DNA (ceDNA) vectors

In some embodiments, LNPs provided by the present disclosure comprise closed-ended DNA (ceDNA).

In some embodiments, the TNA comprises closed-ended linear duplexed (ceDNA) vectors that can express atransgene (e.g., a therapeutic nucleic acid (TNA)). The ceDNA vectors as described herein have no packaging constraints imposed by the limiting space within the viral capsid. ceDNA vectors represent a viable eukaryotically-produced alternative to prokaryote-produced plasmid DNA vectors, as opposed to encapsulated AAV genomes. This permits the insertion of control elements, e.g., regulatory switches as disclosed herein, large transgenes, multiple transgenes etc. ceDNA vectors preferably have a linear and continuous structure rather than a non- continuous structure. The linear and continuous structure is believed to be more stable from attack by cellular endonucleases, as well as less likely to be recombined and cause mutagenesis. Thus, a ceDNA vector in the linear and continuous structure is a preferred embodiment. The continuous, linear, single strand intramolecular duplex ceDNA vector can have covalently bound terminal ends, without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally distinct from plasmids (including ceDNA plasmids described herein), which are circular duplex nucleic acid molecules of bacterial origin. The complimentary strands of plasmids may be separated following denaturation to produce two nucleic acid molecules, whereas in contrast, ceDNA vectors, while having complimentary strands, are a single DNA molecule and therefore even if denatured, it is likely to remain a single molecule. In some embodiments, ceDNA vectors can be produced without DNA base methylation of prokaryotic type, unlike plasmids. Therefore, the ceDNA vectors and ceDNA-plasmids are different both in term of structure (in particular, linear versus circular) and also in view of the methods used for producing and purifying these different objects, and also in view of

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MEl\57916143.vl 131698-30520 their DNA methylation which is of prokaryotic type for ceDNA-plasmids and of eukaryotic type for the ceDNA vector.

Provided herein are non-viral, capsid-free ceDNA molecules with covalently closed ends (ceDNA). These non-viral capsid free ceDNA molecules can be produced in permissive host cells from an expression construct (e.g., a ceDNA-plasmid, a ceDNA-bacmid, a ceDNA-baculovirus, or an integrated cell-line) containing a heterologous gene (e.g., a transgene, in particular a therapeutic transgene) positioned between two different inverted terminal repeat (ITR) sequences, where the ITRs are different with respect to each other. In some embodiments, one of the ITRs is modified by deletion, insertion, and/or substitution as compared to a wild-type ITR sequence (e.g. AAV ITR); and at least one of the ITRs comprises a functional terminal resolution site (trs) and a Rep binding site. The ceDNA vector is preferably duplex, e.g., self-complementary, over at least a portion of the molecule, such as the expression cassette (e.g., ceDNA is not a double stranded circular molecule). The ceDNA vector has covalently closed ends, and thus is resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), e.g., for over an hour at 37°C.

In one aspect, a ceDNA vector comprises, in the 5’ to 3’ direction: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (for example an expression cassette as described herein) and a second AAV ITR. In one embodiment, the first ITR (5’ ITR) and the second ITR (3’ ITR) are asymmetric with respect to each other - that is, they have a different 3D-spatial configuration from one another. As an exemplary embodiment, the first ITR can be a wild-type ITR and the second ITR can be a mutated or modified ITR, or vice versa, where the first ITR can be a mutated or modified ITR and the second ITR a wild- type ITR. In one embodiment, the first ITR and the second ITR are both modified but are different sequences, or have different modifications, or are not identical modified ITRs, and have different 3D spatial configurations. Stated differently, a ceDNA vector with asymmetric ITRs have ITRs where any changes in one ITR relative to the WT-ITR are not reflected in the other ITR; or alternatively, where the asymmetric ITRs have a the modified asymmetric ITR pair can have a different sequence and different three-dimensional shape with respect to each other.

In one embodiment, a ceDNA vector comprises, in the 5’ to 3’ direction: a first adeno- associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (for example an expression cassette as described herein) and a second AAV ITR, where the first ITR (5 ’ ITR) and the second ITR (3 ’ ITR) are symmetric, or substantially symmetrical with respect to each other - that is, a ceDNA vector can comprise ITR sequences that have a symmetrical three-dimensional spatial organization such that their structure is the same shape in geometrical space, or have the same A, C- C’ and B-B’ loops in 3D space. In such an embodiment, a symmetrical ITR pair, or substantially symmetrical ITR pair can be modified ITRs (e.g., mod-ITRs) that are not wild-type ITRs. A mod- ITR pair can have the same sequence which has one or more modifications from wild-type ITR and are reverse complements (inverted) of each other. In one embodiment, a modified ITR pair are

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MEl\57916143.vl 131698-30520 substantially symmetrical as defined herein, that is, the modified ITR pair can have a different sequence but have corresponding or the same symmetrical three-dimensional shape. In some embodiments, the symmetrical ITRs, or substantially symmetrical ITRs can be wild type ITRs (WT- ITRs) as described herein. That is, both ITRs have a wild-type sequence from the same AAV serotype. In some other embodiments, the two wild-type ITRs can be from different AAV serotypes. For example, one WT-ITR can be from one AAV serotype, and the other WT-ITR can be from a different AAV serotype. In such an embodiment, a WT-ITR pair are substantially symmetrical as defined herein, that is, they can have one or more conservative nucleotide modification while still retaining the symmetrical three-dimensional spatial organization.

The wild-type or mutated or otherwise modified ITR sequences provided herein represent DNA sequences included in the expression construct (e.g., ceDNA-plasmid, ceDNA Bacmid, ceDNA- baculovirus) for production of the ceDNA vector. Thus, ITR sequences actually contained in the ceDNA vector produced from the ceDNA-plasmid or other expression construct may or may not be identical to the ITR sequences provided herein as a result of naturally occurring changes taking place during the production process (e.g., replication error).

In one embodiment, a ceDNA vector in the LNPs of the present disclosure comprising the expression cassette with a transgene which is a therapeutic nucleic acid sequence, can be operatively linked to one or more regulatory sequence(s) that allows or controls expression of the transgene. In one embodiment, the polynucleotide comprises a first ITR sequence and a second ITR sequence, wherein the nucleotide sequence of interest is flanked by the first and second ITR sequences, and the first and second ITR sequences are asymmetrical relative to each other, or symmetrical relative to each other.

In one embodiment, an expression cassette is located between two ITRs in the following order with one or more of: a promoter operably linked to a transgene, a posttranscriptional regulatory element, and a polyadenylation and termination signal. In one embodiment, the promoter is regulatable - inducible or repressible. The promoter can be any sequence that facilitates the transcription of the transgene. In one embodiment the promoter is a CAG promoter, or variation thereof. The posttranscriptional regulatory element is a sequence that modulates expression of the transgene, as a non-limiting example, any sequence that creates a tertiary structure that enhances expression of the transgene which is a therapeutic nucleic acid sequence.

In one embodiment, the posttranscriptional regulatory element comprises WPRE. In one embodiment, the polyadenylation and termination signal comprise BGHpolyA. Any cis regulatory element known in the art, or combination thereof, can be additionally used e.g., SV40 late polyA signal upstream enhancer sequence (USE), or other posttranscriptional processing elements including, but not limited to, the thymidine kinase gene of herpes simplex virus, or hepatitis B virus (HBV). In one embodiment, the expression cassette length in the 5 ’ to 3 ’ direction is greater than the maximum length known to be encapsidated in an AAV virion. In one embodiment, the length is greater than 4.6

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MEl\57916143.vl 131698-30520 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Various expression cassettes are exemplified herein.

In one embodiment, the expression cassette can comprise more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides or 50,000 nucleotides, or any range between about 4000-10,000 nucleotides or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 50,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 75,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 10,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 1000 to 10,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 5,000 nucleotides in length. The ceDNA vectors do not have the size limitations of encapsidated AAV vectors, and thus enable delivery of a large-size expression cassette to the host. In one embodiment, the ceDNA vector is devoid of prokaryote -specific methylation.

In one embodiment, the rigid therapeutic nucleic acid can be a plasmid.

In one embodiment, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary uses) or immunogenic polypeptides.

The expression cassette can comprise any transgene which is a therapeutic nucleic acid sequence. In certain embodiments, the ceDNA vector comprises any gene of interest in the subject, which includes protein, enzyme, one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, gRNA, mRNA, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen binding fragments, or any combination thereof.

In one embodiment, the ceDNA expression cassette can include, for example, an expressible exogenous sequence (e.g., open reading frame) that encodes a protein that is either absent, inactive, or insufficient activity in the recipient subject or a gene that encodes a protein having a desired biological or a therapeutic effect. In one embodiment, the exogenous sequence such as a donor sequence can encode a gene product that can function to correct the expression of a defective gene or transcript. In one embodiment, the expression cassette can also encode corrective DNA strands, encode polypeptides, sense or antisense oligonucleotides, or coding RNAs or non-coding RNAs (e.g., siRNAs, guide RNAs, shRNAs, micro-RNAs, and their antisense counterparts (e.g., antagoMiR)). In one embodiment, expression cassettes can include an exogenous sequence that encodes a reporter protein to be used for experimental or diagnostic purposes, such as P-lactamase, P-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

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Accordingly, the expression cassette can include any gene that encodes a protein, polypeptide or RNA that is either reduced or absent due to a mutation or which conveys a therapeutic benefit when overexpressed is considered to be within the scope of the disclosure. The ceDNA vector may comprise a template or donor nucleotide sequence used as a correcting DNA strand to be inserted after a double-strand break (or nick) provided by a nuclease. The ceDNA vector may include a template nucleotide sequence used as a correcting DNA strand to be inserted after a double-strand break (or nick) provided by a guided RNA nuclease, meganuclease, or zinc finger nuclease.

Single-Stranded (ss) Nucleic Acid Molecules

In some embodiments, the TNA comprised in an LNP of the present disclosure may be a single-stranded nucleic acid, e.g., a single -stranded DNA or a single -stranded RNA. In one embodiment, the TNA may be a single -stranded RNA, e.g., mRNA. In another embodiment, the TNA may be a single-stranded DNA (ssDNA) molecule, e.g., a synthetic ssDNA molecule.

3 ’ End Stem -Loop Structure

In some aspects, the TNA is a ssDNA molecule comprising at least one nucleic acid sequence of interest flanked by at least one stem -loop structure at the 3’ end. In some embodiments, the ssDNA molecule may further comprise at least one stem-loop structure at the 5’ end. As described herein, the stem -loop structure at the 3 ’ end may comprise a partial DNA duplex (e.g. , with a free 3 ’ -OH group) to prime replication or transcription. The partial DNA duplex functions, in part, to hold the stem-loop structure together.

According to some embodiments, the partial DNA duplex comprises between 4-500 nucleotides, for example between 4-10 nucleotides, between 4-25 nucleotides, between 4-50 nucleotides, between 4-100 nucleotides, between 4-200 nucleotides, between 4-300 nucleotides, between 4-400 nucleotides, between 20-25 nucleotides, between 20-50 nucleotides, between 20-100 nucleotides, between 20-200 nucleotides, between 20-300 nucleotides, between 20-400 nucleotides, between 20-500 nucleotides, between 50-100 nucleotides, between 50-200 nucleotides, between SO- SOO nucleotides, between 50-400 nucleotides, between 50-500 nucleotides, 150-200 nucleotides, between 150-300 nucleotides, between 150-400 nucleotides, between 150-500 nucleotides, between 200-300 nucleotides, between 200-400 nucleotides, between 200-500 nucleotides, between 250-300 nucleotides, between 250-400 nucleotides, between 250-500 nucleotides, between 300-400 nucleotides, between 300-500 nucleotides, between 350-400 nucleotides, between 350-500 nucleotides, between 400-500 nucleotides, or between 450-500 nucleotides, and at least one loop on the 3’ end. According to some embodiments, the DNA duplex comprises at least 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, 250, 300, 350, 400, 450 or 500 nucleotides, and at least one loop on the 3’ end.

According to some embodiments, the loop structure at the 3 ’ end comprises a minimum of between 3-500 unbound nucleotides, for example between 3-450 nucleotides, between 3-400

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MEl\57916143.vl 131698-30520 nucleotides, between 3-350 nucleotides, between 3-300 nucleotides, between 3-250 nucleotides, between 3-200 nucleotides, between 3-150 nucleotides, between 3-100 nucleotides, between 3-90 nucleotides, between 3-80 nucleotides, between 3-70 nucleotides, between 3-60 nucleotides, between 3-50 nucleotides, between 3-40 nucleotides, between 3-30 nucleotides, between 3-20 nucleotides, between 3-10 nucleotides, between 3-5 nucleotides, between 10-450 nucleotides, between 10-400 nucleotides, between 10-350 nucleotides, between 10-300 nucleotides, between 10-250 nucleotides, between 10-200 nucleotides, between 10-150 nucleotides, between 10-100 nucleotides, between 10- 90 nucleotides, between 10-80 nucleotides, between 10-70 nucleotides, between 10-60 nucleotides, between 10-50 nucleotides, between 10-40 nucleotides, between 10-30 nucleotides, between 10-20 nucleotides, between 50-450 nucleotides, between 50-400 nucleotides, between 50-350 nucleotides, between 50-300 nucleotides, between 50-250 nucleotides, between 50-200 nucleotides, between 50- 150 nucleotides, between 50-100 nucleotides, between 50-90 nucleotides, between 50-80 nucleotides, between 50-70 nucleotides, between 50-60 nucleotides, between 100-450 nucleotides, between 100- 400 nucleotides, between 100-350 nucleotides, between 100-300 nucleotides, between 100-250 nucleotides, between 100-200 nucleotides, between 150-450 nucleotides, between 150-400 nucleotides, between 150-350 nucleotides, between 150-300 nucleotides, between 150-250 nucleotides, between 150-200 nucleotides, between 200-450 nucleotides, between 200-400 nucleotides, between 200-350 nucleotides, between 200-300 nucleotides, between 200-250 nucleotides, between 250-450 nucleotides, between 250-400 nucleotides, between 250-350 nucleotides, between 250-300 nucleotides, between 300-450 nucleotides, between 300-400 nucleotides, between 300-350 nucleotides, between 350-450 nucleotides, between 350-400 nucleotides, or between 400-450 nucleotides.

According to some embodiments, the stem portion of the stem-loop is 4-500 nucleotides in length and the loop portion of the stem-loop is 3-500 nucleotides in length. According to some embodiments, the stem portion of the stem -loop is 4-50 nucleotides in length and the loop portion of the stem-loop is 3-50 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4-20 nucleotides in length and the loop portion of the stem-loop is 3-20 nucleotides in length. According to some embodiments, the stem portion of the stem -loop is 4-10 nucleotides in length and the loop portion of the stem-loop is 3-10 nucleotides in length.

According to some embodiments, the loop further comprises one or more nucleic acids or that are used to stabilize the ends. According to other embodiments, the loop further comprises one or more nucleic acids that may be employed in therapeutic methods. According to other embodiments, the loop further comprises one or more nucleic acids that may be employed in diagnostic methods. According to other embodiments, the loop further comprises one or more nucleic acids that that may be employed for research purposes.

According to some embodiments, the minimal nucleic acid structure that is necessary at the 3 ’ end of the ssDNA is any structure that loops back on itself, z.e., a hairpin structure. However, it is to

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MEl\57916143.vl 131698-30520 be understood that a variety of structures are envisioned at the 3’ end, as long as there is at least one stem and one loop. For example, in some embodiments, the ssDNA described herein may comprise at least one stem -loop structure at the 3’ end. In some embodiments, the ssDNA may comprise at least two stem -loop structures at the 3’ end. In some embodiments, the ssDNA may comprise at least three stem -loop structures at the 3’ end. In some embodiments, the ssDNA may comprise at least four stem -loop structures at the 3’ end. In some embodiments, the ssDNA may comprise at least five stem -loop structures at the 3’ end.

According to some embodiments, the nucleotides at the 3 ’ end form a cruciform DNA structure. A DNA cruciform structure can be formed when both strands form a stem-loop structure at the same location in the molecule, and comprises a four-way junction and two closed hairpin-shaped points.

According to some embodiments, the nucleotides at the 3’ end form a hairpin DNA structure. Hairpin loop structures in nucleic acids consist of a base-paired stem structure and a loop sequence with unpaired or non-Watson-Crick-paired nucleotides.

According to some embodiments, the nucleotides at the 3 ’ end form a hammerhead DNA structure, made up of three base paired helices, separated by short linkers of conserved sequence.

According to some embodiments, the nucleotides at the 3 ’ end form a quadraplex DNA structure. G-quadruplexes are four-stranded DNA secondary structures (G4s) that form from certain guanine-rich sequences.

According to some embodiments, the nucleotides at the 3’ end form a bulged DNA structure.

According to some embodiments, the nucleotides at the 3’ end form a multibranched loop.

According to some embodiments, the nucleotides at the 3 ’ end do not form a 2 stem -loop structure.

According to some embodiments, the stem structure at the 3’ end comprises one or more nucleotides that are modified to be exonuclease resistant. According to some embodiments, the stem structure at the 3’ end comprises two or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 20 or more nucleotides that are modified to be exonuclease resistant.

According to some embodiments, the stem structure at the 3’ end comprises one or more phosphorothioate-modified nucleotides. According to some embodiments, the stem structure at the 3’ end comprises about 2 to about 12 phosphorothioate-modified nucleotides. According to some embodiments, the stem structure at the 3’ end comprises about 4 to about 10 phosphorothioate- modified nucleotides, e.g., about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9,

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MEl\57916143.vl 131698-30520 about 8 to about 10 or about 9 to about 10. According to some embodiments, the stem structure comprises more than 10 phosphorothioate-modified nucleotides.

According to some embodiments, the phosphorothioate-modified nucleotides are located adjacent to each other.

According to some embodiments, the one or more phosphorothioate-modified nucleotides of the 3’ end are resistant to exonuclease degradation. Boranophosphate modified DNA is also resistant to nuclease degradation, and may be considered as an alternative to phosphorothioate modification.

According to further embodiments, the stem structure may comprise at least one functional moiety. In one embodiment, the at least one functional moiety is an aptamer sequence. In further embodiments, the aptamer sequence has a high binding affinity to a nuclear localized protein.

According to some embodiments, the nucleotides in the loop are chemically modified with functional groups in order to alter their properties.

According to some embodiments, the loop further comprises one or more aptamers. According to some embodiments, the aptamer is identified from the Apta-index database of aptamers available to the public (aptagen.com/apta-index).

According to some embodiments, the loop further comprises one or more synthetic ribozymes.

According to some embodiments, the loop further comprises one or more antisense oligonucleotides (ASOs).

According to some embodiments, the loop further comprises one or more short-interfering RNAs (siRNAs).

According to some embodiments, the loop further comprises one or more antiviral nucleoside analogues (ANAs).

According to some embodiments, the loop further comprises one or more triplex forming oligonucleotides.

According to some embodiments, the loop further comprises one or more gRNAs or gDNAs.

According to some embodiments, the loop further comprises one or more molecular probes, for example nucleic acid based fluorescent probes.

According to some embodiments, “click” azide-alkyne cycloaddition (Kolb et al., Angew. Chem. Int. Ed. Engl. 2001, 40, 2004-2021) is used to modify the nucleotides in the loop. Click chemistry was developed to join together organic molecules under mild conditions in the presence of a diverse range of functional groups. Most click-mediated modifications are performed on the nitrogenous bases by introducing novel base analogues, attaching fluorophores or isotopic elements for molecular imaging, forming inter-strand linkages between oligonucleotides, and for the bioconjugation of molecules. The best example of click chemistry is the Cu¹ catalyzed version of Huisgen’s [3 + 2] azide-alkyne cycloaddition reaction (Angew. Chem., Int. Ed. 1963, 2, 633-645),

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MEl\57916143.vl 131698-30520 discovered independently by Sharpless and Meldal (the CuAAC reaction) (Angew. Chem., Int. Ed. 2002, 41, 2596-2599).

According to some embodiments, the introduction of active amino or thiol groups into synthesized oligonucleotides provides acceptors for, e.g., subsequent chemical fluorescent labeling.

According to some embodiments, the stem-loop structure may comprise alternative or modified nucleotides, including, but not limited to, ribonucleic acids (RNA), peptide-nucleic acids (PNA), locked nucleic acids (LNA). According to some embodiments, the loop portion of the stemloop structure may comprise a chemical structure that does not comprise nucleic acids.

IV. Preparation of Lipid Nanoparticles (LNPs)

Lipid nanoparticles (LNPs) can form spontaneously upon mixing of a therapeutic nucleic acid (e.g., ceDNA, ssDNA, synthetic AAV, etc., as described herein) and a pharmaceutically acceptable excipient that comprises a lipid.

Generally, LNPs can be formed by any method known in the art. For example, the LNPs can be prepared by the methods described, for example, in US2013/0037977, US2010/0015218, US2013/0156845, US2013/0164400, US2012/0225129, and US2010/0130588, content of each of which is incorporated herein by reference in its entirety. In some embodiments, LNPs can be prepared using a continuous mixing method, a direct dilution process, or an in-line dilution process. The processes and apparatuses for preparing lipid nanoparticles using direct dilution and in-line dilution processes are described in US2007/0042031, the content of which is incorporated herein by reference in its entirety. The processes and apparatuses for preparing lipid nanoparticles using step- wise dilution processes are described in US2004/0142025, the content of which is incorporated herein by reference in its entirety.

According to some embodiments, the disclosure provides for an LNP comprising a DNA vector, including a ceDNA vector, ssDNA vector, or synthetic AAV, as described herein and an ionizable lipid. For example, a lipid nanoparticle formulation that is made and loaded with therapeutic nucleic acid like ceDNA obtained by the process as disclosed in International Patent Application No. PCT/US2018/050042, fded on September 7, 2018, which is incorporated by reference in its entirety herein. This can be accomplished by high energy mixing of ethanolic lipids with aqueous synthetic AAV at low pH which protonates the ionizable lipid and provides favorable energetics for synthetic AAV/lipid association and nucleation of particles. The particles can be further stabilized through aqueous dilution and removal of the organic solvent. The particles can be concentrated to the desired level.

Generally, the lipid particles are prepared at a total lipid to synthetic AAV (mass or weight) ratio of from about 10: 1 to 30: 1. In some embodiments, the lipid to ssDNA molecule or the dsDNA construct ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1: 1 to about 25: 1, from about 10: 1 to about 14: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1

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MEl\57916143.vl 131698-30520 to about 9: 1, or about 6: 1 to about 9: 1. The amounts of lipids and synthetic AAV can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the lipid particle formulation’s overall lipid content can range from about 5 mg/ml to about 30 mg/mL.

The ionizable lipid is typically employed to condense the nucleic acid cargo at low pH and to drive membrane association and fusogenicity. Generally, ionizable lipids are lipids comprising at least one amino group that is positively charged or becomes protonated under acidic conditions, for example at pH of 6.5 or lower.

In one embodiment, the LNPs can be prepared by an impinging jet process. Generally, the particles are formed by mixing lipids dissolved in alcohol (e.g., ethanol) with ceDNA dissolved in a buffer, e.g., a citrate buffer, a sodium acetate buffer, a sodium acetate and magnesium chloride buffer, a malic acid buffer, a malic acid and sodium chloride buffer, or a sodium citrate and sodium chloride buffer. The mixing ratio of lipids to ceDNA can be about 45-55% lipid and about 65-45% ceDNA.

The lipid solution can contain an ionizable lipid, a ceramide, a lipid-anchored polymer and a sterol (e.g., cholesterol) at a total lipid concentration of 5-30 mg/mL, more likely 5-15 mg/mL, most likely 9-12 mg/mL in an alcohol, e.g., in ethanol. In the lipid solution, mol ratio of the lipids can range from about 25-98% for the cationic lipid, preferably about 35-65%; about 0-15% for the nonionic lipid, preferably about 0-12%; about 0-15% for the PEG or PEG conjugated lipid molecule, preferably about 1-6%; and about 0-75% for the sterol, preferably about 30-50%.

The ceDNA solution can comprise the ceDNA at a concentration range from 0.3 to 1.0 mg/mL, preferably 0.3-0.9 mg/mL in buffered solution, with pH in the range of 3.5-5.

For forming the LNPs, in one exemplary but nonlimiting embodiment, the two liquids are heated to a temperature in the range of about 15-40°C, preferably about 30-40°C, and then mixed, for example, in an impinging jet mixer, instantly forming the LNP. The mixing flow rate can range from 10-600 mL/min. The tube ID can have a range from 0.25 to 1.0 mm and a total flow rate from 10-600 mL/min. The combination of flow rate and tubing ID can have the effect of controlling the particle size of the LNPs between 30 and 200 nm. The solution can then be mixed with a buffered solution at a higher pH with a mixing ratio in the range of 1 : 1 to 1:3 vokvol, preferably about 1 :2 vokvol. If needed this buffered solution can be at a temperature in the range of 15-40°C or 30-40°C. The mixed LNPs can then undergo an anion exchange filtration step. Prior to the anion exchange, the mixed LNPs can be incubated for a period of time, for example 30mins to 2 hours. The temperature during incubating can be in the range of 15-40°C or 30-40°C. After incubating the solution is filtered through a filter, such as a 0.8pm filter, containing an anion exchange separation step. This process can use tubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000 mL/min.

After formation, the LNPs can be concentrated and diafiltered via an ultrafiltration process where the alcohol is removed and the buffer is exchanged for the final buffer solution, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

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The ultrafiltration process can use a tangential flow filtration format (TFF) using a membrane nominal molecular weight cutoff range from 30-500 kD. The membrane format is hollow fiber or flat sheet cassette. The TFF processes with the proper molecular weight cutoff can retain the LNP in the retentate and the filtrate or permeate contains the alcohol; citrate buffer and final buffer wastes. The TFF process is a multiple step process with an initial concentration to a ceDNA concentration of 1-3 mg/mL. Following concentration, the LNPs solution is diafiltered against the final buffer for 10-20 volumes to remove the alcohol and perform buffer exchange. The material can then be concentrated an additional 1-3-fold. The concentrated LNP solution can be sterile filtered.

IV. Pharmaceutical Compositions and Formulations

The present disclosure also provides a pharmaceutical composition comprising the LNPs of the present disclosure and at least one pharmaceutically acceptable excipient.

According to some embodiments, the TNA (e.g., ceDNA) is encapsulated in the LNP. In one embodiment, the LNPs of the disclosure are provided with full encapsulation, partial encapsulation of the therapeutic nucleic acid. In one embodiment, the nucleic acid therapeutics is fully encapsulated in the LNPs to form a nucleic acid containing lipid particle. In one embodiment, the nucleic acid may be encapsulated within the lipid portion of the particle, thereby protecting it from enzymatic degradation.

In one embodiment, encapsulation of TNA (e.g., ceDNA) in the LNPs of the present disclosure can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid, for example, an OLIGREEN® assay or PICOGREEN® assay. Generally, encapsulation is determined by adding the dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent. Detergent- mediated disruption of the lipid bilayer releases the encapsulated TNA (e.g., ceDNA), allowing it to interact with the membrane-impermeable dye. Encapsulation of ceDNA can be calculated as E= (Io - I)/Io, where I and Io refer to the fluorescence intensities before and after the addition of detergent.

Depending on the intended use of the LNPs, the proportions of the components can vary and the delivery efficiency of a particular formulation can be measured using, for example, an endosomal release parameter (ERP) assay.

In one embodiment, the TNA (e.g., ceDNA) can be complexed with the lipid portion of the particle or encapsulated in the lipid position of the lipid particle (e.g., lipid nanoparticle). In one embodiment, the TNA can be fully encapsulated in the lipid position of the lipid particle (e.g., lipid nanoparticle), thereby protecting it from degradation by a nuclease, e.g., in an aqueous solution. In one embodiment, the TNA in the lipid particle (e.g., lipid nanoparticle) is not substantially degraded after exposure of the lipid particle (e.g., lipid nanoparticle) to a nuclease at 37°C. for at least about 20, 30, 45, or 60 minutes. In some embodiments, the TNA in the lipid particle (e.g., lipid nanoparticle) is not substantially degraded after incubation of the particle in serum at 37°C. for at least about 30, 45,

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MEl\57916143.vl 131698-30520 or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.

In one embodiment, the LNPs are substantially non-toxic to a subject, e.g., to a mammal such as a human.

In one embodiment, the pharmaceutical composition comprising LNPs of the disclosure is an aqueous solution. In one embodiment, the pharmaceutical composition comprising LNPs of the disclosure is a lyophilized powder.

According to some aspects, the at least one pharmaceutically acceptable excipient in the pharmaceutical compositions of the present disclosure is a sucrose, tris, trehalose and/or glycine.

In one embodiment, the pharmaceutical composition comprising LNPs of the disclosure are suitable for administration to a subject for in vivo delivery to cells, tissues, or organs of the subject. In some embodiments, the pharmaceutical composition is suitable for a desired route of therapeutic administration (e.g., parenteral administration). The pharmaceutical compositions for therapeutic purposes can be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structure suitable for high TNA (e.g., ceDNA) vector concentration. Sterile injectable solutions can be prepared by incorporating the TNA (e.g., ceDNA) vector compound in the required amount in an appropriate buffer with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

Pharmaceutical compositions comprising LNPs of the disclosure are suitable for topical, systemic, intra-amniotic, intrathecal, intracranial, intraarterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intra-tissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (e.g., extra-orbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, sub-choroidal, intrastromal, intracameral and intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via high pressure intravenous or intraarterial infusion, as well as intracellular injection, such as intranuclear microinjection or intracytoplasmic injection, are also contemplated.

Pharmaceutical compositions for therapeutic purposes typically must be sterile and stable under the conditions of manufacture and storage.

In one embodiment, LNPs are solid core particles that possess at least one lipid bilayer. In one embodiment, the LNPs have a non-bilayer structure, i.e., a non-lamellar (i. e. , non-bilayer) morphology. Without limitations, the non-bilayer morphology can include, for example, three dimensional tubes, rods, cubic symmetries, etc. The non-lamellar morphology (i.e., non-bilayer structure) of the LNPs can be determined using analytical techniques known to and used by those of skill in the art. Such techniques include, but are not limited to, Cryo-Transmission Electron Microscopy (“Cryo-TEM”), Differential Scanning calorimetry (“DSC”), X-Ray Diffraction, and the like. For example, the morphology of the lipid particles (lamellar vs. non-lamellar) can readily be

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MEl\57916143.vl 131698-30520 assessed and characterized using, e.g., Cryo-TEM analysis as described in US2010/0130588, the content of which is incorporated herein by reference in its entirety.

In one embodiment, the LNPs having a non-lamellar morphology are electron dense.

In one embodiment, the LNPs provided by the present disclosure is either unilamellar or multilamellar in structure. In some aspects, the pharmaceutical composition of the disclosure comprises multi-vesicular particles and/or foam-based particles. By controlling the composition and concentration of the lipid components, one can control the rate at which the lipid conjugate exchanges out of the lipid particle and, in turn, the rate at which the LNP becomes fusogenic. In addition, other variables including, for example, pH, temperature, or ionic strength, can be used to vary and/or control the rate at which the LNP becomes fusogenic. Other methods which can be used to control the rate at which the LNP becomes fusogenic will be apparent to those of ordinary skill in the art based on this disclosure. It will also be apparent that by controlling the composition and concentration of the lipid conjugate, one can control the lipid particle size.

According to some embodiments, for ophthalmic delivery, interfering RNA-ligand conjugates and nanoparticle -ligand conjugates may be combined with ophthalmologically acceptable preservatives, co-solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride, or water to form an aqueous, sterile ophthalmic suspension or solution.

Unit Dosage

In one embodiment, the pharmaceutical compositions can be presented in unit dosage form. A unit dosage form will typically be adapted to one or more specific routes of administration of the pharmaceutical composition. In some embodiments, the unit dosage form is adapted for administration by inhalation. In some embodiments, the unit dosage form is adapted for administration by a vaporizer. In some embodiments, the unit dosage form is adapted for administration by a nebulizer. In some embodiments, the unit dosage form is adapted for administration by an aerosolizer. In some embodiments, the unit dosage form is adapted for oral administration, for buccal administration, or for sublingual administration. In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the unit dosage form is adapted for intrathecal or intracerebroventricular administration. In some embodiments, the pharmaceutical composition is formulated for topical administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

V. Methods of Treatment

In some aspects, the present disclosure provides methods of treating a disorder in a subject that comprise administering to the subject an effective amount of an LNP of the disclosure of the

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MEl\57916143.vl 131698-30520 pharmaceutical composition comprising the LNP of the disclosure. In some embodiments, the disorder is a genetic disorder.

As used herein, the term “genetic disease” or “genetic disorder” refers to a disease, partially or completely, directly or indirectly, caused by one or more abnormalities in the genome, including and especially a condition that is present from birth. The abnormality may be a mutation, an insertion or a deletion in a gene. The abnormality may affect the coding sequence of the gene or its regulatory sequence.

Provided herein are methods for treating genetic disorders by administering the LNP of the disclosure or the pharmaceutical composition comprising LNPs of the disclosure. There are a number of inherited diseases in which defective genes are known, and typically fall into two classes: deficiency states, usually of enzymes, which are generally inherited in a recessive manner, and unbalanced states, which may involve regulatory or structural proteins, and which are typically but not always inherited in a dominant manner. For deficiency state diseases, the LNPs and LNP compositions of the disclosure can be used to deliver transgenes to bring a normal gene into affected tissues for replacement therapy, as well, in some embodiments of any of the aspects and embodiments herein, to create animal models for the disease using antisense mutations. For unbalanced disease states, the LNPs and LNP compositions of the disclosure can be used to create a disease state in a model system, which could then be used in efforts to counteract the disease state. Thus, the LNPs or LNP compositions of the disclosure and methods disclosed herein permit the treatment of genetic diseases. As used herein, a disease state is treated by partially or wholly remedying the deficiency or imbalance that causes the disease or makes it more severe.

In general, the LNPs and LNP compositions of the disclosure can be used to deliver any transgene in accordance with the description above to treat, prevent, or ameliorate the symptoms associated with any disorder related to gene expression. Illustrative disease states include, but are not- limited to: cystic fibrosis (and other diseases of the lung), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, epilepsy, and other neurological disorders, cancer, diabetes mellitus, muscular dystrophies (e.g., Duchenne, Becker), Hurler’s disease, adenosine deaminase deficiency, metabolic defects, retinal degenerative diseases (and other diseases of the eye), mitochondriopathies (e.g., Leber’s hereditary optic neuropathy (LHON), Leigh syndrome, and subacute sclerosing encephalopathy), myopathies (e.g., facioscapulohumeral myopathy (FSHD) and cardiomyopathies), diseases of solid organs (e.g., brain, liver, kidney, heart), and the like. In some embodiments of any of the aspects and embodiments herein, the ceDNA vectors as disclosed herein can be advantageously used in the treatment of individuals with metabolic disorders (e.g., ornithine transcarbamylase deficiency).

In one embodiment of any of the aspects or embodiments herein, the LNPs of the disclosure or the pharmaceutical composition comprising the LNPs of the disclosure can be used to treat,

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MEl\57916143.vl 131698-30520 ameliorate, and/or prevent a disease or disorder caused by mutation in a gene or gene product. Exemplary diseases or disorders that can be treated with the LNPs or the LNP compositions of the disclosure include, but are not limited to, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage disease); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis Type II (MPSII; Hunter syndrome)); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC); blood diseases or disorders (e.g., hemophilia A and B, thalassemia, and anemia); cancers and tumors, and genetic diseases or disorders (e.g, cystic fibrosis).

In one embodiment, the LNPs or LNP compositions of the disclosure may be employed to deliver a heterologous nucleotide sequence in situations in which it is desirable to regulate the level of transgene expression (e.g., transgenes encoding hormones or growth factors).

In one embodiment of any of the aspects or embodiments herein, the LNPs or LNP compositions of the disclosure can be used to correct an abnormal level and/or function of a gene product (e.g., an absence of, or a defect in, a protein) that results in the disease or disorder. The LNPs or LNP compositions of the disclosure can produce a functional protein and/or modify levels of the protein to alleviate or reduce symptoms resulting from, or confer benefit to, a particular disease or disorder caused by the absence or a defect in the protein. For example, treatment of OTC deficiency can be achieved by producing functional OTC enzyme; treatment of hemophilia A and B can be achieved by modifying levels of Factor VIII, Factor IX, and Factor X; treatment of PKU can be achieved by modifying levels of phenylalanine hydroxylase enzyme; treatment of Fabry or Gaucher disease can be achieved by producing functional alpha galactosidase or beta glucocerebrosidase, respectively; treatment of MFD or MPSII can be achieved by producing functional arylsulfatase A or iduronate-2-sulfatase, respectively; treatment of cystic fibrosis can be achieved by producing functional cystic fibrosis transmembrane conductance regulator; treatment of glycogen storage disease can be achieved by restoring functional G6Pase enzyme function; and treatment of PFIC can be achieved by producing functional ATP8B1, ABCB11, ABCB4, or TJP2 genes.

In another embodiment, autoimmune diseases or disorders that may be treated using the stealth LNPs or LNP compositions described herein. Autoimmune diseases or disorders may include, but are not limited, to, rheumatoid arthritis, multiple sclerosis, type I diabetes, systemic lupus erythematosus, psoriasis, psoriatic arthritis, Sjogren’s syndrome, Crohn’s disease, Celiac disease, ulcerative colitis, Graves’ disease, Hashimoto’s thyroiditis, Addison’s disease, dermatomyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome, myasthenia gravis, autoimmune vasculitis, pernicious anemia, vitiligo, systemic sclerosis, scleroderma, diffuse scleroderma, limited scleroderma, linear scleroderma, localized scleroderma, hemolytic anemia, inflammatory bowel disease (IBD), achantholysis, acute disseminated encephalomyelitis, adult-onset Still disease, allergic glomerulonephritis, ANCA associated vasculitis, ankylosing spondylitis, anti-

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MEl\57916143.vl 131698-30520 glomerular basement membrane disease, anti-N-methyl-D-aspartate receptor encephalitis, antiphospholipid syndrome, antisynthetase syndrome, autoimmune gastritis, autoimmune hypophysitis, autoimmune liver disease, autoimmune lung disease, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, autoimmune skin disease, autoimmune thyroiditis, autoimmune uveitis, Behcet diseae, bullous pemphigoid, Churg Strauss syndrome, dermatitis herpetiformis, endocrine ophthalmopathy, erythematous pemphigus, Felty syndrome, Giant cell arteritis, IgA glomerulonephritis, membranous glomerulonephritis, Goodpasture syndrome, Granulomatosis with polyangiitis, Graves’ ophthalmopathy, Hailey Hailey syndrome, heparin induced thrombocytopenia, autoimmune hepatitis, idiopathic hypoparathryroidism, idiopathic thrombocytopenic purpura, IgA pemphigus, immune complex nephritis, immunoglobulin A nephropathy juvenile rheumatoid arthritis, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, linear IgA bullous dermatosis, lupus nephritis, lupus vasculitis, microscopic polyangiitis, morphea, mucous membrane phemphigoid, myasthenia gravis, nonarticular rheumatism, sympathetic ophthalmia, opsoclonous myoclonus syndrome, paraneoplastic pemphigus, pemphigoid, pemphigoid gestationis, pemphigus, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, autoimmune polyendocrinopathies, polyradiculoneuropathy, postpartum thyroiditis, primary biliary cirrhosis, rheumatic disease, rheumatic heart disease, rheumatoid polymyalgia, rheumatoid nodule, rheumatoid vasculitis, stiff-person syndrome, CREST syndrome, systemic juvenile idiopathic arthritis, sarcoidosis, and uveomeningoencephalitic syndrome.

In some embodiments, the LNPs or LNP compositions of the disclosure can be used to provide a DNA-based therapeutic to a cell in vitro or in vivo. Examples of DNA-based therapeutics include, but are not limited to, minicircle DNA, minigene, viral DNA (e.g., Lentiviral or AAV genome) or non-viral synthetic DNA vectors, closed-ended linear duplex DNA (ceDNA / CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimalistic immunological-defmed gene expression (MIDGE) -vector, nonviral ministring DNA vector (linear-covalently closed DNA vector), or dumbbell-shaped DNA minimal vector (“dumbbell DNA”).

In one embodiment of any of the aspects or embodiments herein, exemplary transgenes encoded by ceDNA in the LNPs or LNP compositions of the disclosure include, but are not limited to: X, lysosomal enzymes (e.g., hexosaminidase A, associated with Tay-Sachs disease, or iduronate sulfatase, associated, with Hunter Syndrome/MPS II), erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, as well as cytokines (e.g, a interferon, -interferon, interferon-y, interleukin-2, interleukin-4, interleukin 12, granulocytemacrophage colony stimulating factor, lymphotoxin, and the like), peptide growth factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), neurotrophic factor-3 and 4, brain-derived neurotrophic factor (BDNF), glial derived growth factor (GDNF), transforming growth factor-a and -b, and the like), receptors (e.g., tumor necrosis

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MEl\57916143.vl 131698-30520 factor receptor). In some exemplary embodiments, the transgene encodes a monoclonal antibody specific for one or more desired targets. In some exemplary embodiments, more than one transgene is encoded by the ceDNA vector. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments of any of the aspects and embodiments herein, the transgene encodes an antibody, including a full-length antibody or antibody fragment, as defined herein. In some embodiments of any of the aspects and embodiments herein, the antibody is an antigen-binding domain or an immunoglobulin variable domain sequence, as that is defined herein. Other illustrative transgene sequences encode suicide gene products (thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor), proteins conferring resistance to a drug used in cancer therapy, and tumor suppressor gene products.

In one embodiment of any of the aspects or embodiments herein, this disclosure provides a method of providing anti-tumor immunity in a subject, the method comprising administering to the subject an effective amount of any embodiment of an LNP contemplated herein or any embodiment of a pharmaceutical composition comprising an LNP contemplated herein. Furthermore, this disclosure provides a method of treating a subject having a disease, disorder or condition associated with an elevated expression of a tumor antigen, the method comprising administering to the subject an effective amount of any embodiment of an LNP contemplated herein or any embodiment of a pharmaceutical composition comprising an LNP contemplated herein. In some embodiments, the TNA is retained in the spleen for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after dosing of an LNP of this disclosure, for example, via intravenous or intratumoral administration. In some embodiments, the amount (z.e., number of copies) of the TNA at the start of a 12, 18, or 24-hour time window post-dosing and the amount of the TNA at the end of the time window are within the same order of magnitude (e.g., 10^-7 copies, IO^-6 copies, IO^-5 copies, 10^-4 copies, KF³ copies, IO^-2 copies, IO ¹ copies, 10° copies, 10¹ copies, 10² copies, 10³ copies, etc. or any other suitable therapeutic levels). In other words, there is minimal reduction in concentrations of the TNA in the spleen within a 12, 18, or 24-hour time window post-dosing. In some embodiments, the TNA is a messenger RNA (mRNA).

Examples of solid tumors treatable with an LNP disclosed herein or a pharmaceutical composition comprising the same include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. According to some embodiments, the tumor or cancer is a melanoma, e.g., an

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MEl\57916143.vl 131698-30520 advanced stage melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the disclosure. Examples of other solid tumors or cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers.

In further embodiments, the present disclosure provides a method of treating a blood disease, disorder or condition in a subject, the method comprising administering to the subject an effective amount of any embodiment of an LNP contemplated herein or any embodiment of a pharmaceutical composition comprising an LNP contemplated herein. Non-limiting examples of the blood disease, disorder or condition include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Hodgkin lymphoma (HL), multiple myeloma, a myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), adrenoleukodystrophy (ALD), Hurler syndrome, Krabbe disease (Globoid-cell leukodystrophy or GLD), metachromatic leukodystrophy (MLD), severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), sickle cell disease (SCD), thalassemia, Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, essential thrombocytosis, Panconi anemia, hemophagocytic lymphohistiscytosis (HLH), juvenile myelomonocytic leukemia (JMML), myelofibrosis, polycythemia vera, and a combination thereof. In some embodiments, the TNA is a messenger RNA (mRNA). In some embodiments, the TNA is retained in the bone marrow for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after dosing of an LNP of this disclosure, for example, via intravenous or intratumoral administration. In some embodiments, the amount (i.e. number of copies) of the TNA at the start of a 12, 18, or 24-hour time window post-dosing and the number of the TNA at the end of the time window are within the same order of magnitude (e.g., 10^-7 copies, IO^-6 copies, IO^-5 copies, 10^-4 copies, IO^-3 copies, IO^-2 copies, 10^-1 copies, 10° copies, 10¹ copies, 10² copies, 10³ copies, etc. or any other suitable therapeutic levels) or are reduced for less than one order of magnitude. In other words, there is minimal or insignificant reduction in concentrations

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MEl\57916143.vl 131698-30520 of the TNA in the bone marrow within a 12, 18, or 24-hour time window post-dosing. In some embodiments, the TNA is a messenger RNA (mRNA).

Administration

In some embodiments, an LNP or an LNP composition of the disclosure can be administered to an organism for transduction of cells in vivo. In some embodiments, an LNP or an LNP composition of the disclosure can be administered to an organism for transduction of cells ex vivo.

Generally, administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. Exemplary modes of administration of an LNP or an LNP composition of the disclosure include oral, rectal, transmucosal, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular (including administration to skeletal, diaphragm and/or cardiac muscle), intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, and the like, as well as direct tissue or organ injection (e.g., to liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle or brain).

Administration of the LNP or LNP compositions of the disclosure can be to any site in a subject, including, without limitation, a site selected from the group consisting of the brain, a skeletal muscle, a smooth muscle, the heart, the diaphragm, the airway epithelium, the liver, the kidney, the spleen, the pancreas, the skin, and the eye. In some embodiments, an LNP or LNP composition as described herein may be targeted to a blood cell or cell fragment, including, but not limited to, T cells (e.g., CD8+ cells, CD4+ cells), B cells, natural killer cells (NK cells), dendritic cells, macrophages, red blood cells, platelets, and megakaryocytes. In some embodiments, an LNP or LNP composition as described herein may be targeted to a stem cell or progenitor cell, including, but not limited to, hematopoietic stem cells (HSCs), a hematopoietic stem or progenitor cell (HSPCs), and CD34+ cells. The most suitable route in any given case will depend on the nature and severity of the condition being treated, ameliorated, and/or prevented and on the nature of the particular ceDNA LNP that is being used. Additionally, ceDNA permits one to administer more than one transgene in a single vector, or multiple ceDNA vectors (e.g., a ceDNA cocktail).

In one embodiment of any of the aspects or embodiments herein, the LNPs or LNP compositions of the disclosure can be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intra-ocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular

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(e.g., intra-vitreous, sub-retinal, anterior chamber) and peri -ocular (e.g., sub-Tenon’s region) delivery as well as intramuscular delivery with retrograde delivery to motor neurons.

In some embodiments, the LNPs of the disclosure or the pharmaceutical compositions comprising the LNPs of the disclosure, when administered to a subject, is characterized by a lower immunogenicity than a reference LNP or a pharmaceutical composition comprising a reference LNP. In some embodiments, the immunogenicity of the LNP of the disclosure or the pharmaceutical composition comprising the LNP of the disclosure may be measured by measuring levels of one or more proinflammatory cytokines. Accordingly, in some embodiments, the LNPs of the disclosure or the pharmaceutical compositions comprising the LNPs of the disclosure, when administered to a subject, elicits a lower pro-inflammatory cytokine response than a reference LNP or a pharmaceutical composition comprising a reference LNP. The term “elicits a lower pro-inflammatory cytokine response than a reference LNP or a pharmaceutical composition comprising a reference LNP”, as used herein, means that the LNP of the disclosure or the pharmaceutical composition comprising the LNP of the disclosure, when administered to a subject, causes a smaller increase in the levels of one or more pro-inflammatory cytokines as compared to a reference LNP or a pharmaceutical composition comprising a reference LNP. Exemplary pro-inflammatory cytokines include, but are not limited to, granulocyte colony stimulating factor (G-CSF), interleukin 1 alpha (IL- la), interleukin 1 beta (IL-1 P), interleukin 6 (IL-6), interleukin 8 (IL-8 or CXCL8), interleukin 11 (IL-11), interleukin 17 (IL-17), interleukin 18 (IL- 18), interferon a (IFN-a), interferon (IFN-P), interferon y (IFN-y), C-X-C motif chemokine ligand 10 (CXCL10 or IP-10), monocyte chemoattractant protein 1 (MCP-1), CD40L, CCL2, CCL3, CCL4, CCL5, CCL11, tumor necrosis factor a (TNF-a), and combinations thereof.

Dosing regimens

In some embodiments, an LNP or LNP composition of the disclosure may be administered according to different regimens. As described below in Example 15 and in FIG. 11A and 11B, LNPs comprising lipid-conjugated polyglycerol avoid antibody-mediated clearance from the blood after multiple doses, in contrast to LNPs containing, for example, polyethylene glycol (PEG), which are rapidly cleared from the blood after multiple doses.

Accordingly, an LNP or LNP composition of the disclosure may be administered as a single dose, or may be administered multiple times. In some embodiments, an LNP or LNP composition of the disclosure may be administered in 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, or 10 or more doses, without decreasing the effectiveness of the LNP or the cargo it carries.

In some embodiments, each dose may be separated by a time period of about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, about 60 days, about 65 days,

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In some embodiments, each dose may be separated by a time period of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 20 weeks, about 25 weeks, about 30 weeks, about 35 weeks, about 40 weeks, about 45 weeks, about 50 weeks, about 55 weeks, about 60 weeks, about 65 weeks, about 70 weeks, about 75 weeks, about 80 weeks, about 85 weeks, about 90 weeks, about 95 weeks, about 100 weeks, about 1-100 weeks, about 10-90 weeks, about 20-80 weeks, about 30-70 weeks, about 40-60 weeks, about 1-2 weeks, about 2-3 weeks, about 3-4 weeks, about 4-5 weeks, about 5-6 weeks, about 6-7 weeks, about 7-8 weeks, about 8-9 weeks, about 9-10 weeks, about 10-11 weeks, about 11-12 weeks, about 12-15 weeks, about 15-20 weeks, about 20-25 weeks, about 25-30 weeks, about 30-35 weeks, about 35-40 weeks, about 40-45 weeks, about 45-50 weeks, about 50-55 weeks, about 55-60 weeks, about 60-65 weeks, about 65-70 weeks, about 75-80 weeks, about 80-85 weeks, about 85-90 weeks, about 90-95 weeks, about 95-100 weeks, about 1-10 weeks, about 10-20 weeks, about 20-30 weeks, about 30-40 weeks, about 40-50 weeks, about 50-60 weeks, about 60-70 weeks, about 70-80 weeks, about 80-90 weeks, about 90-100 weeks, about 1-25 weeks, about 25-50 weeks, about 50-75 weeks, about 75-100 weeks, about 1-50 weeks, or about 50-100 weeks.

In some embodiments, each dose may be separated by a time period of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36

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MEl\57916143.vl 131698-30520 months, about 1-2 months, about 2-3 months, about 3-4 months, about 4-5 months, about 5-6 months, about 6-7 months, about 7-8 months, about 8-9 months, about 9-20 months, about 10-11 months, about 11-12 months, about 12-13 months, about 13-14 months, about 14-15 months, about 15-16 months, about 16-17 months, about 17-18 months, about 18-19 months, about 19-20 months, about 20-21 months, about 21-22 months, about 22-23 months, about 23-34 months, about 1-4 months, about 4-8 months, about 8-12 months, about 12-16 months, about 16-20 months, about 20-24 months, about 1-24 months, about 2-20 months, about 3-16 months, about 4-12 months, about 5-8 months, about 1-12 months, or about 12-24 months.

In some embodiments, each dose may be separated by a time period of about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 1-2 years, about 2-3 years, about 3-4 years, about 4-5 years, about 5-6 years, about 6-7 years, about 7-8 years, about 8-9 years, about 9-10 years, about 1-4 years, about 4-6 years, about 6-8 years, about 8-10 years, about 1-5 years, or about 5-10 years.

In some embodiments, each dose may be separated by a time period of at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 125 days, at least 150 days, at least 175 days, at least 200 days, at least 225 days, at least 250 days, at least 275 days, at least 300 days, at least 325 days, at least 350 days, at least 375 days, at least 400 days, at least 425 days, at least 450 days, at least 475 days, or at least at least 500 or more days.

In some embodiments, each dose may be separated by a time period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 20 weeks, at least 25 weeks, at least 30 weeks, at least 35 weeks, at least 40 weeks, at least 45 weeks, at least 50 weeks, at least 55 weeks, at least 60 weeks, at least 65 weeks, at least 70 weeks, at least 75 weeks, at least 80 weeks, at least 85 weeks, at least 90 weeks, at least 95 weeks, at least 100 weeks.

In some embodiments, each dose may be separated by a time period of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, or at least 24 or more months.

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In some embodiments, each dose may be separated by a time period of at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 or more years.

As used herein, the term “separated by a time period”, when used in the context of multiple doses of an LNP or LNP composition as described herein, refers to the amount of time that has elapsed since the administration of a prior dose.

In some embodiments, at least three or more doses are administered, and the time period between each dose is the same (which may be referred to herein as a “period schedule”). In some embodiments, at least three or more doses are administered, and the time period between each dose is different (which may be referred to herein as a “variable schedule”). In some embodiments, a variable schedule may comprise at least four doses, wherein some of the time periods between doses are the same, and wherein some of the time periods between doses are different. For example, in one embodiment, a subject may be administered first doses separated by a short time period (e.g., a few days or weeks), followed by a third dose after a longer time period (e.g. , a few months or years).

In some embodiments, a subject may be administered a dose of an LNP or LNP composition as described herein on a periodic or variable schedule indefinitely. In some embodiments, a subject may be administered a dose of an LNP or LNP composition based on need, and a subject may be monitored periodically to determine if additional dose(s) are needed, based on, e.g., clinical presentation and/or diagnostic tests.

In some embodiments, each dose of the LNP or LNP compositions of the disclosure may be about 0.01 mg/kg, 0.05 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.25 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.75 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1.0 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.25 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.75 mg/kg, about 1.8 mg/kg, about 1.9 mg/kg, about 2.0 mg/kg, about 2.1 mg/kg, about 2.2 mg/kg, about 2.25 mg/kg, about 2.3 mg/kg, about 2.4 mg/kg, about 2.5 mg/kg, about 2.6 mg/kg, about 2.7 mg/kg, about 2.75 mg/kg, about 2.8 mg/kg, about 2.9 mg/kg, about 3.0 mg/kg, about 3.1 mg/kg, about 3.2 mg/kg, about 3.25 mg/kg, about 3.3 mg/kg, about 3.4 mg/kg, about 3.5 mg/kg, about 3.6 mg/kg, about 3.7 mg/kg, about 3.75 mg/kg, about 3.8 mg/kg, about 3.9 mg/kg, about 4.0 mg/kg, about 4.1 mg/kg, about 4.2 mg/kg, about 4.25 mg/kg, about 4.3 mg/kg, about 4.4 mg/kg, about 4.5 mg/kg, about 4.6 mg/kg, about 4.7 mg/kg, about 4.75 mg/kg, about 4.8 mg/kg, about 4.9 mg/kg, about 5.0 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 0.01-10 mg/kg, about 0.1-9 mg/kg, about 0.25-8 mg/kg, about 0.5-7 mg/kg, about 0.75-6 mg/kg, about 1.0-5.0 mg/kg, about 1.25-4.0 mg/kg, about 1.5-3.5 mg/kg, about 1.75-3.0 mg/kg, about 2.0-2.5 mg/kg, about 0.1-1.0 mg/kg, about 1.0-2.0 mg/kg, about 2.0-3.0 mg/kg, about 3.0-4.0 mg/kg, about 4.0-5.0 mg/kg, about 5.0-6.0 mg/kg, about 6.0-7.0 mg/kg, about 7.0-8.0 mg/kg, about 8.0-9.0 mg/kg, about 9.0-10.0 mg/kg, about 0. 1-2.0 mg/kg, about 2.0-4.0 mg/kg, about 4.0-6.0 mg/kg, about 6.0-8.0 mg/kg, about 8.0-10.0

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MEl\57916143.vl 131698-30520 mg/kg, about 0. 1-2.5 mg/kg, about 2.5-5.0 mg/kg, about 5.0-7.5 mg/kg, about 7.5-10.0 mg/kg, about 0.1-5.0 mg/kg, about 5.0-10.0 mg/kg, or about 0.1-10.0 mg/kg.

In some embodiments, each dose of the LNP or LNP compositions of the disclosure may be at least 0.01 mg/kg, at least 0.05 mg/kg, at least 0.1 mg/kg, at least 0.2 mg/kg, at least 0.25 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at least 0.5 mg/kg, at least 0.6 mg/kg, at least 0.7 mg/kg, at least 0.75 mg/kg, at least 0.8 mg/kg, at least 0.9 mg/kg, at least 1.0 mg/kg, at least 1.1 mg/kg, at least 1.2 mg/kg, at least 1.25 mg/kg, at least 1.3 mg/kg, at least 1.4 mg/kg, at least 1.5 mg/kg, at least 1.6 mg/kg, at least 1.7 mg/kg, at least 1.75 mg/kg, at least 1.8 mg/kg, at least 1.9 mg/kg, at least 2.0 mg/kg, at least 2. 1 mg/kg, at least 2.2 mg/kg, at least 2.25 mg/kg, at least 2.3 mg/kg, at least 2.4 mg/kg, at least 2.5 mg/kg, at least 2.6 mg/kg, at least 2.7 mg/kg, at least 2.75 mg/kg, at least 2.8 mg/kg, at least 2.9 mg/kg, at least 3.0 mg/kg, at least 3.1 mg/kg, at least 3.2 mg/kg, at least 3.25 mg/kg, at least 3.3 mg/kg, at least 3.4 mg/kg, at least 3.5 mg/kg, at least 3.6 mg/kg, at least 3.7 mg/kg, at least 3.75 mg/kg, at least 3.8 mg/kg, at least 3.9 mg/kg, at least 4.0 mg/kg, at least 4.1 mg/kg, at least 4.2 mg/kg, at least 4.25 mg/kg, at least 4.3 mg/kg, at least 4.4 mg/kg, at least 4.5 mg/kg, at least 4.6 mg/kg, at least 4.7 mg/kg, at least 4.75 mg/kg, at least 4.8 mg/kg, at least 4.9 mg/kg, at least 5.0 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, or at least 10 mg/kg or more.

In some embodiments, each dose of the LNP or LNP compositions of the disclosure may be less than 0.05 mg/kg, less than 0. 1 mg/kg, less than 0.2 mg/kg, less than 0.25 mg/kg, less than 0.3 mg/kg, less than 0.4 mg/kg, less than 0.5 mg/kg, less than 0.6 mg/kg, less than 0.7 mg/kg, less than 0.75 mg/kg, less than 0.8 mg/kg, less than 0.9 mg/kg, less than 1.0 mg/kg, less than 1.1 mg/kg, less than 1.2 mg/kg, less than 1.25 mg/kg, less than 1.3 mg/kg, less than 1.4 mg/kg, less than 1.5 mg/kg, less than 1.6 mg/kg, less than 1.7 mg/kg, less than 1.75 mg/kg, less than 1.8 mg/kg, less than 1.9 mg/kg, less than 2.0 mg/kg, less than 2. 1 mg/kg, less than 2.2 mg/kg, less than 2.25 mg/kg, less than 2.3 mg/kg, less than 2.4 mg/kg, less than 2.5 mg/kg, less than 2.6 mg/kg, less than 2.7 mg/kg, less than 2.75 mg/kg, less than 2.8 mg/kg, less than 2.9 mg/kg, less than 3.0 mg/kg, less than 3.1 mg/kg, about 3.2 mg/kg, less than 3.25 mg/kg, less than 3.3 mg/kg, less than 3.4 mg/kg, less than 3.5 mg/kg, about 3.6 mg/kg, less than 3.7 mg/kg, less than 3.75 mg/kg, less than 3.8 mg/kg, about 3.9 mg/kg, about 4.0 mg/kg, less than 4. 1 mg/kg, less than 4.2 mg/kg, less than 4.25 mg/kg, less than 4.3 mg/kg, about 4.4 mg/kg, less than 4.5 mg/kg, less than 4.6 mg/kg, less than 4.7 mg/kg, less than 4.75 mg/kg, about 4.8 mg/kg, less than 4.9 mg/kg, less than 5.0 mg/kg, less than 6 mg/kg, less than 7 mg/kg, less than 8 mg/kg, less than 9 mg/kg, or less than 10 mg/kg.

In some embodiments, the amount of the LNP or LNP composition may be the same in each of multiple doses. In some embodiments, the amount of the LNP or LNP composition may be different in each of multiple doses. In some embodiments, the dosage of the LNP or LNP composition may be adjusted based on the level of the therapeutic nucleic acid (TNA) needed by the subject.

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In some embodiments, the TNA persists in the subject’s blood at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours after the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or subsequent dose.

According to some embodiments, the LNP comprises a half-life (ti/2) in blood in vivo of between about 3 hours and 48 hours. According to some embodiments, the LNP comprises a half-life (ti/2) in blood in vivo of greater than 4 hours. According to some embodiments, the LNP comprises a half-life (ti/2) in blood in vivo of greater than 3 hours. According to some other embodiments, the LNP comprises an in vivo (ti/2) in vivo that is prolonged in the subject’s blood as compared to the in vivo half-life of an LNP that is not conjugated to PG or a PG derivative. According to other embodiments, the in vivo half-life of the LNP is increased by at least a factor of about two or more as compared to the in vivo half-life of an LNP that is not conjugated to PG or a PG derivative. According to other further embodiments, the in vivo half-life of the LNP is increased by at least a factor of about three or more as compared to the in vivo half-life of an LNP that is not conjugated to PG or a PG derivative.

The prolonging of LNP half-life can be assessed by measuring the pharmacokinetics, or as described in any of the examples below.

According to some embodiments, the LNP or LNP compositions of the disclosure may be used to prolong the half-life of a therapeutic nucleic acid in a subject.

In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 3 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 4 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 5 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 6 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 7 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 8 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 9 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 10 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 11 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 12 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 14 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 16 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 18 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 20 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 22 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 24 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 28 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 32 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 36 hours. In one

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MEl\57916143.vl 131698-30520 embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 40 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 44 hours. In one embodiment, the half-life (ti/2) of the LNP in blood in vivo is greater than 48 hours.

The administration of multiple doses of the LNP or LNP compositions of the disclosure may be used for a variety of purposes, including, but not limited to, maintaining a level of a therapeutic nucleic acid (e.g., mRNA, siRNA, ceDNA, ssDNA, or other TNAs described herein), increasing the level of a TNA, or decreasing the level of a target gene (e.g. , using an inhibitory TNA such as an siRNA).

REFERENCES

All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology

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MEl\57916143.vl 131698-30520 described herein. These publications are provided solely for their disclosure prior to the fding date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

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EXAMPLES

The following examples are provided by way of illustration, not limitation.

Example 1. Synthesis of polymer-conjugated lipids

The goal of this experiment was to synthesize exemplary polymer-conjugated lipids for use in LNPs. In this experiment, the polymer-conjugated lipids synthesized were dioctadecylamine (DODA) conjugated to polyglycerol containing 34, 41, and 46 monomeric subunits (DODA-PG34, DODA- PG41 and DODA-PG46, respectively) in accordance with Scheme 1 as shown in FIG. ID.

In an oven dried round bottom flask, freshly prepared suspension of aluminum chloride (1.7 g, 12.75 mmol) and chloroform (10 mL) was cooled down to 0°C, then triethylamine (2.3 mL in 5.4 mL chloroform) was added to it and allowed to stir under N2 atmosphere. After addition of trimethylamine, the reaction temperature was raised to the room temperature. In a separate pressure vial, DODA (4.0 g, 7.7 mmol) was dissolved in chloroform (75 mL) and transferred to the oil bath (preset temperature of 55 °C). To the solution of DODA, valerolactone (0.51 g, 5.1 mmol) and AICL suspension (dropwise) was added consequently. Reaction was allowed to stir for 3 hours at 55-60°C. After completion, the reaction mixture was cooled down to room temperature and quenched with 30 mL of H₂O. The organic layer was washed with H2O (2 x 150 mL) and brine (150 mL), dried over anhydrous Na2SC>4, and evaporated under reduced pressure at rotovap to obtain crude (3.4 g, 84%). The product was used for the next step without purification.

'HNMR (300 MHz, Chloroform- d₃) 5 ppm: 3.60 (m, 2H), 3.14 -3.32 (m, 4H), 2.41 (s, 1H), 2.33 (t, J = 6.8 Hz, 2H), 1.40- 1.77 (m, 8H), 1.09 -1.33 (m, 68H), 0.87 (t, J = 6.6 Hz, 6H).

2,3-Epoxy-l-(l-ethoxyethoxy)propane (EEGE) was dried before use by co-evaporation with toluene three times. Compound DODA-1 was also co-evaporated with toluene to azeotrope off any water present and kept over P2O5 overnight on high vacuum line. The reaction was carried out under inert atmosphere and super dry conditions. To a solution of DODA_1 (0.1 g, 0.16 mmol) in toluene in a Schlenk line tube, was added phosphazene base P4-t-Bu solution (0.2 mL, 0.8 M in hexane) under argon atmosphere, and allowed to stir for 15 minutes. Subsequently, 2,3 -epoxy- 1-(1- ethoxy ethoxy )propane) (EEGE) (1.1 g, 7.5 mmol) was added dropwise to the reaction mixture and

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MEl\57916143.vl 131698-30520 was allowed to stir overnight under argon atmosphere. The reaction was quenched with 0.1 g benzoic acid and the solvent was evaporated using a rotavapor. The crude was purified using C4 column using H2O and MeOH as eluent to afford DODA_2 (0.32 g, 36%).

MALDI-TOF: 3980 (MW(n=34)+Na+) 'HNMR (300 MHz, d-chloroform) 5 ppm: 5 4.69 (q, J = 5.4 Hz, 38H), 3.83-3.95(m, 2H), 3.42-3.65 (m, 272H), 3.24-3.31 (m, 2H), 3.15-3.19 (m, 2H), 1.59- 1.63(m, 27H), 1.17- 1.28 (m, 305H), 0.87 (t, J = 6.0 Hz, 6H).

Synthesis ofDODA 2 (n= 41)

To a solution of DODA_1 (0.17 g, 0.28 mmol) in toluene in a Schlenk line tube under argon atmosphere, was added phosphazene base P4-t-Bu solution (0.2 mb, 0.8 M in hexane) and stirred for 15 minutes. Subsequently, 2,3-epoxy-l-(l-ethoxyethoxy)propane) (EEGE) (1.1 g, 7.5 mmol) was added dropwise to the reaction mixture and stirred overnight under argon atmosphere The reaction was quenched with 0.1 g benzoic acid and concentrated using rotavapor. The crude was purified using C4 column using H₂O and MeOH as eluent to afford DODA_2 (0.32 g, 36%).

'HNMR (300 MHz, CDCWi)) 5 ppm: 5 4.69 (q, J= 5.4 Hz, 38H), 3.83-3.95 (m, 2H), 3.42- 3.65 (m, 272H), 3.24-3.31 (m, 2H), 3.15-3.19 (m, 2H), 1.59-1.63(m, 27H), 1.17- 1.28 (m, 305H), 0.87 (t, J = 6.0 Hz, 6H).

EEGE was azeotrope with toluene and desiccated with P2O5 before the rection. DODA l was also desiccated with P2O5.

Synthesis ofDODA 2A (n= 46)

To a solution of DODA l (0.4 g, 0.06 mmol) in toluene in a Schlenk line tube under argon atmosphere was added phosphazene base P4-t-Bu solution (0.2 mb, 0.8 M in hexane) and stirred for 15 minutes. Then, 2,3 -epoxy- 1-(1 -ethoxyethoxy )propane) (EEGE) (1.1 g, 7.5 mmol) was added dropwise to the reaction mixture and stirred overnight under argon atmosphere. The reaction was quenched with 0.1 g benzoic acid and concentrated using rotovap. The crude was purified using C4 column using H₂O and MeOH as eluent to afford DODA_2A (0.38 g, 36%).

MALDI-TOF: 7365 (MW(n=46)+Na⁺) ’H NMR (300 MHz, CDC1₃-A) 5 ppm: 4.64-4.85 (m, 48 H), 3.22-3.35 (m,392 H), 1,10-1.40 (m, 393 H), 0.8 (m, 6H).

To a solution of DODA 2 in MeOH was added HC1 (0.1 mb, IM in ethyl acetate) dropwise and stirred for 4 hours at room temperature. Subsequently, the reaction mixture was evaporated at rotavapor to obtain white solid. Further, trituration was done by dissolving the white solid in a

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MEl\57916143.vl 131698-30520 minimum amount of methanol and adding Et2O (chilled). After addition of Et2O, solid crashed out, and then the mixture was centrifuged (4.4 RPM for 10 min) to separate the product DODA 3 (0.16 g, 88%).

HPLC/ELSD: >98.6%. MALDI-TOF: 3533 (MW+23(Na)). ’H NMR (300 MHz, DMSO- ₆)) 5 ppm: 5 3.72 - 4.59 (m, 42H), 3.27- 3.64 (m, 233 H), 3.13-3.22 (m, 7H), 1.38-1.58 (m, 10H), 1.23 (s, 61H), 0.85 (t, J= 5.9 Hz, 6H). FIG. 1A is a MALDI-TOF spectrum of DODA-PG34.

Synthesis of DODA-PG41

To a solution of DODA 2 in MeOH was added HC1 (0.1 mL, IM in ethyl acetate) dropwise and stirred for 4 hours at room temperature. Subsequently, the reaction mixture was concentrated via rotavapor. The white solid formed and was dissolved in a minimum amount of MeOH, and ice cold Et2O was added to precipitate out the product. The mixture was centrifuged (4.4 RPM for 10 min) to separate the product DODA-PG41.

HPLC/ELSD: >99%. MALDI-TOF: 3682.36 (MW+23(Na)). ’H NMR (300 MHz, DMSO- d₆)) 5 ppm: 5 3.72- 4.59 (m, 42H), 3.37-3.53 (m, 240H), 3.22 - 3.13 (m, 7H), 2.49-2.51 (m, 50H), 2.12-2.23 (m, 2H), 1.58 - 1.38 (m, 9H), 1.23 (s, 61H), 0.85 (t, J = 5.9 Hz, 6H).

Synthesis ofDODA-PG46

To a solution of DODA_2A in MeOH was added HC1 (0. 1 mL, IM in ethyl acetate) dropwise and stirred for 4 hours at room temperature. Subsequently, the reaction mixture was concentrated via rotovapor. The white solid was formed and was dissolved in a minimum amount of MeOH, and cooled Et2O was added to precipitate out the product. The mixture was centrifuged (4.4 RPM for 10 min) to separate the product (DODA-PG46). (0.22 g, 88%). HPLC/ELSD: >98%. MALDI-TOF: 4051.93 (MW+23(Na)).

'HNMR (300 MHz, DMSO L)) 5 ppm: 5 3.37 - 3.54 (m, 598 H), 2.20-2.33 (m, 6H), 1. SSLS 8 (m, 6H), 1.23 (s, 56H), 1.07-1.11 (m, 56 H), 0.85 (t, J= 5.9 Hz, 6H).

Example 2. Alternative Synthesis of Polymer-Conjugated Lipids

The goal of this experiment was to synthesize exemplary polymer-conjugated lipids for use in LNPs using a synthesis method that is different from the synthesis method described in Example 1. In this experiment, the polymer-conjugated lipids synthesized were dioctadecylamine (DODA) conjugated to polyglycerol containing 45 and 58 monomeric subunits (DODA-PG45 and DODA- PG58, respectively) in accordance with Scheme 2 as shown in FIG. IE.

Scheme 2

Synthesis of 5-(henzyloxy)pentanoic acid (2)

Compound 1 (5.02 g, 25.8 mmol) was dissolved in 70 mL of acetone, cooled to 0°C and treated with 42 mL (85.1 mmol) of Jones reagent (2.0 M CrO, in H2SO4). The reaction mixture was stirred for 2 hours at ambient temperature and quenched by addition of 15 mL of i-PrOH at 0°C. The

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MEl\57916143.vl 131698-30520 mixture was diluted with 200 mL of EtOAc and washed twice with water, brine and dried over Na2SC>4. The solvents were distilled off, providing 5.2 grams of compound 2, which was used in the next step without further purification.

'HNMR (300 MHz, d-chloroform) 5 ppm: 7.25-7.40 (m, 5H), 4.50 (2H), 3.49 (t, J = 6.3 Hz, 2H), 2.31 - 2.44 (m, 2H), 1.60-1.80 (m, 4H).

Synthesis of 5-hydroxy-N,N-dioctadecylpentanamide (3)

Compound 2 (1.37 g, 6.58 mmol) was dissolved in 50 mL of chloroform, and DIPEA (3.35 mL, 24.8 mmol) was added, followed by DMAP (0.19 g 1.5 mmol), DODA (3.26 g, 6.21 mmol) and HATU (2.9 g, 7.45 mmol). The coupling reaction was run at 48-50°C overnight. The reaction mixture was cooled to ambient temperature, diluted with dichloromethane and washed with NaHC'CL (sat), water and brine. The organic layer was dried over Na2SC>4, filtered and concentrated. The crude material was purified by normal phase column chromatography (Hexanes-EtOAc), providing 3.6 g (81% yield) of amide 3.

'HNMR (300 MHz, d-chloroform) 5 ppm:7.25-7.40 (m, 5H), 4.50 (2H), 3.49 (t, J = 6.3 Hz, 2H), 3.22-3.35 (m, 2H), 3.12-3.25 (m, 2H), 2.25-2.35 (m, 2H), 1.60-1.80 (m, 4H), 1.40-1.55 (m, 4H), 1.10-1.33 (m, 65H), 0.85-0.95 (m, 6H).

Synthesis of 5-hydroxy-N,N-dioctadecylpentanamide (DODA-1) (4)

Compound 3 (3.6 g, 5.1 mmol) was dissolved in MeOH/EtOAC (70 mL/100 mL) mixture and underwent deprotection reaction using 0.5 g of Pd/C in a Parr reactor under 40 psi. The conversion was quantitative, providing 3.1 grams of DODA-1 which was used for the next step without further purification.

'HNMR (300 MHz, d-chloroform) 5 ppm: 3.61 (t, J = 6.0 Hz, 2H), 3.22-3.35 (m, 2H), 3.12- 3.25 (m, 2H), 2.30-2.40 (m, 2H), 2.15 (br s, 1H), 1.70-1.83 (m, 2H), 1.40-1.75 (m, 6H), 1. 1-1.4 (m, 63H), 0.85-0.95 (m, 6H).

Synthesis of DODA 2 (n= 45)

2,3-Epoxy-l-(l -ethoxyethoxypropane) was dried before use by co-evaporation with toluene three times. Compound DODA-1 was also co-evaporated with toluene to azeotrope off any water present and kept over P2O5 overnight on high vacuum line. The reaction was carried under inert atmosphere and very dry conditions.

Compound DODA-1 (0.2 g, 0.32 mmol, 1 eq.) was dissolved in 2 mL of dry toluene and a catalytic amount of P4-tBu (0.4 mL, 0.8 M in hexane) was added. The reaction mixture was stirred for 20-30 minutes at ambient temperature, followed by addition of dry 2,3 -Epoxy- 1-(1- ethoxyethoxypropane) (4.49 g, 30.7 mmol, 96 eq.) in 1 mL of toluene. The stirring continued for 16 hours and then polymerization reaction was stopped by quenching with solid benzoic acid (~ 300 mg), stirred for 20 min, concentrated and kept on a vacuum line for 1 hour. The crude was purified by RP chromatography (C4 -40g, H2O-i-PrOH) following ELSD signal.

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MALDI-TOF revealed formation of 0.5 g oligomer with n=9. This material (0.5 g, 0.26 mmol) was mixed with 1.5 mL of toluene and subjected to further polymerization under conditions described above: P4-tBu (0.36 mL, 0.8 M in hexane), dry 2,3-Epoxy-l-(l-ethoxyethoxypropane) (2.7 g, 18.5 mmol, 71 eq) in 1 mL of toluene. After reaction was run overnight it was quenched with 0.26 grams of benzoic acid, stirred for 15 minutes, concentrated and purified by RP chromatography (C4 - 40g, LLO-i-PrOH), providing 1.7 g (73%) of polymer with n=45.

MALDI-TOF: 7302 (MW(n=45)+Na+) 'HNMR (300 MHz, d-chloroform) 5 ppm: 4.64-4.85 (m, 47 H), 3.22-3.35 (m, 378 H), 1,10-1.40 (m, 393 H), 0.8 (m, 6H).

Synthesis of DODA 2A (n = 58)

2,3-Epoxy-l-(l-ethoxyethoxypropane) and compound DODA-1 was dried the way as described above.

The synthesis of DODA 2A (n= 58) was done stepwise. Initially DODA with n=23 was synthesized using 60 eq excess of 2,3 -epoxy- 1-(1 -ethoxyethoxypropane) according to procedure written above. DODA with 23 units (0.72 grams, 1.6 mmol) was dissolved in 2 mL of dry toluene and treated with 0.3 mL of P4-tBu (0.8M/hexanes) stirring for 20 minutes before 2,3 -epoxy- 1-(1- ethoxyethoxypropane) was added (2.3 grams, 144 mmol) in 0.5 mL of toluene. The reaction was stirred overnight and then quenched with 160 mg of benzoic acid and the crude was purified by RP chromatography (C4 -40g, H2O-i-PrOH) following ELSD signal. 1.7 g of DODA 2A (n= 58) was isolated.

MALDI-TOF: 9126 (MW+23(Na)).’H NMR (300 MHz, d-chloroform) 5 ppm: 4.64-4.85 (m, 65 H), 3.22-3.35 (m, 490H), 1,10-1.40 (m, 483 H), 0.8 (m, 6H).

Synthesis ofDODA-PG45

DODA 2 (1.7 g, 0.24 mmol) was dissolved in MeOH (44mL) and treated with of IN HCl/EtOAc (0.45 mL, 0.45 mmol) and stirred for 4 hours at ambient temperature. The reaction mixture was concentrated dissolved in 4 mL of MeOH and treated with 35 mL of ice-cold Et2O.

The cloudy-oily mixture was centrifuged at 4.4xl0³ x g for 10 min, the solvents were decanted and sonication procedure was repeated two times using 35 ml of Et2O. After decanting the last supernatant. a light brown oil residue was obtained which was filtered under N2-blanket, washed with ice cold ether and kept under vacuum over P2O5, providing 910 mg (95%) of off-white solid - DODA-PG45

HPLC/ELSD: >98.6%. MALDI-TOF: 3980 (MW+23(Na)). ’H NMR (300 MHz, d- chloroform) 5 ppm: 3.30-4.50 (m, 390 H), 2.20-2.33 (m, 2H), 1.40-1.50 (m, 8 H), 1.15-1.30 (m, 63 H), 0.8 (m, 6H). FIG. IB is a MALDI-TOF spectrum of DODA-PG45.

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Synthesis ofDODA-PG58

DODA 2A (1.5 g, 0.19 mmol) was dissolved in MeOH (40 mL) and treated with IN HCl/EtOAc (0.4 mL, 0.4 mmol) and stirred for 4h at ambient temperature. The reaction mixture was concentrated, dissolved in 3 mL of MeOH and treated with 30 mL of ice-cold Et2O.

The cloudy-oily mixture was centrifuged at 4.4xl0³ x g for 10 minutes, the solvents were decanted, and the sonication procedure was repeated the same way as described for the analog above providing 770 mg (93%) of DODA-PG58.

HPLC/ELSD: >98%. MALDI-TOF: 4938 (MW+23(Na)). ’H NMR (300 MHz, d- chloroform) 5 ppm: 3.80-4.75 (br s, 88H), 3.20-4.60 (m, 383 H), 2.20-2.30 (m, 2H), 1.40-1.50 (m, 8 H), 1.15-1.30 (m, 63 H), 0.8 (m, 6H). FIG. 1C is a MALDI-TOF spectrum of DODA-PG58.

Example 3. Preparation of LNPs comprising polymers PEG and PG conjugated lipids

The goal of this experiment was to prepare LNP formulations using different anchored polymers. LNP formulations were prepared using polymer-conjugated lipids such as DSPE-PEG2K- OH, DODA-PG45, and DSPE-PMPC50, in the same way. The specific formulations that were prepared in this experiment are shown and described in Table 8.

Generally, LNPs were prepared as follows: a lipid composition described in Table 8 dissolved in ethanol was mixed with an aqueous solution of DNA at pH 4. The resulting mixture was exhaustively dialyzed against a phosphate buffered saline (PBS) solution and then concentrated using spin filtration. The LNP was characterized using dynamic light scattering to measure size and polydispersity index (PDI), and Picogreen fluorescent method was used to quantify encapsulation efficiency (EE). For dosing of animals, the solutions were diluted to the desired concentration using phosphate buffered saline solution.

Table 8

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Example 4. In vivo expression of nucleic acids in LNP formulations containing different anchored polymers

The goal of this study was to evaluate the in vivo expression of nucleic acids that are formulated and comprise of LNPs with various anchored polymers, such as DSPE-PEG2K-OH or DODA-PG45. To this end, CD-I mice (males) were intravenously (IV) injected with ceDNA nucleic acid carrying a firefly luciferase reporter construct that was formulated in LNPs comprising DSPE- PEG2K-OH or DODA-PG45 (composition in Table 8) at a dose of 0.5 mg/kg (0 day).

Whole-body luciferase bioluminescence was measured by In vivo Imaging System (IVIS) at Day 4 and Day 7. FIG. 2A shows the total flux measured by the total photon counts per the region of interest, i. e. , the liver, measured by IVIS at Day 4 post-dosing for tested LNPs and for a negative control (PBS) injected with saline instead of formulated ceDNA. FIG. 2B shows the total flux measured for tested LNPs and negative control at Day 7 post-dosing. FIG. 2C shows the total flux measured for tested LNPs and negative control across two collection days (Day 4 and Day 7). The results shown in FIG. 2C indicate that administration of formulated LNPs with different anchored polymers in combination with a targeting ligand, z.e., GalNAc3 (Formulations 180, 182, and 184) resulted in higher expression of luciferase as compared to untargeted LNPs (Formulations 179, 181, and 183) at both Day 4 and Day 7. FIG. 2D shows the percentage change in body weight (BW) of mice at Day 1 post-dosing. The results indicate that the tested LNPs with targeting ligand GalNAc3 (Formulations 180, 182, and 184), caused a smaller change in body weight in mice as compared to untargeted LNPs.

The results presented in Example 4 demonstrate that a GalNAc3 targeted LNP of the disclosure comprising anchored polymers (DSPE-PEG2K-OH or DODA-PG45) when delivered in vivo supported the expression of nucleic acids without triggering any major tolerability issues and other adverse events in mice.

Unexpectedly, the data presented in Example 4 demonstrate that only half the amount (1.5 mol%) of DODA-PG45 in an untargeted LNP formulated with PG-containing anchored polymer (Formulation 181), resulted in about the same level of transgene expression as compared to double the amount (3.0 mol%) of DSPE-PEG2K-OH in an untargeted LNP formulated with PEG-containing anchored polymer (Formulation 179). The significance of this unexpected discovery on achieving advantageous stealth/endosomal tradeoff for PG-containing LNP formulations relative to PEG- containing LNP formulations is further explored in the subsequent Examples.

Example 5. Analysis of luciferase mRNA expression in mRNA formulated LNP

The goal of this study was to quantify the expression of a luciferase mRNA formulated with LNPs containing different anchored polymers. LNP formulations used in this study are shown in Table 9. Freshly isolated mouse hepatocytes were seeded on collagen-coated plates at a cell density of 25,000 per well. The assay plate was then incubated for 4 hours at 37°C, 5% CO2 in a humidified

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MEl\57916143.vl 131698-30520 incubator to allow for cell attachment. Following the attachment period, each well was treated for 1 hour with 100 ng of mRNA formulated LNPs containing no mouse serum. Following treatment, the plate was washed twice in lx dPBS, and maintenance media was added to each well. The following morning, viability readouts were collected using CelltiterFluor, and expression readout was collected using “One-Step” Luciferase Assay on a SpectraMax spectrometer. The final analytics were plotted as RLU values of Luciferase Activity, which represent Luciferase expression normalized by viability. The negative control in this experiment was a high expressing LNP (Formulation 070), which was either uninhibited or competitively inhibited with 55 pM of Free TetraGalNAc.

FIG. 3 is a bar graph showing luciferase activity for the tested LNP formulations containing different lipid-anchored polymers. The results are shown in FIG. 3 and indicate that an LNP formulated with helper lipid DSPC and anchored polymers DODA-PG34 and DSPE-PEG2K- GalNAc3 (Formulation 227) showed higher luciferase activity than uninhibited control.

Table 9

Ionizable Lipid Z (structure not shown) belongs to a different class of ionizable lipids as compared to Ionizable Lipid 87, where both the headgroup and lipid tail moieties are structurally different from those of Ionizable Lipid 87.

Example 6. Evaluation of opsonization-driven uptake of LNPs in primary mouse hepatocytes

The goal of this assay was to screen for “stealthy” LNPs via differential uptake of the fluorophore DiD in primary mouse hepatocytes via mouse serum opsonization. Without being bound by a specific theory, it is hypothesized that stealthy untargeted LNPs bind to minimal proteins from the serum, thus leading to minimal primary hepatocyte uptake, whereas non-stealthy LNPs bind to serum proteins, leading to a high cell uptake. This hypothesis is depicted as a schematic in FIG. 4A.

A schematic of the assay is shown in FIG. 4B. Briefly, freshly isolated hepatocytes were seeded on collagen-coated plates at a cell density of 25,000 per well, after which the assay plate was incubated for 4 hours at 37°C, 5% CO2, in a humidified incubator. After the attachment period, each

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MEl\57916143.vl 131698-30520 well was treated with 500 ng of DiD-labeled LNP containing 10% mouse serum for 1 hour. LNP formulations evaluated in this study are shown in Table 10. Following treatment, the plate was washed twice in lx dPBS and maintenance media was added to each well. The following morning, an image of the plate was obtained on a Phenix confocal microscope using a 20x water objective. Image analysis was performed and the data were plotted as DiD fluorescence area normalized to area of live nuclei.

FIG. 4C is a bar graph showing DiD fluorescence normalized to area of live nuclei measured for the various LNP formulations containing different lipid-anchored polymers. The results shown in FIG. 4C indicate that LNP formulations comprising the DODA-PG45 anchored polymer showed minimal primary hepatocyte uptake. These results suggest that among all anchored polymers tested, PG anchored polymers performed the best in inhibiting opsonization-driven uptake.

Table 10

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Example 7. Evaluation of the effect of anchored polymer composition on opsonization-driven LNP uptake in primary mouse hepatocytes

The aim of this study was to screen for “stealthy” LNP formulations comprising polymer- conjugated lipids of the present disclosure that varied in their identity and percentage composition, wherein a stealthy LNP is defined as one that has minimal uptake into cells in the absence of a targeting ligand. The benchmark LNP was prepared using 47.5% Lipid Z, 10% DSPC, 39% cholesterol, 3% DSG-PEG2K, and 0.5% DiD. As the % of polymer is decreased, the amount of cholesterol is increased to compensate. Freshly isolated hepatocytes were seeded on collagen-coated plates at a cell density of 25,000 per well, after which the assay plate was incubated for 4 hours at 37°C, 5% CO2, in a humidified incubator. After the attachment period, each well was treated with 500 ng of DiD-labeled LNP containing 10% Mouse Serum for 1 hour. Following treatment, the plate was washed twice in lx dPBS and maintenance media was added to each well. The following morning, an image of the plate was obtained on a Phenix confocal microscope using a 20x water objective.

FIG. 5 is a bar graph showing DiD fluorescence area normalized to area of live nuclei for the tested LNP formulations containing different amounts of polyglycerol-conjugated lipids, wherein it is apparent that the use of 1.8% DODA-PG within the LNP provides a comparable level of stealth protection compared to 3% PEG. Additionally, as the amount of DODA-PG is increased, the level of non-targeted uptake is decreased compared to the 3% PEG benchmark, indicating that one could use a lower amount of PG and maintain a similar level of stealthiness. The results shown in FIG. 5 indicate that significantly lower opsonization-driven uptake is observed for LNPs containing 2.8-6.8 percent (%) of PG as compared to LNPs containing 3% PEG, thereby suggesting that PG may provide better shielding to the LNP base composition than 3% PEG.

Example 8. Evaluation of endosomal escape of LNP formulations

The aim of this study was to evaluate the efficiency of endosomal escape of LNP formulations comprised of anchored polymers that varied in their identity and percentage composition. The benchmark LNP was prepared using 47.5% Lipid Z, 10% DSPC, 39% cholesterol, 2.95% DSG-PEG2K, 0.05% DSPE-PEG77-GalNAc3, and 0.5% DiD. As the % of polymer is decreased, the amount of cholesterol is increased to compensate. All compositions used 0.05% GalNAc3 as a targeting ligand for active targeting to primary hepatocytes through the ASGPR uptake pathway, regardless of polymer %. Freshly isolated mouse hepatocytes were seeded on collagen- coated plates at a cell density of 25,000 per well, after which the assay plate was incubated for 4 hours at 37°C, 5% CO2, in a humidified incubator. After the attachment period, each well was treated with 500 ng of DiD-labeled LNP containing 10% mouse serum for 1 hour. Following treatment, the plate was washed twice in lx dPBS and maintenance media was added to each well. The following morning, viability readouts were collected using CelltiterFluor and expression readout was collected

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MEl\57916143.vl 131698-30520 using “One-Step” Luciferase Assay on a SpectraMax spectrometer. DiD uptake was quantified from images obtained on a Phenix confocal microscope using a 20x water objective. The results were plotted as Luciferase (mLuc) expression normalized to DiD uptake, which represents expression normalized by viability and DiD uptake. When the uptake and mLuc expression are compared as a ratio, one can indirectly infer the ability of each composition to escape the endosome.

FIG. 6 is a bar graph showing the amount of endosomal escape measured as the amount of luciferase expression normalized to DiD uptake in mouse hepatocytes treated with LNP formulations containing different amounts of polyglycerol -conjugated lipids and a control. It was demonstrated in FIG. 6 that by increasing the amount of DODA-PG, the ability for the LNP to escape the endosome is reduced. This is important when the data from FIG. 5 is considered where less PG can result in a higher level of stealthiness. Thus, an LNP containing less DODA-PG can be used to achieve a similar level of stealth while also enhancing the endosomal escape potential of the LNP.

Taken together, the results described in Examples 6, 7 and 8, along with a comparison of FIGs. 4, 5 and 6, suggest that PG-containing LNPs display a more advantageous stealth/endosomal escape trade off compared to PEG-containing LNPs. In particular, FIG. 5 shows that LNPs formulated with 2.8% of PG was significantly more stealthy compared to LNPs formulated with a similar amount (3%) of PEG. FIG. 5 also shows that LNPs formulated with -1.5% PG, as indicated by the arrow, would have about the same level of stealthiness of LNPs formulated with 3% PEG, as indicated by the horizontal line. In contrast, FIG. 6 shows the inverse relationship between the amount of a polymer-conjugated lipid in an LNP formulation and the level of endosomal escape. Specifically, FIG. 6 shows that LNPs formulated with a relatively low amount (1.45%) of PG maintained a relatively high level of endosomal escape, as compared to LNPs formulated with significantly higher amount (2.95%) of PG or PEG. The ability of LNPs formulated with PG- containing anchored polymer to achieve this advantageous stealth/endosomal tradeoff as compared to LNPs formulated with PEG-containing anchored polymer is further supported by FIG. 4 wherein the stealthiness of LNPs formulated with PEG suffers as the amount of PEG decreases, in contrast to LNPs formulated with PG for which the stealthiness does not suffer as the amount of PG decreases.

Example 9. Analysis of whole blood clearance of LNPs formulated with ionizable lipid: Lipid Z, and different polymer-conjugated lipids

The goal of this study was to measure and compare the pharmacokinetic (PK) properties of novel LNPs formulations containing the ionizable lipid Lipid Z, along with DSPC, cholesterol, and different polymer-conjugated lipids, as described in Table 11, with a control LNP formulated with ionizable Lipid 87, cholesterol, and DSG-PEG2K-OMe (Formulation 829). Formulations of control LNP and Lipid Z carrying LNPs were injected via IV bolus in the tail vein of CD-I mice. Whole blood samples were collected for qPCR at 2 min, 1 hour, 3 hour and 6-hour time-points, and K2EDTA

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MEl\57916143.vl 131698-30520 was added as an anticoagulant at 50 pL/aliquot. Body weight, mortality, and clinical observations were recorded.

The whole blood clearance of the Control LNP, and the different Lipid Z carrying LNPs are shown in FIG. 7, while individual pharmacokinetic parameters are reported in Table 11. Whole blood concentrations of ceDNA in mice treated with the control LNP and LNPs carrying Lipid Z, DSPC, cholesterol, and either 3% DSPE-PEG2K-OMe, 1.5% DSPE-PMPC50, or 5% DODA-PG45, as measured by the AUCl_ast, and ti/2 values, were observed to show no significant differences. These results indicate that the higher retention of ceDNA in the bloodstream, and hence the less rapid clearance, of the ceDNA-luciferase cargo from the bloodstream as delivered by 3.0% DSPE-PEG, 1.5% DSPE-PMPC50, and 5% DODA-PG45 -containing LNPs (Formulations 023, 777, and 678) could be beneficial in the reduction of off-target delivery to non-target cells, including, but not limited to, blood cells such as leukocytes, neutrophils, eosinophils, basophils, macrophages, and monocytes, or to immune cells such as T-cells, B-cells, and macrophages.

Table 11

Example 10. In vivo expression of nucleic acids in LNP formulations containing ceramides and anchored polymers

The goal of this study was to evaluate the in vivo expression of nucleic acids formulated as LNPs with ceramides as the helper lipid, in combination with various anchored polymers such as DSPE-PEG2K-OH or DODA-PG45. To this end, CD-I mice (males) were intravenously (IV) injected with ceDNA nucleic acid carrying firefly luciferase reporter construct formulated as in the disclosure with LNPs comprising various helper lipids (ceramide and DSPC) in combination with different anchored polymers (DSPE-PEG2K-OH or DODA-PG45) at 2 different doses of either 1 mg/kg or 2.0 mg/kg (0 day). The LNPs used in the experiment are shown in Table 12.

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Whole-body luciferase bioluminescence was measured by In vivo Imaging System (IVIS) at Day 7. FIG. 8A shows the total flux quantified by total photon counts per the region of interest, z.e., the liver, measured by IVIS at Day 7 post-dosing for tested LNPs and for a negative control (DPBS) injected with saline instead of formulated ceDNA. The results shown in FIG. 8A indicate that administration of formulated LNPs with helper lipid DSPC and polymer conjugated lipid DSPE- PEG2K-OH (formulation no: 002) resulted in a dose -dependent expression of nucleic acid at Day 7, while other LNPs formulated with either C2 ceramide and DSPE-PEG2K-OH or Cl 8: 1 ceramide and DODA-PG45 did not show a dose-dependent increase. The percentage change in body weight of mice at Day 1 is shown in FIG. 8B. These results indicate that the tested GalNAc3 targeted LNPs with either DSPC or ceramide helper lipids and different anchored polymers caused a milder body weight change in mice as compared to an untargeted LNP with ceramide helper lipid and DODA-PG45.

Overall, the results presented in this example demonstrate that a GalNAc3 targeted LNP of the disclosure comprising different helper lipids and anchored polymers, when delivered in vivo, could support expression of nucleic acids without triggering any major tolerability issues or other adverse events in mice that could be clinically observed (e.g., rough hair coat, facial swelling).

Table 12

Example 11. Evaluating immunogenicity of exemplary LNPs of the disclosure

The goal of this study was to evaluate the immunogenicity of exemplary formulated LNPS comprised of DSPC or ceramide helper lipids, and DSG-PEG2K-OH or DODA-PG45 polymer conjugated lipids. The immunogenicity profiles of LNPs containing helper lipids DSPC, C2 ceramide or Cl 8: 1 ceramide were compared. The formulations of the LNPs evaluated in this study are shown in Table 13. Blood serum was collected at 6 hours post-dosing, and the levels of cytokines implicated in the regulation of innate immune response, i.e., IFN-alpha, IL-6, IFN-gamma, TNF-alpha, and IL- 18, were measured for each animal. The results are shown in FIGs. 9A-9D, respectively, and indicate that at a dosage of 2.0 mg/kg, serum levels of IFN-alpha, IL-6, IFN-gamma, TNF-alpha, and IL-18

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MEl\57916143.vl 131698-30520 were lower for the C2 and C18: 1 ceramide-containing LNPs, as compared to DSPC-containing LNPs. These results also show that some cytokine levels trend lower for PG-containing LNPs, and higher for PEG-containing LNPs, especially in the case of IFN-alpha.

These results suggest that the identity of the helper lipid, as well as the identity of the polymer in the anchor lipid-conjugated polymer, directly affected the immunogenicity of LNPs formulated as in the disclosure.

Table 13

Example 12. Preparation of DSPE-PEG5k-Mal-Protein

This example describes a method for the preparation of an LNP conjugated to a protein ligand of interest, which requires the inclusion of an additional cysteine residue not present in the native protein sequence. The protein ligand of interest is initially reduced with 10 molar equivalents of TCEP for 30 minutes at 23°C. After reduction, TCEP is removed using a Zeba spin column. The reduced ligand is then incubated for 3 hours at 23°C with LNPs formulated with DSPE-PEG5k- Maleimide using a mole percentage of 0.5%. The ratio of ligand to DSPE-PEG5k-maleimide is varied from 0.3 down to 0.02. SDS-PAGE is used to confirm whether the conjugation occurred and to what extent.

Example 13. Preparation of DSPE-PEG5k-DBCO-Protein

This example describes a method for the preparation of an LNP -conjugated to a protein ligand of interest, which requires the inclusion of an additional cysteine residue not present in the native protein sequence. The protein ligand of interest is initially reduced with 10 molar equivalents of TCEP for 30 minutes at 23°C. After reduction, TCEP is removed using a Zeba spin column. The reduced ligand is then incubated with 10 molar equivalents of Sulfo DBCO-PEG4-maliemide for 3 hours at 23°C. The excess DBCO reagent is then removed using a Zeba spin column. The extent of labelling and overall protein purity is confirmed using a UPLC-QTOF.

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The DBCO labelled protein is then incubated for 16 hours at 23 °C with LNPs formulated with DSPE-PEG5K-Ns using a mole percentage of 0.5%. The ratio of ligand to DSPE-PEG5K-Ns is varied from 0.3 down to 0.02. SDS-PAGE is used to confirm whether the conjugation occurred and to what extent.

Example 14. Improved shielding of LNPs by DODA-PG

FIG. 10 is a bar graph showing DiD fluorescence area normalized to the area of live nuclei for the tested LNP formulations containing different amounts of polyglycerol-conjugated lipids, formulated with DSPE-PEG5K-Ns at a mole percentage of 0.5%. The formulations of the LNPs evaluated in this study are shown in Table 14. As shown in FIG. 10, as the amount of DODA-PG was increased, the level of non-targeted uptake was decreased, as compared to the 3% PEG benchmark. Specifically, significantly lower opsonization-driven uptake was observed for LNPs containing 2.8-6.8 percent (%) of PG, as compared to LNPs containing 3% PEG, suggesting that PG may provide better shielding to the LNP base composition than 3% PEG. These results further indicate that a lower percentage of PG may be used to maintain a similar level of stealthiness, even in LNPs formulated with DSPE-PEG5K-Ns (whereby the LNPs can be readily conjugated with a protein-based targeting ligand as described above).

Table 14

Example 15. LNPs with polyglycerol-conjugated lipids avoid antibody-mediated clearance upon redosing

This example describes the ability of LNPs containing polyglycerol-conjugated lipids to avoid antibody-mediated clearance after redosing. LNPs containing luciferase mRNA cargo and 3% DSG- PEG2000, 3% DODA-PG45, or 3% DODG-PG45 were prepared as set forth below in Table 15. Each LNP was dosed in two groups of mice. One group underwent a multi -dose regimen of three

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MEl\57916143.vl 131698-30520 weekly doses (every 7 days) on days 0, 7, and 14. The second group was administered a single dose on day 14. In both groups, LNPs were dosed at 0.1 mg/kg per dose. Blood samples were taken at 2 min, 1 hr, 6 hr, and 24 hr for all groups after the day 14 dose for pharmacokinetic (PK) analysis. Subsequently, whole blood was processed for mRNA isolation and qPCR analysis to quantify the concentration of mRNA present at each time point.

As shown in both FIG. 11A and FIG. 11B, while LNPs containing 3% DSG-PEG2000 (Formulation 282) showed persistence in the blood after a single dose, these LNPs were rapidly cleared from the bloodstream after the third dose (as indicated by the sharp decrease in the level of mRNA cargo in the blood), which indicated the development of antibodies to PEG (a known response to PEG). However, LNPs containing DODA-PG45 (FIG. 11 A; Formulation 283) or DODG-PG (FIG. 11B; Formulation 284) showed persistence in the blood long after administration of the third dose. The data from this experiment indicated that polyglycerol -conjugated lipids did not induce antibodies that cleared the LNPs from the blood after multiple doses, which would have precluded their effectiveness.

Table 15

The experiment was repeated with repeat dosing of 2% and 4% polyglycerol -conjugated LNPs, as described in Table 16, below.

Table 16

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LNPs containing luciferase mRNA cargo and 3% DSG-PEG2000, 2% DODA-PG34, or 4% DODA-PG34 were prepared as set forth below in Table 17. Each LNP was dosed in two groups of mice (n=3 per group). One group underwent a multi -dose regimen of weekly doses (every 7 days) for 3 doses, on days 0, 7, and 14. The second group was administered a single dose on day 14. In both groups, LNPs were dosed at 0. 1 mg/kg per dose. Blood samples were taken at 2 min and 6 hr for all groups after the day 14 dose for pharmacokinetic (PK) analysis. Subsequently, whole blood was processed for mRNA isolation and qPCR analysis to quantify the concentration of mRNA present at each time point in the blood.

Table 17

As shown in FIG. 12, repeat dosing of 2% and 4% PG LNPs did not induce an antibody response and maintained an extended blood circulation profile as compared to LNPs containing 3% DSG-PEG2000, which were rapidly cleared from the bloodstream after the third dose (as indicated by the sharp decrease in the level of mRNA cargo in the blood). 2% PG34 being stealthy and redosable allowed lower anchored polymer content, being less of a hindrance to endosomal escape.

Example 16. LNPs with 3% polyglycerol-conjugated lipids avoid antibody-mediated clearance upon redosing with all tested ionizable lipids

This example describes the ability of LNPs containing 3% polyglycerol -conjugated lipids with various ionizable lipids to avoid antibody-mediated clearance after redosing.

The ability to achieve specific delivery of genetic medicine has been a primary challenge in the nucleic acid therapeutics space and LNPs are one of the most advanced solutions. Over the past two decades, LNP technology has primarily been used to deliver nucleic acids to the liver via intravenous administration. First-generation LNPs reach the liver by taking advantage of serumbinding proteins known as opsonins that drive about half of every dose to the liver and the other half to cells of the reticuloendothelial system, predominantly in the spleen. While this rapid clearance from

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MEl\57916143.vl 131698-30520 circulation and biodistribution is favorable for delivery to the liver and spleen, it has prevented LNPs from efficiently reaching target cells and tissues elsewhere. Second-generation LNP technologies incorporate novel lipid components that improve potency, increase tolerability and are biodegradable, but the inclusion of these elements has failed to change the overwhelming clearance by the liver and spleen. The vast majority of the LNPs dosed are still deposited in the liver and in macrophages/monocytes because the core LNP still interacts with the serum-binding proteins that drive this distribution profile. The consequence is broad, non-selective biodistribution that does not efficiently deliver payload to the cell of interest. This inefficient delivery can challenge potency, translation to higher species, and off-target payload activity.

The redosable ctLNP delivery system described herein is designed to address the limitations of existing technologies and comprises three elements. First, the core LNP incorporates proprietary technology referred to as “stealth” that prevents our LNP from interacting with serum-binding proteins and reduces clearance by the liver and spleen to less than 1% of the administered systemic dose in both mice and non-human primates. Second, the LNP utilizes format targeting ligand against a receptor uniquely expressed on the target cell to achieve highly potent and selective delivery to specific tissues and cell types of interest. Finally, bioconjugated linkers are developed to enable modular attachment of the targeting ligands to the core stealth LNP, resulting in ctLNP that can achieve highly efficient, redosable, and selective delivery to target cells and tissues. The ctLNP delivery system is compatible with nucleic acid payloads such as siRNA and mRNA and can be configured to reach new cell types with modular identification and attachment of new targeting ligands to the base stealth LNP.

LNPs containing luciferase mRNA cargo and 3% DSG-PEG2000 or 3% DODA-PG50 were prepared as set forth below in Table 18. Each LNP was dosed in two groups of mice (n=3 per each group). One group underwent a multi -dose regimen of weekly doses (every 7 days) for 3 doses, on days 0, 7, and 14. The second group was administered a single dose on day 14. In both groups, LNPs were dosed at 0.1 mg/kg per dose. Blood samples were taken at 2 min and 6 hr for all groups after the day 14 dose for pharmacokinetic (PK) analysis. Subsequently, whole blood was processed for mRNA isolation and qPCR analysis to quantify the concentration of mRNA present at each time point.

As shown in FIG. 13, while LNPs containing 3% DSG-PEG2000 (Formulation 1007) show persistence in the blood after a single dose, these LNPs were rapidly cleared from the bloodstream after the third dose (as indicated by the sharp decrease in the level of mRNA cargo in the blood), indicating the development of antibodies to PEG (a known response to PEG). However, LNPs containing DODA-PG50 (FIG. 13) showed persistence in the blood long after administration of the third dose for all tested ionizable lipids. The data from this experiment indicated that polyglycerol- conjugated lipids with a variety of different ionizable lipids did not induce antibodies that clear the LNPs from the blood after multiple doses, which would preclude their effectiveness.

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Table 18

Example 17. In vivo expression of mRNA in LNP formulations containing polyglycerol- conjugated lipids with two ionizable lipids (Lipid 87 and L-319) The goal of this study was to evaluate the expression of formulated mRNA in vivo. LNP formulations used in this study are shown in Table 19 below. LNPs with several PG derivatives and either Lipid 87 or L-319 were compared to anchored DSPE-PEG2K-OMe. All LNPs were targeted to liver using a targeting ligand, i.e., A05 VHH. CD-I mice (males) were intravenously (IV) injected with LNPs containing luciferase mRNA cargo at a dose of 0.05 mg/kg (0 day). Whole-body luciferase bioluminescence was measured by In vivo Imaging System (IVIS) 24hrs post dose. FIG. 14 shows the total flux measured by the total photon counts per the region of interest, i. e. , the liver, measured by IVIS 24 hours post-dosing for tested LNPs and for a negative control (PBS) injected with saline instead of formulated mRNA. The results shown in FIG. 14 indicate that administration of LNPs containing L-319 and PG both resulted in a high level of luciferase expression.

Table 19

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Example 18. In vivo expression of iqDNA in LNP formulations containing polyglycerol- conjugated lipids with two ionizable lipids (Lipid 87 and L-319) The goal of this study was to evaluate the expression of formulated ssDNA (nucleic acid carrying a firefly luciferase reporter construct) in vivo. LNP formulations used in this study are shown in Table 20 below. LNPs containing DODA-PG50 or DSG-PEG2000 with Lipid 87 and L- 319 were tested. All LNPs were targeted to liver using a targeting ligand, i.e., A05 VHH. CD-I mice (males) were intravenously (IV) injected LNPs containing luciferase mRNA cargo at a dose of 0.5 mg/kg (0 day). Whole-body luciferase bioluminescence was measured by IVIS at Day 7 post dose.

FIG. 15 shows the total flux measured by the total photon counts per the region of interest, z.e., the liver, measured by IVIS Day 7 post-dosing for tested LNPs and for a negative control (PBS) injected with saline instead of formulated iqDNA). The results shown in FIG. 15 indicate that administration of LNPs containing L-319/PG or L-319/DODA-PG and DSG-PEG2000 resulted in similar expression of luciferase transgene .

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Table 20

Example 19. LNP formulations with only PG-lipids have no detrimental effect on targeted expression of mRNA in vivo

This example describes the comparison of in vivo mRNA expression cell-targeted LNPs (ctLNPs) comprising either polyethylene glycol (PEG) or polyglycerol (PG).

Materials and Methods

LNP Formulation & Conjugation: mRNA encoding firefly luciferase was diluted in 20 mM sodium citrate buffer, to an mRNA concentration of 0.115 mg/mL. The lipids (according to the molar ratios described below in Table 21) were dissolved in ethanol to a total lipid concentration of 7.96 mg/mL. A 3: 1 ratio of aqueous mRNA solution and organic lipid solution were mixed using a microfluidic mixer to formulate the lipid nanoparticles (LNPs). The mixed LNPs were dialyzed against TBS saline solution using a 10 kDa dialysis membrane, and further concentrated using a 100 kDa ultracentrifugation filter. After a final sterile syringe filtration (0.2 rm), a filtered sucrose solution was added to 8.5% by volume. The LNP encapsulation efficiency and concentration of mRNA were measured using a Ribogreen intercalating dye, and the LNP size and PDI were measured using a dynamic light scattering (DLS) instrument.

The conjugation of a protein-based targeting ligand specific for the ASGPR receptor of hepatic cells was done as follows. mRNA-ctLNPs were prepared by mixing reduced ligands with LNPs at a 12.8 molar ratio (0.1 mol% of lipids conjugated) in TBS with 1 mM EDTA and 8.5% sucrose at a cargo concentration of 0.5 mg/mL for 2 hours at room temperature. Conjugation efficiency was assessed via SDS-PAGE under non-reducing conditions, stained with Coomassie blue, and quantified using densitometry to compare the band density ratio of ligand-lipid conjugates to free

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MEl\57916143.vl 131698-30520 ligand (iBright, Thermo Fisher). ctLNP concentration (based on cargo) was quantified using Ribogreen intercalating dye (Thermo Fisher), and size was quantified using plate-based DLS (Wyatt). Size growth post ligand conjugation was quantified by comparing parental LNP size (without ligand) to ctLNP size (with ligand). ctLNPs were frozen at -80°C on the same day as conjugation and used in vitro or in vivo after one freeze/thaw cycle.

Table 21

IVIS Methods:

Whole-body luciferase bioluminescence was measured by In vivo Imaging System (IVIS) 24 hours (DI) after dose administration via tail-vein IV injection into CD-I mice. The total flux is recorded; represented as total photon count per second, across the region of interest, z.e., the liver, measured by IVIS at DI for tested LNPs and PBS, a negative control injected with saline solution.

Results

As shown in FIG. 16, in vivo luciferase mRNA expression (as measured by IVIS) was comparable for a single dose of ctLNPs comprising either PEG or PG. These results demonstrated that ctLNPs comprising only PG (and no PEG), and in particular PG conjugated to A05, a liverspecific antibody, were fully capable of delivering mRNA cargo that is expressed at the same level as ctLNPs comprising PEG.

Example 20. PG-containing LNPs carrying siRNAs are potent and durable and resulted in robust, dose dependent target knockdown

T cells are central regulators and effectors of the immune system. When inappropriately activated against autoantigens, naive T cells expand and differentiate, driving various effects in other cell types and ultimately leaving the circulation to drive tissue inflammation and damage. This pathophysiology is separate and distinct from autoreactivity and inflammation caused by B cells, for which B cell depleting approaches involving T cells, such as chimeric antigen receptor (CAR)-T cell therapies, have emerged.

Reducing or eliminating T cell activity with current therapeutic approaches, namely small molecules and antibodies, has been challenging for two primary reasons. First, T cell targets that drive

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MEl\57916143.vl 131698-30520 autoimmune disease processes often perform critical functions in other immune and non-immune cell types. Thus, engaging these targets with small molecules outside of T cells can lead to safety concerns and limit the therapeutic index. Small molecule approaches can also be difficult to modulate, and as a result, they can cause immune deficiency by suppressing all T cell activity. Second, targeting surface molecules on T cells can result in unwanted activation of T cells and/or a range of effects beyond the desired target engagement, limiting the effectiveness of antibody approaches.

By combining technologies to modulate T cells that drive disease pathology selectively, ctLNP-siRNA therapeutics can block target function with sequence-level specificity while sparing the broader immune system. Further, redosable ctLNP-siRNA therapies enable reprograming of T cells in vivo, expanding treatment possibilities for T cell-driven diseases.

Redosable ctLNPs, e.g., ctLNP implementing polyglycerol (PG) as a polymer in every lipid- anchored polymer is designed to address the limitations of existing technologies and comprises three elements. First, the core LNP incorporates proprietary technology referred to as “stealth” that prevents the LNP from interacting with serum-binding proteins and reduces clearance by the liver and spleen to, e.g., less than 1% of the administered systemic dose in both mice and non-human primates largely due to the presence of lipid polymers securely anchored in the LNP (lipid-anchored polymer). Second, the LNP utilizes a conjugated targeting ligand against a receptor uniquely expressed on the target cell to achieve highly potent and selective delivery to specific cell types of interest. Third, the polymer in lipid-anchored polymer present in the ctLNP is singularly made of PG which allows the repeat dosing without triggering a rapid clearance by an immune response. The ctLNP delivery system is compatible with nucleic acid payloads such as siRNA and can be configured to reach new cell types with modular identification and attachment of new targeting ligands to the base stealth LNP.

First, to examine whether redosable ctLNP can be targeted to T cell with siRNA as a cargo designed to knock down the expression of B2M mRNA, several experiments were performed with PG vs. PEG containing ctLNPs carrying siRNA cargos with a common sequence designed to target human beta-2 -microglobulin (hB2M) mRNA with two different chemical modification patterns (“Modification Pattern Nos. 1 and 2”; siRNA216 and siRNA217; see FIG. 17A). siRNA directed to B2M mRNA having a random modification pattern was used as a negative control (“Control mods” or “scramble”).

In vitro siRNA Knockdown of hB2M

Specifically, redosable ctLNPs were employed to assess ctLNP delivery of siRNA to T cells in vitro. These experiments were also conducted to assess the functional impact of replacing a conventional second lipid-anchored polymer, “PEG-Mal,” with another second lipid-anchored polymer comprising polyglycerol (PG) as a polymer, “PG-Mal,” in a standard T cell hB2M siRNA knockdown assay. ctLNPs having DODA-PG34 as a first lipid-anchored polymer (2.8 Mol %) and DSPE-PEG5k-Mal as a second lipid anchored polymer (0.2 Mol %) (“PEG-Mal LNP”; LNP No. 354)

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MEl\57916143.vl 131698-30520 was used as a benchmark for redosable ctLNP having DODA-PG34 as a first lipid-anchored polymer (2.8 Mol %) and DODA-PG68-Mal as a second lipid anchored polymer (0.2 Mol %) (“PG-Mal LNP”; LNP NO. 355). The physiochemical characteristics and molecular compositions of these LNPs are shown below in Table 22, while the sequences of the siRNA are shown below in Table 23, as well as in FIG. 17A

Table 22

For the in vitro T cell B2M knockdown assay, frozen human PB Pan T-cells were thawed and washed with a cell medium. The washed cells were seeded in a round-bottom 96-well plate. The LNP formulations were pre-incubated in complement conserved human serum at 37°C for 1 hour. The LNP/serum mix was pipetted onto the seeded cells and incubated for 30 minutes at 37°C, after which the plate was centrifuged, and the supernatant was discarded. The cell pellet was washed with the cell medium, suspended in a cell growth medium. After a 24-hour incubation, the cells were stained with an antigen specific dye and analyzed on an Attune cytometer.

The results are shown in FIG. 17B and FIG. 17C. FIG. 17B is a graph that shows ctLNP delivery increased in a dose dependent manner for both ctLNP PEG-Mal and PG-Mal formulations, but not for the cell-only control (y-axis shows DiD labelling (gMFl)). FIG. 17C is a graph that shows there was dose-dependent target knockdown in non-activated T cells in vitro for both PEG-Mal and PG-Mal formulations, but not for the cell-only control and siRNA with a random modification (scramble) pattern. These results show that replacement of PEG-Mal with an overall 100% PG polymer in an LNP (e.g., PG-Mal ctLNP) had no effect on potency of siRNA ctLNP formulation in non-activated T cells in vitro.

In vivo siRNA Knockdown of B2M

This Example describes the robust uptake and inhibition of housekeeping gene beta-2 - microglobulin (B2M) expression in T cells in humanized mice (NSG-hCD34). Humanized NSG mice were administered intravenously at 0. 1 mg/kg with two groups of ctLNP -PEG-Mal (N/P ratio 3 and 6)

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MEl\57916143.vl 131698-30520 carrying negative control siRNA with a random modification pattern; ctLNP-PEG-Mal carrying siRNA with modification pattern no. 1 (siRNA216); ctLNP-PG-Mal carrying siRNA with modification pattern no. 1 (siRNA216); two groups of ctLNP-PEG-Mal (N/P ratio 3 and 6) carrying siRNA with modification pattern no. 2 (siRNA217). Blood samples were taken at Day 2, 6, 10, 14 and 17 for cytometry analysis.

As shown in FIG. 18A, both B2M siRNAs (siRNA 216 and 217; modification pattern nos. 1 and 2, respectively) carried by ctLNP containing PEG-Mal or PG-Mal were more potent and durable than the B2M siRNA with a random chemical modification pattern. The y-axis in FIG. 18A shows percent (%) knockdown (KD), over time in days (x-axis) where the B2M siRNA with modification pattern no. 1 and the B2M siRNA with modification pattern no. 2 demonstrated higher levels of knockdown than the negative control B2M siRNA with a random chemical modification pattern at day 2 (D2). The negative control’s knockdown levels significantly dropped by Day 6 (D6) and fell to -15% knockdown level by Day 10 (D10), while siRNA with modification pattern no. 1 or no. 2 drove persistent knockdown through day 17 (DI 7). FIG. 18B shows percent knockdown of B2M at Day 2 (D2), 6 (D6), 10 (D10), 14 (D14) and 17 (D17) in bar graphs.

In summary, ctLNP delivery of siRNA to T cells described herein resulted in robust, dosedependent target knockdown in vitro. Using the siRNAs described above, it was demonstrated the ctLNP-siRNA successfully knocked down expression of B2M, a protein involved in cellular processes in primary (resting) human T cells. In primary resting T cells, the ctLNP half-maximal inhibitory concentration (IC50) was low, approximately 2nM (FIG. 17C), suggesting very efficient delivery and knockdown of B2M protein. In humanized mouse models, potent and durable knockdown of B2M expression (approximately 50-80% knockdown) was achieved in circulating T cells at 0.1 mg/kg dose through Day 17.

Example 21. LNPs with only PG-conjugated lipids avoid antibody-mediated clearance upon redosing

This example describes the persistence of mRNA cargo delivered by LNPs comprising only PG-conjugated lipids, as compared to LNPs comprising varying amounts of PEG-conjugated lipids.

Methods:

LNPs were prepared as described below in Table 23. Formulations 357, 358, and 359 comprised different percentages of 3% DODA-PG39 as the first lipid-anchored polymer, with either DODA-PG68-Mal (Formulations 357 and 358) or DSPE-PEG5k-Mal (Formulation 359) as the second lipid anchored polymer. Formulation 356 comprised only DODA-PG39, while Formulation 360 comprised only DSG-PEG-2k.

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Table 23

Each LNP was dosed in two groups of mice. One group underwent a multi -dose regimen of weekly doses (every 7 days) for up to 3 doses, on day 0, day 7 and day 14 of the study. The second group was dosed on the final study day - day 14. Whole blood samples were taken at 2 min, 1 hr, 6 hr, and 24 hr for all groups after the day 14 dose. Subsequently, whole blood pharmacokinetics (PK) was performed using qPCR to quantify the concentration of drug substance (mRNA) at each time point.

FIG. 19 shows the concentration of mRNA cargo 2 minutes after dosing, out to 24 hours. Each data point represents a group of 3 animals. As shown in FIG. 19, the first dose of all tested LNP formulations had persistence in circulation for the 6 hours observed. After three doses, LNP formulations with DSPE-PEG5k-Mal or DSG-PEG2k were rapidly cleared from circulation. However, LNP formulations with PG-lipids, either 3% DODA-PG39 or DODA-PG39 + DODA- PG68-Mal, had minimal clearance of the third dose, allowing for redosing of LNP formulations without rapid loss of the drug product in circulation.

In a second study, LNPs comprising mRNA cargo and only PG-conjugated lipids with different PG lengths were tested. LNP composition was as described below in Table 24.

Table 24

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CD-I mice were dosed intravenously on day 0, day 7, and day 14 of the study with 0. 1 mg/kg of each LNP. Whole blood samples were taken at 2 minutes and 6 hours after the day 14 dose (third dose). Subsequently, whole blood pharmacokinetics (PK) was performed using qPCRto quantify the concentration of drug substance cargo (mRNA) remaining in circulation 6 hours after the third dose.

FIG. 20 shows the concentration of mRNA cargo 2 minutes after dosing and 6 hours after dosing. As shown in FIG. 20, after three doses, LNP formulations with DSG-PEG2k were rapidly cleared from circulation. However, LNP formulations with PG-lipids of varying lengths (PG34, PG39, PG45, and PG50) all had minimal clearance of the third dose, allowing for redosing of LNP formulations without rapid loss of the drug product in circulation.

Example 22. Alternative Synthesis of Polymer-Conjugated Lipids

The goal of this experiment was to synthesize exemplary polymer-conjugated lipids for use in LNPs using a synthesis method that is different from the synthesis method described in Example 1 and Example 2. In this experiment, the polymer-conjugated lipids synthesized were dioctadecylamine (DODA) conjugated to polyglycerol containing 39 monomeric subunits (DODA-PG39) in accordance with Scheme 3 as shown in FIG. 21.

To a solution of dioctadecylamine (13.10 g, 1 eq, 25.10 mmol) in chloroform (80 ml) was added DIPEA (4.217 g, 5.68 mL, 1.3 eq, 32.62 mmol), followed by dihydro-2H-pyran-2,6(3H)-dione (2.749 g, 0.96 eq, 24.09 mmol). The resulting mixture was stirred at RT for 2 hours, and then at 30- 40°C for 3 hours. MS showed the desired product ([M+l] 636.7). The reaction mixture was poured

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MEl\57916143.vl 131698-30520 into IM aq. HC1 (150 mL) and extracted with DCM/CHCE (2x250mL). The organic phase was filtered through celite and washed with CHC13/DCM, and concentrated to afford crude DODA-A1 (15.71 g) which was used for the next step without further purification. 'H NMR (CDCI3, 400Mhz, ppm: 53.25-3.30 (m, 2H); 3.20-3.25 (m, 2H); 2.35-2.45 (m, 4H), 1.92-2.0 (m, 2H), 1.50-1.63 (m, 6H), 1.23-1.34 (m, 63 H), 0.85-0.95 (m, 6H). APCI: 634.7 [MW-1],

A mixture of 5 -(dioctadecylamino) -5 -oxopentanoic acid DODA-A1 (15.71 g, 1 eq, 24.70 mmol), O,N-dimethyl-hydroxylamine HC1 (7.227 g, 3 eq, 74.09 mmol), EDCI (7.102 g, 1.5 eq, 37.05 mmol), DMAP (1.509 g, 0.5 eq, 12.35 mmol), DIPEA (17.56 g, 23.7 mL, 5.5 Eq, 135.8 mmol) in chloroform (65 mL) was stirred at RT o/n. APCI/MS showed product and no starting material (acid). The reaction mixture was diluted with DCM, washed with water, IN HC1, brine, dried over Na4SC>4, filtered, concentrated. The crude was purified by column chromatography (Hexanes-EtOAc) to DODA-A2 (14.83 g, 21.84 mmol, 88.41 % for two steps). 'HNMR (CDC1₃, 400 MHz, ppm: 53.68 (s, 3H), 3.25-3.30 (m, 2H), 3.15-3.25(m, 2H), 3.18(s, 3H), 2.50-2.60 (m, 2H), 1.95-2.05 (m, 2H), 1.20-1.30 (m, 61H), 0.85-0.95 (m, 6H). APCI: 679.6 [MW+1]⁺.

DODA-A2 (14.67 g, 1 eq, 21.60 mmol) was dissolved in a mixture of THF (120 mL) and MeOH (44 mL). To the mixture was added sodium tetrahydroborate (4.15 g, 3.88 mL, 5.08 eq, 110 mmol) at 0°C. The reaction mixture was stirring at RT for 16 hours. MS showed the target and significant (-40% by MS) amount of SM. To the reaction mixture was added 1.0 g (1.2 eq) ofNaBH4 and stirred at 40-50 °C for 3 hours to achieve complete conversion to the target. The reaction mixture was quenched by 100 mL of NH4Cl(sat) and stirred at RT for 30 min, partially concentrated on a rotovap and extracted with EtOAc. The organic phase was washed with brine and dried over sodium sulfate. The crude was purified on silica, 0-100% EtOAc in DCM. DODA-1 was obtained (12.75 g, 20.49 mmol, 94.88 %) as white solid. 'HNMR (CDCI3, 400 Mhz, ppm: 53.55-3.65(m, 2H), 3.25- 3.35 (m, 2H), 3.15-3.25(m, 2H), 2.30-2.40 (m, 2H), 1.70-1.83 (m, 2H), 1.45-1.65 (m, 6H), 1.20-1.32 (m, 61H), 0.85-0.95 (m, 6H). APCI: 623.4 [MW+1]⁺.

Synthesis of DODA-PG-OH

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DODA-1 (3.05 g, 1.00 Eq, 4.90 mmol) was dissolved in Toluene (18.0 mL) and 0.8 M Phosphazene base P4-t-Bu solution (3.29 g, 6.50 mL, 0.8 molar, 1.060 Eq, 5.20 mmol)) was added under Argon. The reaction mixture was stirred for 30 minutes at RT. 2-((l- ethoxyethoxy)methyl)oxirane (35.8 g, 50.00 Eq, 245 mmol) was added dropwise at RT over 16 minutes. The reaction mixture was stirred at RT overnight under Ar The reaction was quenched with 888 mg (~1.5 eq) of benzoic acid stirred for 30 min until benzoic acid dissolved and concentrated using rotovap. The crude was purified using C4 column using H2O and i-PrOH as eluent to afford DODA-PG40-OH (28.1 g, 90%). ’HNMR (400 MHz, d-chloroform) 54.69 (m, 39H), 4.09 (m, 1H), 3.92-3.42 (m, 292H), 3.25 (t, J = 7.4 Hz, 2H), 3.17 (t, J = 7.4 Hz, 2H), 2.27 (t, J = 7.4 Hz, 2H), 1.69-1.62 (m, 2H), 1.56-1.42 (m, 6H), 1.32-1.11 (m, 314H), 0.85 (t, J = 6.0 Hz, 6H).

DODA-PG-OH (28.0 g, 1 Eq, 4.33 mmol) was dissolved in MeOH (180.0 mL) and treated with hydrogen chloride (2.19 g, 20.0 mL, 3.0 molar, 13.9 Eq, 60.0 mmol) (3M in MeOH) overnight. The reaction mixture was concentrated. The crude was triturated/sonicated in Et2O, dried in oven at 40-45 °C under high vacuum for 24 hours providing 15.1 g (97% yield) of DODA-PG39. 'HNMR (400 MHz, d4-methanol) 5 3.75-3.45 (m, 220H), 2.36 (t, J = 7.2 Hz, 2H), 1.69-1.45 (m, 8H), 1.32-1.16 (m, 60H), 0.87 (t, J = 6.0 Hz, 6H). MALDI-TOF: 3534 (M+23(Na)).

Example 23. Alternative Synthesis of Polymer-Conjugated Lipids Containing a Reactive Species

The goal of this experiment was to synthesize exemplary polymer-conjugated lipids that also contain a reactive species, such as maleimide, which can, in turn, be conjugated to a targeting moiety. In this experiment, the polymer-conjugated lipids synthesized were dioctadecylamine (DODA) conjugated to polyglycerol containing 68 monomeric subunits and also containing maleimide (DODA-PG-Mal) in accordance with Scheme 4A as shown in FIG. 22A and Scheme 4B as shown in FIG. 22B

Synthesis of DODA-A1, DODA-A2, and DODA-1 was carried out using the synthetic procedures described in Example 22.

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Synthesis of DODA-PG-OH

Synthesis of DODA-PG-OH was carried out using the synthetic procedure described in Example 22, but was conducted on a larger scale. DODA-1 (0.475 g, 1 eq, 764 pmol) was dissolved in Toluene (4.0 m ) and 0.8 M Phosphazene base P4-t-Bu solution (532 mg, 1.05 m , 0.8 molar, 1.1 eq, 840 pmol) was added under Argon. The reaction mixture was stirred for 30 minutes at RT. 2-((l- ethoxyethoxy)methyl)oxirane (14.3 g, 128 eq, 97.8 mmol) was added dropwise at RT over 16 minutes. The reaction mixture was stirred at RT overnight under Ar The reaction was quenched with 190 mg of benzoic acid stirred for 30 min until benzoic acid dissolved and concentrated using rotovap. The crude was purified using C4 column using H₂O and i-PrOH as eluent to afford DODA-PG-OH (5.9 g, 73%). 'H NMR (400 MHz, d-chloroform) 5 4.69 (q, J = 5.4 Hz, 68H), 3.75-3.42 (m, 480H), 3.31-3.24 (m, 2H), 3.19-3.15 (m, 2H), 2.26 (t, J = 6.0 Hz, 2H), 1.69-1.35 (m, 8H), 1.32-1.11 (m, 490H), 0.87 (t, J = 6.0 Hz, 6H).

Synthesis ofDODA-PG-Ll

DODA-PG-OH (1.42 g, 1 eq, 140 pmol) was dissolved in 4.5 mb of THE and cooled to 0°C. Sodium hydride (19.2 mg, 60% Wt, 3.5 eq, 480 pmol) was added. The reaction mixture was stirred for Ih at 0°C, then tert-butyl (4-(bromomethyl)benzyl)carbamate (420 mg, 10 eq, 1.4 mmol) was added neat (solid) and the reaction mixture was left stirring and slowly warming to RT o/n.

The reaction mixture was diluted with DCM and quenched with NH4CI (sat). Organic layer was separated and dried over Na₂SO4 and concentrated. Crude ’H NMR showed product. The crude was dissolved in DCM (90 mb) and the cloudy solution was filtered through celite, purified by ISCO, C4, water-I-PrOH (20 g) to give DODA-PG-E1 (1.26 g, 83%). ’H NMR (400 MHz, d4-methanol) 5 7.32-7.21 (m, 4H), 4.69 (m, 70H), 4.19 (s, 2H), 3.8-3.2 (m, 515H), 2.36 (t, J = 6.0 Hz, 2H), 1.69-1.35 (m, 8H), 1.32-1.11 (m, 490H), 0.87 (t, J = 6.0 Hz, 6H).

Synthesis of DODA-PG-L2

DODA-PG-E1 (1.45 g, 1 eq, 151 pmol) was dissolved in MeOH (7.0 mb) and hydrogen chloride (0.33 g, 3.0 mb, 3.0 N, 9.0 mmol) in MeOH was added to it at RT. The reaction mixture was stirring at RT o/n and concentrated. The residue was washed with (8 mbx2) of ether to give the desired DODA-PG-E2 (745 mg, 98%). ’H NMR (400 MHz, d4-methanol) 5 7.40-7.51 (m, 4H), 4.70 (s, 2H), 4.11 (s, 2H), 3.8-3.37 (m, 340H), 3.42-3.27 (m, 4H), 2.36 (t, J = 6.0 Hz, 2H), 1.69-1.45 (m, 6H), 1.32-1.21 (m, 66H), 0.87 (t, J = 6.0 Hz, 6H).

Synthesis of DODA-PG-Mal

DODA-PG-E2 (140 mg, 1 eq, 24.1 pmol) was suspended in ethanol (2.0 mb). To the mixture was added 2,5-dioxopyrrolidin-l-yl 4-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)butanoate (10.1 mg, 1.5

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MEl\57916143.vl 131698-30520 eq, 36.1 pmol) and Triethylamine (7.31 mg, 10.1 pL, 3 eq, 72.2 pmol). The resulting mixture was stirred at rt for 2-3 h, then concentrated. ’H NMR showed the desired product with the starting NHS- Maleimide. The crude product was washed with DCM (3 x 2 ml) and dried to give DODA-PG-Mal (140 mg, 97%). 'H NMR showed only the desired product. 'H NMR (400 MHz, d4-methanol) 5 7.35- 7.24 (m, 4H), 6.77 (s, 2H), 5.12 (1H), 4.63 (s, 2H), 4.31 (s, 2H), 4.25-4.08 (m, 2H), 3.8-3.3 (m, 340H), 3.42-3.27 (m, 4H), 2.36 (t, J = 6.0 Hz, 2H), 2.21 (t, J = 6.0 Hz, 2H), 1.88 (t, J = 6.0 Hz, 2H), 1.69-1.45 (m, 6H), 1.32-1.21 (m, 66H), 0.87 (t, J = 6.0 Hz, 6H).

Example 24. Knockdown of P-2-microglobulin (B2M) gene in T and NK cells in vivo using siRNA delivered with ctLNPs targeting CD7

To evaluate the capacity of ctLNPs to deliver siRNA and knockdown gene expression in T and NK cells in vivo, cynomolgus monkeys (Macaca fasciculciris,' also referred to herein as “NHP”) were used as a model system, and siRNA targeting p -2 -microglobulin (B2M), a component of MHC Class I that is broadly expressed across T and NK cells, or siRNA targeting Activator of Hsp90 ATPase Activity 1 (AHSA1), a Hsp90 co-chaperone, was utilized as a cargo. The ctLNP (composition as provided in Table 25, below) was conjugated to a CD7-targeting antibody (CD7-005 scFv, or a cynomolgus CD7 VHH) to specifically target T and NK cells which express CD7 on their surface. All LNPs had the following composition: 47.5% Lipid 87, 10% DSPC, 39.45% Cholesterol, 2.8% DODA-PG39, 0.2% DODA-PG68-Mal, 0.05% DiD.

Table 25

The study design and treatment groups are shown in Table 26, below. The cynomolgus monkeys were acclimated at the study site and peripheral blood mononuclear cells (PBMCs) were isolated from blood draws prior to dosing. The level of B2M expressed in PBMCs prior to dosing was utilized as a baseline for B2M expression to normalize post-treatment B2M expression on an animal- by-animal basis.

Table 26

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Cryopreserved PBMC aliquots from cynomolgus monkeys in the study were thawed, washed in cell culture medium, normalized to a consistent cell density, a viability dye was applied and then washed out, blocked and the cells were then labeled with a panel of fluorescently labeled antibodies against non-human primate CD45 (BD Biosciences), CD3 (BD Biosciences), CD20 (BioLegend), CD4 (BioLegend), CD8 (BioLegend), CD159a (Miltenyi), and B2M (BioLegend) or CD7 (BD Biosciences), fixed in 3.2% formaldehyde and the samples were run on an Attune NXT Cytometer (ThermoFisher Scientific) following appropriate compensation.

T and NK cells are cell types which express high CD7 relative to circulating B cells. To test whether ctLNPs conjugated to a CD7-targeting antibody differentially knocked down B2M expression in T and NK cells relative to circulating B cells, the median fluorescence value of the antibody against B2M at 48 hours following dose was converted to a ratio relative the median fluorescence value for that animal pre-dose for each cell type. Cell types were defined by flow cytometric analysis as: CD3+ T cells (Live/CD45+CD3+CD20-), CD4+ T cells (Live/CD45+CD3+CD20-CD4+CD8-), CD8+ T Cells (Live/CD45+CD3+CD20-CD4-CD8+), NK cells (Live/CD45+CD3-CD20-CD159a+), and B cells (Live/CD45+CD3-CD20+). As shown in FIG. 23, siRNA against B2M achieved more than 80% knock-down of B2M expression in T and NK cells when delivered using a ctLNP conjugated to a CD7 antibody. The percent knockdown of B2M expression in T and NK cells was significantly higher than in B cells.

To determine the efficacy of the ctLNP conjugated to a CD7-targeting antibody in targeting cells positive for CD7 expression, the percentage of cells that were positive for DiD (1,1’- Dioctadecyl-3,3,3’3’-Tetramethylindodicarbocyanide Perchlorate) staining at 48 hours post-dose was compared with the percentage of each cell type that was positive for CD7 expression in each of the animals pre-dose sample. As shown in FIG. 24, the percentage of cells expressing CD7 at pre-dose was tightly correlated with the percentage of cells that were stained with the DiD dye delivered by the formulated LNP at 48 hours.

In order to evaluate the efficacy of the conjugated ctLNP in delivering B2M siRNA and reducing the expression of B2M protein at 48 hours post-dosing at 0.5 mg/kg, B2M knockdown was

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MEl\57916143.vl 131698-30520 evaluated for groups treated either with unconjugated parental ctLNP without a targeting moiety, ctLNP conjugated to human anti-CD7 scFv (CD7-005 scFv), and ctLNP conjugated to a cynomolgus anti-CD7 VHH , both of the latter targeting cynomolgus CD7. FIG. 25 shows the ratio of median B2M fluorescence at 48 hours post-dose to pre-dose. The reduction in B2M expression (relative to each animal’s pre-dose B2M level) was only seen in animals that were administered the conjugated LNPs with a CD7-targeting moiety (n=3/group).

In order to evaluate the durability of knockdown, animals dosed with ctLNP conjugated to a scFv targeting CD7 were evaluated for B2M expression at pre-dose, and at days 2, 6, 10, 15, 19, 23, and 28 post-dose by flow cytometry. FIG. 26 shows the percentage expression relative to each animal’s pre-dose B2M geometric mean (gMFI) in PBMC samples. B2M expression in total T cells, CD4+ T cells, CD8+ T cells and NK cells showed average gMFI decrease to an average of 31%, 25%, 35% and 23% of pre-dose level through day 15 post-dose, respectively. It was shown that there was no consistent change in B2M expression in B cells over 28 days post dose. The minimum B2M gMFI relative to pre-dose was 17%, 14%, 19%, and 8% for CD3+ T cells, CD4+ T cells, CD8+ T cells, and NK cells, respectively.

Next, in order to determine the distribution of knockdown in T cells, the gMFI distribution of phycoerythrin (PE) fluorescence was gated as a percentage of the pre-dose B2M gMFI for all cells with less than 50%, 60%, 70%, 80% and 90% B2M expression. FIG. 27 shows the distribution of B2M gMFI reduction or knockdown relative to predose over time in PBMC samples. The grey region corresponds to the range of 48-hour post-dose percentage of DiD+ CD3+ T cells in n=3 animals and thus the maximum expected percentage of cells achievable post-dose. Nearly all targeted T cells had at least 50% knockdown of B2M for 10 days.

FIG. 28 shows the ratio of post dose DiD+ or DiD- B2M gMFI to predose total B2M gMFI for each immune cell type in PBMC samples over four weeks post-dose. For animals dosed with ctLNP conjugated to CD7-targeting scFv carrying B2M targeting siRNA (n=3), DiD+ cells, corresponding to engagement with the LNP, showed greater reduction in B2M gMFI relative to the predose total B2M gMFI for targeted cell types, whereas DiD- cells post-dose exhibited no consistent change in B2M gMFI.

FIG. 29 is a summary of safety and tolerability data for the NHP study and shows that CD7- ctLNP was well -tolerated in non-human primates (NHP) with no significant clinical pathology changes after multiple doses. ULN refers to upper limit of normal, and LLOQ refers to lower limit of quantification.

Clinical pathology markers include the following:

Liver Enzymes: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase Hematology: RBC, Hemoglobin, Platelets, Neutrophil, Lymphocyte, Monocyte, Eosinophil, Basophil

Coagulation: Activated Partial Thromboplastin Time, Prothrombin Time, Fibrinogen

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Cytokines & Complement: INF-y, IL-10, IL-12/IL-23p4, IL-ip, IL-2, IL-4, IL-6, IL-8, TNF-a,

MCP-1, Bb, C3a, sC5b-9 and C4d

Lipids: Total cholesterol, Triglycerides

Serum Tolerability Markers: Gamma glutamyl transferase, Bilirubin, Total protein, Albumin, Globulin, Glucose, Blood urea nitrogen, Creatinine, C-Reactive Protein, Lactate dehydrogenase

Table 27. Exemplary Target Genes

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Claims

EPO <DP n="209"/> 131698-30520 CLAIMS What is Claimed is:

1. A method of treating a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject at least a first dose and at least a second dose of an effective amount of a lipid nanoparticle (LNP) comprising a therapeutic nucleic acid (TNA), a first lipid-anchored polymer, and a second lipid-anchored polymer, wherein the first lipid-anchored polymer comprises:

(i) a first polymer, wherein the first polymer comprises a first polyglycerol (PG) or PG derivative;

(ii) a first lipid moiety; and

(iii) an optional first linker conjugating the first PG or PG derivative to the first lipid moiety, and wherein the second lipid-anchored polymer comprises:

(i) a second polymer;

(ii) a second lipid moiety;

(iii) an optional second linker, wherein the second polymer is conjugated to the second lipid moiety via the second linker; and

(iv) a reactive species conjugated to the second polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety.

2. A method of inhibiting gene expression in an immune cell, the method comprising: administering to a subject at least a first dose and at least a second dose of an effective amount of a lipid nanoparticle (LNP) comprising a therapeutic nucleic acid (TNA), a first lipid- anchored polymer, and a second lipid-anchored polymer, wherein the first lipid-anchored polymer comprises:

(ii) a first lipid moiety; and

(i) a second polymer;

(ii) a second lipid moiety;

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(iv) a reactive species conjugated to the second polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety., thereby inhibiting expression of the gene in the immune cell.

3. The method of claim 2, wherein the immune cell is a T cell.

4. The method of claim 3, wherein the T cell is a naive T cell.

5. The method of claim 3, wherein the T cell is a CD8+ T cell.

6. The method of claim 3 or 4, wherein the T cell is a CD4+ T cell.

7. The method of claim 3, wherein the T cell is an autologous T cell.

8. The method of claim 3, wherein the T cell is an allogeneic T cell.

9. The method of claim 2, wherein the immune cell is a natural killer (NK) cell.

10. The method of any one of claims 2-9, wherein the gene is a target gene selected from a target gene in Table 27.

11. The method of claim 2, wherein the gene expression is inhibited by at least about 50%, about 60%, 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

12. The method of any one of claims 2-11, wherein the cell is in a subject.

13. The method of claim 12, wherein the subject is a human.

14. The method of claim 13, wherein the subject is suffering from an autoimmune disease or disorder.

15. The method of claim 14, wherein the autoimmune disease or disorder is selected from the group consisting of rheumatoid arthritis juvenile idiopathic arthritis, autoimmune hepatitis, sarcoidosis, giant cell arteritis, Sjogren’s syndrome, systemic sclerosis, systemic lupus erythematosus, inflammatory bowel disease, primary biliary cirrhosis, dermatomyositis, multiple sclerosis, type I diabetes, psoriasis, psoriatic arthritis, Crohn’s disease, Celiac disease, ulcerative colitis, Graves’ disease, Hashimoto’s thyroiditis, Addison’s disease, dermatomyositis, chronic inflammatory

209

MEl\57916143.vl 131698-30520 demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome, myasthenia gravis, autoimmune vasculitis, pernicious anemia, vitiligo, systemic sclerosis, scleroderma, diffuse scleroderma, limited scleroderma, linear scleroderma, localized scleroderma, hemolytic anemia, achantholysis, acute disseminated encephalomyelitis, adult-onset Still disease, allergic glomerulonephritis, ANCA associated vasculitis, ankylosing spondylitis, anti-glomerular basement membrane disease, anti-N- methyl-D-aspartate receptor encephalitis, antiphospholipid syndrome, antisynthetase syndrome, autoimmune gastritis, autoimmune hypophysitis, autoimmune liver disease, autoimmune lung disease, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, autoimmune skin disease, autoimmune thyroiditis, autoimmune uveitis, Behcet disease, bullous pemphigoid, Churg Strauss syndrome, dermatitis herpetiformis, endocrine ophthalmopathy, erythematous pemphigus, Felty syndrome, Giant cell arteritis, IgA glomerulonephritis, membranous glomerulonephritis, Goodpasture syndrome, Granulomatosis with polyangiitis, Graves’ ophthalmopathy, Hailey syndrome, heparin induced thrombocytopenia, autoimmune hepatitis, idiopathic hypoparathryroidism, idiopathic thrombocytopenic purpura, IgA pemphigus, immune complex nephritis, immunoglobulin A nephropathy, juvenile rheumatoid arthritis, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, linear IgA bullous dermatosis, lupus nephritis, lupus vasculitis, microscopic polyangiitis, morphea, mucous membrane phemphigoid, myasthenia gravis, nonarticular rheumatism, sympathetic ophthalmia, opsoclonous myoclonus syndrome, paraneoplastic pemphigus, pemphigoid, pemphigoid gestationis, pemphigus, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, autoimmune polyendocrinopathies, polyradiculoneuropathy, postpartum thyroiditis, rheumatic disease, rheumatic heart disease, rheumatoid polymyalgia, rheumatoid nodule, rheumatoid vasculitis, stiff-person syndrome, CREST syndrome, sarcoidosis, and uveomeningoencephalitic syndrome.

16. The method of claim 15, wherein the autoimmune disease or disorder is selected from the group consisting of: autoimmune hepatitis, juvenile idiopathic arthritis, sarcoidosis, giant cell arteritis, Sjogren’s Syndrome, systemic sclerosis, systemic lupus erythematosus, inflammatory bowel disease, primary biliary cirrhosis, and dermatomyositis.

17. The method of claim 16, wherein the juvenile idiopathic arthritis (JIA) is systemic onset JIA, oligoarticular JIA, polyarticular JIA, enthesitis-related JIA, or psoriatic arthritis.

18. The method of any one of claims 2-17, wherein the cell is in vitro, ex vivo, or in vivo.

19. A method of modulating T cell activation or activity, the method comprising: administering to the subject at least a first dose and at least a second dose of an effective amount of a lipid nanoparticle (LNP) comprising a therapeutic nucleic acid (TN A), a first lipid-

210

MEl\57916143.vl 131698-30520 anchored polymer, and a second lipid-anchored polymer, wherein the first lipid-anchored polymer comprises:

(ii) a first lipid moiety; and

(i) a second polymer;

(ii) a second lipid moiety;

(iv) a reactive species conjugated to the second polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety., thereby inhibiting expression of the gene in the immune cell, thereby modulating T cell activation or activity.

20. The method of claim 19, wherein the decrease is by at least 50%, at least 60%, at least 70% or at least 80% at day 15 compared to a T cell contacted with a control TNA.

21. The method of any one of claims 1-20, further comprising administering to the subject at least a third dose of an effective amount of the LNP.

22. The method of any one of claims 1-21, further comprising administering to the subject at least a fourth, fifth, sixth, seventh, eighth, ninth, tenth, or subsequent dose of an effective amount of the LNP.

23. The method of any one of claims 1-22, wherein each dose is formulated as a pharmaceutical composition comprising the LNP and a pharmaceutically effective carrier.

24. The method of any one of claims 1-23, wherein the second polymer is a second PG or PG derivative.

25. The method of any one of claims 1-24, wherein the first PG derivative is a carboxylated PG.

26. The method of claim 25, wherein the carboxylated PG is a glutarylated PG.

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27. The method of claim 26, wherein the glutarylated PG is 3 -methyl glutarylated PG.

28. The method of claim 25, wherein the carboxylated PG is 2-carboxycyclohexane-l- carboxylated PG.

29. The method of any one of claims 1-28, wherein the first PG or PG derivative is linear or branched.

30. The method of any one of claims 1-29, wherein the first lipid moiety is represented by Formula (I):

R²

Z ^Rl³ (1). or a pharmaceutically acceptable salt thereof, wherein:

R³ is a hydrophobic tail comprising 10-30 carbon atoms, wherein o njxr i_n Formula (I) is a bond conjugating the lipid moiety and the linker, when present.

31. The method of claim 30, wherein R¹ is absent, and wherein R² and R³ are each independently a hydrophobic tail comprising 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

32. The method of claim 31, wherein R² and R³ are each independently a hydrophobic tail comprising 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

33. The method of claim 32, wherein R² and R³ are each independently a hydrophobic tail comprising 18 carbon atoms, and wherein the lipid moiety is dioctadecylamine (DODA).

34. The method of any one of claims 1-33, wherein the first lipid moiety conjugated to the first linker is represented by the following structure:

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35. The method of any one of claims 1-33, wherein the first lipid moiety is selected from the group consisting of l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1 -palmitoyl -2 -oleoyl - glycero-3 -phosphocholine (POPC), 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), 1 -palmitoyl -2 -oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (POPG), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), l,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dielaidoyl-sn-phosphatidylethanolamine (DEPE), 1 -stearoyl -2 -oleoyl-sn-glycero-3- phosphoethanolamine (SOPE), l,2-dioleoyl-sn-glycero-3 -phosphoglycerol (DOPG), 1,2-dipalmitoyl- sn-glycero-3 -phosphoglycerol (DPPG), 18-1-trans PE, l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), l,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), dihexadecylamine, distearoyl -rac -glycerol (DSG), 1,2-dipalmitoyl-rac -glycerol (DPG), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1,2-O-dioctadecyl-sn-glycerol (DODG), and a derivative of any of the foregoing.

36. The method of any one of claims 1-35, wherein the first lipid moiety is selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, DODG, and a derivative of any of the foregoing.

37. The method of any one of claims 1-36, wherein the first PG or PG derivative comprises an average of about 5-100 monomeric units.

38. The method of any one of claims 1-37, wherein the first PG or PG derivative comprises an average of about 30-80 monomeric units.

39. The method of any one of claims 1-38, wherein the first PG or PG derivative comprises an average of about 5-100, about 10-100, about 15-100, about 20-100, about 25-100, about 27-100, about 30-100, about 34-100, about 35-100, about 39-100, about 40-100, about 45-100, about 46-100, about 50-100, about 55-100, about 58-100, about 60-100, about 65-100, about 68-100, about 70-100, about 75-100, about 80-100, about 85-100, about 90-100, or about 95-100 monomeric units.

40. The method of any one of claims 1-39, wherein the first PG or PG derivative comprises an average of at least about 20, at least about 25, at least about 27, at least about 30, at least about 34, at least about 35, at least about 39, at least about 40, at least about 45, at least about 46, at least about 50, at least about 55, at least about 58, at least about 60, at least about 65, at least about 68, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 monomeric units.

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41. The method of any one of claims 1-40, wherein the first PG or PG derivative comprises an average of about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100 monomeric units.

42. The method of any one of claims 1-41, wherein the first PG or PG derivative comprises an average of about 5-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 monomeric units.

43. The method of any one of claims 1-42, wherein the first PG or PG derivative comprises an average of about 5-25, 25-50, 50-75, or 75-100 monomeric units.

44. The method of any one of claims 1-43, wherein the first PG or PG derivative comprises an average of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,

56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,

83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 monomeric units.

45. The method of any one of claims 1-44, wherein the first PG or PG derivative comprises an average of about 8, 27, 34, 39, 45, 46, 50, 58, or 68 monomeric units.

46. The method of any one of claims 1-45, wherein the first PG or PG derivative comprises an average of about 8 monomer units.

47. The method of any one of claims 1-45, wherein the first PG or PG derivative comprises an average of about 27 monomer units.

48. The method of any one of claims 1-45, wherein the first PG or PG derivative comprises an average of about 34 monomeric units.

49. The method of any one of claims 1-45, wherein the first PG or PG derivative comprises an average of about 39 monomeric units.

50. The method of any one of claims 1-45, wherein the first PG or PG derivative comprises an average of about 45 monomeric units.

51. The method of any one of claims 1-45, wherein the first PG or PG derivative comprises an average of about 46 monomeric units.

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52. The method of any one of claims 1-45, wherein the first PG or PG derivative comprises an average of about 50 monomeric units.

53. The method of any one of claims 1-45, wherein the first PG or PG derivative comprises an average of about 58 monomeric units.

54. The method of any one of claims 1-45, wherein the first PG or PG derivative comprises an average of about 68 monomeric units.

55. The method of any one of claims 1-54, wherein the first linker is absent.

56. The method of any one of claims 1-54, wherein the first linker, when present, is selected from the group consisting of an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker, an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, and any combination thereof.

57. The method of any one of claims 1-54 or 56, wherein the first linker, when present, is selected from the group consisting of -(CH₂)_n-, -C(O)(CH₂)_n-, -C(O)O(CH₂)_n, -OC(O)(CH₂)_nC(O)O-, and -NH(CH₂)_nC(O)O-, wherein n is an integer ranging from 1 to 20.

58. The method of any one of claims 1-54 or 56-57, wherein the first linker, when present, is a glutaryl linker or a succinyl linker.

59. The method of any one of claims 1-58, wherein the first linker, when present, is -C(O)(CH₂)_n-, and wherein n is 2, 3, 4, 5, or 6.

60. The method of any one of claims 57-59, wherein n is 4.

61. The method of any one of claims 1-45, 50, or 55-60, wherein the first lipid-anchored polymer is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

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62. The method of any one of claims 1-45, 51, or 55-60, wherein the first lipid-anchored polymer and/or the second lipid-anchored polymer are represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

63. The method of any one of claims 1-45, 53, or 55-60, wherein the first lipid-anchored polymer and/or the second lipid-anchored polymer arerepresented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

64. The method of any one of claims 1-45, 48, or 55-60, wherein the first lipid-anchored polymer and/or the second lipid-anchored polymer arerepresented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

65. The method of any one of claims 1-45, 49, or 55-60, wherein the first lipid-anchored polymer and/or the second lipid-anchored polymer arerepresented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

66. The method of any one of claims 1-45, or 54-60, wherein the first lipid-anchored polymer and/or the second lipid-anchored polymer arerepresented by the following structure:

216

67. The method of any one of claims 1-45 or 55-60, wherein the first lipid-anchored polymer and/or the second lipid-anchored polymer arerepresented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

68. The method of any one of claims 1-67, wherein the reactive species is a click chemistry reagent, maleimide, or thiol.

69. The method of any one of claims 1-68, wherein the reactive species is maleimide or thiol.

70. The method of any one of claims 1-68, wherein the click chemistry reagent is selected from the group consisting of a dibenzocyclooctyne (DBCO) reagent, a transcylooctene (TCO) reagent, a tetrazine (Tz) reagent, an alkyne reagent, and an azide reagent.

71. The method of any one of claims 1-70, further comprising a targeting moiety conjugated to the second polymer derivative via the reactive species.

72. The method of claim 71, wherein the targeting moiety is conjugated to the second polymer via a dibenzocyclooctyne (DBCO)-azide conjugation, an azide-alkyne conjugation, a TCO-Tz conjugation, or a thiol-maleimide conjugation.

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73. The method of any one of claims 71-72, wherein the targeting moiety is conjugated to the second polymer via a thiol-maleimide conjugation.

74. The method of any one of claims 71-73, wherein the targeting moiety is capable of binding to a liver cell.

75. The method of claim 74, wherein the liver cell is a hepatocyte.

76. The method of any one of claims 71-75, wherein the targeting moiety is N-acetyl galactosamine (GalNAc) or a GalNAc derivative.

77. The method of any one of claims 71-76, wherein the targeting moiety is a tri-antennary GalNAc conjugate or a tetra-antennary GalNAc conjugate.

78. The method of claim 71-75, wherein the targeting moiety is selected from the group consisting of an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, and a fragment or a variant thereof.

79. The method of any one of claims 71-75, wherein the targeting moiety is an antibody or an antibody fragment.

80. The method of claim 79, wherein the antibody or the antibody fragment is capable of specifically binding an antigen present on the surface of a cell.

81. The method of claim 79 or claim 80, wherein the antibody or the antibody fragment is a monoclonal antibody (mAb), a single chain variable fragment (scF_v), a heavy chain antibody (he Ab), a nanobody (Nb), a heavy-chain-only immunoglobulin (HCIg), an immunoglobulin new antigen receptor (IgNAR), variable domain of immunoglobulin new antigen receptor (VNAR), a single-domain antibody, or a variable heavy chain-only antibody (VHH).

82. The method of any one of claims 79-81, wherein the antibody or antibody fragment is a VHH.

83. The method of any one of claims 79-81, wherein the antibody or antibody fragment is an scF_v.

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84. The method of any one of claims 71-75 or 79-82, wherein the second lipid-anchored polymer is represented by the following structure: wherein n is 65, 66, 67, 68, 69, or 70; or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

85. The method of claim 84, wherein n is 68.

86. The method of any one of claims 71-73 or 79-85, wherein the targeting moiety is capable of binding to an antigen on a cell or cell fragment selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a macrophage, a red blood cell, a platelet, a megakaryocyte, a hematopoietic stem cell (HSC), a hematopoietic stem or progenitor cell (HSPC), a CD34+ cell, a muscle cell, a brain cell, a nerve cell, a lung cell, an endothelial cell, an epithelial cell, a kidney cell, a spleen cell, an ovarian cell, a testicular cell, a uterine cell, a placental cell, a vascular cell, a skin cell, and an endocrine cell.

87. The method of claim 86, wherein the cell is a T cell.

88. The method of claim 87, wherein the T cell is a naive T cell.

89. The method of claim 87 or claim 88, wherein the T cell is a CD8+ T cell or a CD4+ T cell.

90. The method of any one of claims 71-89, wherein the targeting moiety binds to an antigen expressed on a T cell selected from the group consisting of a CD4+ T cell-specific antigen, a CD8+ T cell-specific antigen, and a CD3+ T cell-specific antigen.

91. The method of any of claims 71-90, wherein the targeting moiety binds to an antigen expressed on a T cell selected from the group consisting of CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD 10, CD 11, PD-1, and TCR.

92. The method of claim 91, wherein the targeting moiety binds to CD7.

93. The method of claim 86, wherein the cell is an NK cell.

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94. The method of claim 86, wherein the cell is a hematopoietic stem cell (HSC), a hematopoietic stem or progenitor cell (HSPC), or a CD34+ cell.

95. The method of any one of claims 1-94, wherein the LNP further comprises:

(i) a therapeutic nucleic acid (TNA);

(ii) an ionizable lipid; and

(iii) a sterol.

96. The method of any one of claims 1-95, wherein the LNP further comprises a helper lipid.

97. The method of claim 96, wherein the helper lipid comprises a phospholipid.

98. The method of claim 96 or claim 97, wherein the helper lipid is selected from the group consisting of l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), hydrogenated soybean PC (HSPC), phosphatidylserine (PS), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2-dipalmitoyl-sn- glycero-3 -phosphocholine (DPPC), 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2- dilauroyl-sn-glycero-3 -phosphocholine (DLPC), 1-margaroyl -2 -oleoyl-sn-glycero-3 -phosphocholine (MOPC), 1 -palmitoyl -2 -linoleoyl-sn-glycero-3 -phosphocholine (PLPC), 1 -stearoyl -2 -myristoyl-sn- glycero-3 -phosphocholine (SMPC), l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), 1,2- dihexanoyl-sn-glycero-3 -phosphocholine (DHPC), 1 ,2-dierucoyl-sn-glycero-3 -phosphocholine (DEPC), 1 -palmitoyl -2 -oleoyl -glycero-3 -phosphocholine (POPC), and l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE).

99. The method of any one of claims 96-98, wherein the helper lipid is DSPC.

100. The method of any one of claims 96-99, wherein the helper lipid is represented by Formula (II): or a pharmaceutically acceptable salt or an ester thereof, wherein:

'' is a single bond or a double bond;

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R¹ is C1-C17 alkyl or C2-C17 alkenyl;

R² is C1-C22 alkyl or C2-C22 alkenyl;

R³ is hydrogen or C1-C2 alkyl; and R⁴ is hydrogen or C1-C2 alkyl.

101. The method of claim 100, wherein '' is a double bond.

102. The method of claim 100 or claim 101, wherein R¹ is C10-C20 alkenyl, R² is C10-C20 alkyl and R³ is hydrogen.

103. The method of any one of claims 96-102, wherein the helper lipid represented by Formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

104. The method of any one of claims 96-102, wherein the helper lipid represented by Formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

105. The method of any one of claims 96-102, wherein the helper lipid represented by Formula (II) is:

221

106. The method of any one of claims 96-102 wherein the helper lipid represented by Formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

107. The method of any one of claims 96-102, wherein the helper lipid represented by Formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

108. The method of any one of claims 96-102, wherein the helper lipid represented by Formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

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109. The method of any one of claims 95-108, wherein the sterol is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and a derivative thereof.

110. The method of any one of claims 95-109, wherein the sterol is cholesterol.

111. The method of any one of claims 95-110, wherein the ionizable lipid is represented by: a) Formula (A):

Formula (A), or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R¹ are each independently optionally substituted linear or branched C1-3 alkylene;

R² and R² are each independently optionally substituted linear or branched C1-6 alkylene;

R³ and R³ are each independently optionally substituted linear or branched C1-6 alkyl; or alternatively, when R²is optionally substituted branched C1-6 alkylene, R² and R³, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R² is optionally substituted branched C1-6 alkylene, R² and R³ , taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl;

R⁴ and R⁴ are each independently -CR^a, -C(R^a)2CR^a, or -[C(R^a)2]2CR^a;

R^a, for each occurrence, is independently H or C1-3 alkyl; or alternatively, when R⁴is -C(R^a)2CR^a, or -[C(R^a)2]2CR^a and when R^a is C1-3 alkyl, R³ and R⁴, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R⁴ is -C(R^a)2CR^a, or -[C(R^a)2]2CR^a and when R^a is C1-3 alkyl, R³ and R⁴ , taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl;

R⁶ and R⁶ , for each occurrence, are independently C1-20 alkylene, C3-20 cycloalkylene, or C2-20 alkenylene; and m and n are each independently an integer selected from 1, 2, 3, 4, and 5; b) Formula (B):

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Formula (B), or a pharmaceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10;

R¹ is absent or is selected from (C2-C2o)alkenyl, -C(0)0(C2-C2o)alkyl, and cyclopropyl substituted with (C2-C2o)alkyl; and

R² is (C2-C₂o)alkyl; c) Formula (C):

Formula (C), or a pharmaceutically acceptable salt thereof, wherein:

R² and R² are each independently (Ci-C2)alkylene;

R³ and R³ are each independently (Ci-Ce)alkyl optionally substituted with one or more groups selected from R^b; or alternatively, R² and R³ and/or R² and R³ are taken together with their intervening N atom to form a 4- to 7-membered heterocyclyl;

R⁴ and R⁴’ are each a (C2-Ce)alkylene interrupted by -C(O)O-;

R⁵ and R⁵’ are each independently a (C2-C3o)alkyl or (C2-C3o)alkenyl, each of which are optionally interrupted with -C(O)O- or (C3-Ce)cycloalkyl; and

R^a and R^b are each halo or cyano;

224

MEl\57916143.vl 131698-30520 d) Formula (D):

Formula (D), or a pharmaceutically acceptable salt thereof, wherein:

R’ is absent, hydrogen, or Ci-Ce alkyl; provided that when R’ is hydrogen or Ci-Ce alkyl, the nitrogen atom to which R’, R¹, and R² are all positively charged;

R¹ and R² are each independently hydrogen, Ci-Ce alkyl, or C2-C6 alkenyl;

R³ is C1-C12 alkylene or C2-C12 alkenylene;

R⁴ is Ci-Cis unbranched alkyl, C2-C18 unbranched alkenyl, or ; wherein:

R⁵ is absent, Ci-Cs alkylene, or C2-C8 alkenylene;

R^a, for each occurrence, is independently hydrogen or Ci-Ce alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6; e) Formula (E):

Formula (E), or a pharmaceutically acceptable salt thereof, wherein:

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R¹ and R² are each independently hydrogen or C1-C3 alkyl;

R³ is C3-C10 alkylene or C3-C10 alkenylene;

R⁴ is Ci-Cie unbranched alkyl, C2-C16 unbranched alkenyl, ; wherein:

R⁵ is absent, Ci-Ce alkylene, or C2-C6 alkenylene;

R^6a and R^6b are each independently C7-C14 alkyl or C7-C14 alkenyl;

X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(R^a)=N-,

-N=C(R^a)-, -C(R^a)=NO-, -O-N=C(R^a)-, -C(=O)NR^a-, -NR^aC(=O)-, -NR^aC(=O)NR^a-,

-OC(=O)O-, -OSi(R^a)₂O-, -C(=O)(CR^a)₂C(=O)O-, or OC(=O)(CR^a ₂)C(=O)-; wherein:

R^a, for each occurrence, is independently hydrogen or Ci-Ce alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6; or f) an ionizable lipid selected from the group consisting of: any of the ionizable lipids in Table 1, 4, 5, 6, or 7.

112. The method of any one of claims 95-111, wherein the ionizable lipid is Lipid 87, represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

113. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

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114. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

115. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

116. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

117. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

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118. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

119. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

120. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

121. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

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122. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

123. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

124. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

125. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

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126. The method of any one of claims 95-111, wherein the ionizable lipid is Lipid A, represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

127. The method of any one of claims 95-111, wherein the ionizable lipid is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.

128. The method of any one of claims 1-127, wherein the first lipid-anchored polymer and the second lipid-anchored polymer are the same.

129. The method of any one of claims 1-127, wherein the first lipid-anchored polymer and the second lipid-anchored polymer are different.

130. The method of any one of claims 24-129, wherein the second PG derivative is a carboxylated PG.

131. The method of claim 130, wherein the carboxylated PG is a glutarylated PG or 2- carboxy cyclohexane- 1 -carboxylated PG.

132. The method of claim 131, wherein the glutarylated PG is 3-methyl glutarylated PG.

133. The method of any one of claims 24-132, wherein the second PG or PG derivative is linear or branched.

134. The method of any one of claims 24-133, wherein the second PG or PG derivative comprises an average of about 5-100 monomeric units.

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135. The method of any one of claims 24-134, wherein the second PG or PG derivative comprises an average of about 30-80 monomeric units.

136. The method of any one of claims 24-135, wherein the second PG or PG derivative comprises an average of about 5-100, about 10-100, about 15-100, about 20-100, about 25-100, about 27-100, about 30-100, about 34-100, about 35-100, about 39-100, about 40-100, about 45-100, about 46-100, about 50-100, about 55-100, about 58-100, about 60-100, about 65-100, about 68-100, about 70-100, about 75-100, about 80-100, about 85-100, about 90-100, or about 95-100 monomeric units.

137. The method of any one of claims 24-136, wherein the second PG or PG derivative comprises an average of at least about 20, at least about 25, at least about 27, at least about 30, at least about 34, at least about 35, at least about 39, at least about 40, at least about 45, at least about 46, at least about 50, at least about 55, at least about 58, at least about 60, at least about 65, at least about 68, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 monomeric units.

138. The method of any one of claims 24-137, wherein the second PG or PG derivative comprises an average of about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60- 65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100 monomeric units.

139. The method of any one of claims 24-138, wherein the second PG or PG derivative comprises an average of about 5-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 monomeric units.

140. The method of any one of claims 24-139, wherein the second PG or PG derivative comprises an average of about 5-25, 25-50, 50-75, or 75-100 monomeric units.

141. The method of any one of claims 24-140, wherein the second PG or PG derivative comprises an average of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,

55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,

82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 monomeric units.

142. The method of claim 141, wherein the second PG or PG derivative comprises an average of about 8, 27, 34, 39, 45, 46, 50, 58, or 68 monomeric units.

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143. The method of any one of claims 24-142, wherein the second PG or PG derivative comprises an average of about 8 monomer units.

144. The method of any one of claims 24-142, wherein the second PG or PG derivative comprises an average of about 27 monomer units.

145. The method of any one of claims 24-142, wherein the second PG or PG derivative comprises an average of about 34 monomeric units.

146. The method of any one of claims 24-142, wherein the second PG or PG derivative comprises an average of about 39 monomeric units.

147. The method of any one of claims 24-142, wherein the second PG or PG derivative comprises an average of about 45 monomeric units.

148. The method of any one of claims 24-142, wherein the second PG or PG derivative comprises an average of about 46 monomeric units.

149. The method of any one of claims 24-142, wherein the second PG or PG derivative comprises an average of about 50 monomeric units.

150. The method of any one of claims 24-142, wherein the second PG or PG derivative comprises an average of about 58 monomeric units.

151. The method of any one of claims 24-142, wherein the second PG or PG derivative comprises an average of about 68 monomeric units.

152. The method of any one of claims 24-45, 49, 55-142, or 151, wherein the first PG or PG derivative comprises an average of about 39 monomeric units, and the second PG or PG derivative comprises an average of about 68 monomeric units.

153. The method of any one of claims 24-152, further comprising a targeting moiety conjugated to the second PG or PG derivative via the reactive species.

154. The method of claim 153, wherein the targeting moiety is conjugated to the second PG or PG derivative via a dibenzocyclooctyne (DBCO)-azide conjugation, an azide-alkyne conjugation, a TCO- Tz conjugation, or a thiol-maleimide conjugation.

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155. The method of claim 153 or claim 154, wherein the targeting moiety is conjugated to the second PG or PG derivative via a thiol-maleimide conjugation.

156. The method of any one of claims 153-155, wherein the targeting moiety is capable of binding to a liver cell.

157. The method of claim 156, wherein the liver cell is a hepatocyte.

158. The method of any one of claims 153-157, wherein the targeting moiety is N-acetyl galactosamine (GalNAc) or a GalNAc derivative.

159. The method of any one of claims 151-158, wherein the targeting moiety is a tri-antennary GalNAc conjugate or a tetra-antennary GalNAc conjugate.

160. The method of any one of claims 151-157, wherein the targeting moiety is selected from the group consisting of an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, and a fragment or a variant thereof.

161. The method of any one of claims 151-157, wherein the targeting moiety is an antibody or an antibody fragment.

162. The method of claim 161, wherein the antibody or the antibody fragment is capable of specifically binding an antigen present on the surface of a cell.

163. The method of claim 162, wherein the antigen on the surface of the cell is a T cell antigen.

164. The method of any one of claims 161-163, wherein the cell is positive for the cell surface marker CD7+.

165. The method of any one of claims 161-164, wherein the antibody or the antibody fragment is a monoclonal antibody (mAb), a single chain variable fragment (scF_v), a heavy chain antibody (he Ab), a nanobody (Nb), a heavy-chain-only immunoglobulin (HCIg), an immunoglobulin new antigen receptor (IgNAR), variable domain of immunoglobulin new antigen receptor (VNAR), a single-domain antibody, or a variable heavy chain-only antibody (VHH).

166. The method of any one of claims 161-165, wherein the antibody or antibody fragment is a VHH.

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167. The method of any one of claims 161-165, wherein the antibody or antibody fragment is an scF_v.

168. The method of any one of claims 153-155 or 161-167, wherein the targeting moiety is capable of binding to an antigen on a cell or cell fragment selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a macrophage, a red blood cell, a platelet, a megakaryocyte, a hematopoietic stem cell (HSC), a hematopoietic stem or progenitor cell (HSPC), a CD34+ cell, a muscle cell, a brain cell, a nerve cell, a lung cell, an endothelial cell, an epithelial cell, a kidney cell, a spleen cell, an ovarian cell, a testicular cell, a uterine cell, a placental cell, a vascular cell, a skin cell, and an endocrine cell.

169. The method of claim 168, wherein the cell is a T cell.

170. The method of claim 169, wherein the cell is a naive T cell.

171. The method of any one of claims 168-170, wherein the T cell is a CD8+ T cell or a CD4+ T cell.

172. The method of claim 168, wherein the cell is a NK cell.

173. The method of claim 168, wherein the cell is a hematopoietic stem cell (HSC), a hematopoietic stem or progenitor cell (HSPC), or a CD34+ cell.

174. The method of any one of claims 1-173, wherein the first linker and the second linker are the same or are both absent.

175. The method of any one of claims 1-174, wherein the first linker and the second linker are different, wherein the first linker is absent and the second linker is present, or wherein the first linker is present and the second linker is absent.

176. The method of any one of claims 1-175, wherein the second linker, when present, is selected from the group consisting of an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker, an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, and any combination thereof.

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177. The method of any one of claims 1-176, wherein the second linker, when present, is selected from the group consisting of -(CH2)_n-, -C(O)(CH2)_n-, -C(O)O(CH2)_n-, -OC(O)(CH2)_nC(O)O-, and - NH(CH2)_nC(O)O-, wherein n is an integer ranging from 1 to 20.

178. The method of any one of claims 1-177, wherein the second linker, when present, is a glutaryl linker or a succinyl linker.

179. The method of claim 175 or claim 178, wherein the second linker, when present, is - C145O)(CH₂)_n-, and wherein n is 2, 3, 4, 5, or 6.

180. The method of claim 177-179, wherein n is 4.

181. The method of any one of claims 1-180, wherein the second lipid moiety is represented by Formula (I) or a pharmaceutically acceptable salt thereof, wherein:

R³ is a hydrophobic tail comprising 10-30 carbon atoms, wherein arcrtr i_n Formula (I) is a bond conjugating the lipid moiety, when present, and the linker.

182. The method of claim 181, wherein R¹ is absent, and wherein R² and R³ are each independently a hydrophobic tail comprising 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

183. The method of claim 181 or claim 182, wherein R² and R³ are each independently a hydrophobic tail comprising 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

184. The method of any one of claims 181-183, wherein R² and R³ are each independently a hydrophobic tail comprising 18 carbon atoms, and wherein the lipid moiety is dioctadecylamine (DODA).

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185. The method of any one of claims 1-184, wherein the second lipid moiety comprises a moiety selected from the group consisting of l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1- palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1 -palmitoyl -2 -oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phospho-( 1 '-rac-glycerol) (POPG), l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-phosphatidylethanolamine (DEPE), 1 -stearoyl -2- oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), l,2-dioleoyl-sn-glycero-3 -phosphoglycerol (DOPG), l,2-dipalmitoyl-sn-glycero-3 -phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn- glycero-3-phospho-L-serine (DOPS), l,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), dihexadecylamine, distearoyl -rac-glycerol (DSG), 1,2-dipalmitoyl -rac- glycerol (DPG), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2-O-dioctadecyl-sn- glycerol (DODG), and a derivative thereof.

186. The method of any one of claims 1-185, wherein the second lipid moiety is selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, DODG, and a derivative of any of the foregoing.

187. The method of any one of claims 1-186, wherein the second lipid moiety comprises DODA.

188. The method of any one of claims 1-187, wherein the second lipid moiety comprises DSPE.

189. The method of any one of claims 1-188, wherein the first lipid moiety and the second lipid moiety are the same.

190. The method of any one of claims 1-188, wherein the first lipid moiety and the second lipid moiety are different.

191. The method of any one of claims 95-190, wherein the ionizable lipid is present in the LNP in an amount of about 20 mol% to about 60 mol% of the total lipid present in the LNP.

192. The method of any one of claims 95-191, wherein the ionizable lipid is present in the LNP in an amount of about 35 mol% to about 50 mol% of the total lipid present in the LNP.

193. The method of any one of claims 95-192, wherein the ionizable lipid is present in the LNP in an amount of about 40 mol% to about 50 mol% of the total lipid present in the LNP.

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194. The method of any one of claims 95-193, wherein the ionizable lipid is present in the LNP in an amount of about 45 mol% to about 50 mol% of the total lipid present in the LNP.

195. The method of any one of claims 95-194, wherein the ionizable lipid is present in the LNP in an amount of about 47.5 mol% of the total lipid present in the LNP.

196. The method of any one of claims 95-195, wherein the sterol is present in the LNP in an amount of about 20 mol% to about 50 mol% of the total lipid present in the LNP.

197. The method of any one of claims 95-196, wherein the sterol is present in the LNP in an amount of about 30 mol% to about 45 mol% of the total lipid present in the LNP.

198. The method of any one of claims 95-197, wherein the sterol is present in the LNP in an amount of about 38 mol% to about 42 mol% of the total lipid present in the LNP.

199. The method of claim 95-198, wherein the sterol is present in the LNP in an amount of about 39 mol% to about 40 mol% of the total lipid present in the LNP.

200. The method of any one of claims 95-199, wherein the sterol is present in the LNP in an amount of about 39.5 mol% of the total lipid present in the LNP.

201. The method of any one of claims 95-200, wherein the helper lipid is present in the LNP in an amount of about 1 mol% to about 40 mol% of the total lipid present in the LNP.

202. The method of any one of claims 95-201, wherein the helper lipid is present in the LNP in an amount of about 5 mol% to about 15 mol% of the total lipid present in the LNP.

203. The method of any one of claims 95-202, wherein the helper lipid is present in the LNP in an amount of about 7 mol% to about 13 mol% of the total lipid present in the LNP.

204. The method of any one of claims 95-203, wherein the helper lipid is present in the LNP in an amount of about 9 mol% to about 11 mol% of the total lipid present in the LNP.

205. The method of any one of claims 95-204, wherein the helper lipid is present in the LNP in an amount of about 10 mol% of the total lipid present in the LNP.

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206. The method of any one of claims 95-205, wherein the first lipid-anchored polymer is present in the LNP in an amount of about 0.5 mol% to about 5 mol% of the total lipid present in the LNP.

207. The method of any one of claims 95-206, wherein the first lipid-anchored polymer is present in the LNP in an amount of about 1.5 mol% to about 3 mol% of the total lipid present in the LNP.

208. The method of any one of claims 1-207, wherein the first lipid-anchored polymer is present in the LNP in an amount of about 2 mol% to about 3 mol% of the total lipid present in the LNP.

209. The method of any one of claims 1-208, wherein the first lipid-anchored polymer is present in the LNP in an amount of about 2.6 mol% to about 3 mol% of the total lipid present in the LNP.

210. The method any one of claims 1-209, wherein the first lipid-anchored polymer is present in the LNP in an amount of about 2.8 mol% of the total lipid present in the LNP.

211. The method of any one of claims 1-210, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0.05 mol% to about 5 mol% of the total lipid present in the LNP.

212. The method of any one of claims 1-211, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0. 1 mol% to about 1 mol% of the total lipid present in the LNP.

213. The method of any one of claims 1-212, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0.15 mol% to about 0.5 mol% of the total lipid present in the LNP.

214. The method of any one of claims 1-213, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0.15 mol% to about 0.3 mol% of the total lipid present in the LNP.

215. The method of any one of claims 1-214, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0.2 mol% of the total lipid present in the LNP.

216. The method of any one of claims 1-215, wherein the nanoparticle (LNP) comprises:

(i) a therapeutic nucleic acid (TNA);

(ii) an ionizable lipid, wherein the ionizable lipid is heptadecan-9-yl 9-((4- (dimethylamino)butanoyl)oxy)hexadecanoate, having the following structure:

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(iii) a sterol, wherein the sterol is cholesterol;

(iv) a helper lipid, wherein the helper lipid is DSPC;

(v) a first lipid-anchored polymer, wherein the first lipid-anchored polymer comprises DODA conjugated to a linear PG via a linker; and

(vi) a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises DODA conjugated to PG, and further comprising a reactive species conjugated to the PG, wherein the reactive species is functionalized to be conjugated to a targeting moiety.

217. The method of any one of claims 95-216, wherein: the ionizable lipid is present in the LNP in an amount of about 35 mol% to about 50 mol% of the total lipid present in the LNP; the sterol is present in the LNP in an amount of about 30 mol% to about 45 mol% of the total lipid present in the LNP; the helper lipid is present in the LNP in an amount of about 5 mol% to about 15 mol% of the total lipid present in the LNP; the first lipid-anchored polymer is present in the LNP in an amount of about 1.5 mol% to about 3 mol% of the total lipid present in the LNP; and the second lipid-anchored polymer is present in the LNP in an amount of about 0.1 mol% to about 1 mol% of the total lipid present in the LNP.

218. The method of any one of claims 1-217, wherein the TNA is selected from the group consisting of a minigene, a plasmid, a minicircle, a small interfering RNA (siRNA), a microRNA (miRNA), a guide RNA (gRNA), an antisense oligonucleotide (ASO), a ribozyme, a deoxyribozyme, a closed-ended DNA (ceDNA), a single-stranded DNA (ssDNA), a substantially single -stranded DNA, a ministring, a doggybone™, a protelomere closed ended DNA, a dumbbell linear DNA, a dicer-substrate dsRNA, a small hairpin RNA (shRNA), an asymmetrical interfering RNA (aiRNA), an mRNA, a tRNA, an rRNA, a gRNA, a DNA viral vector, a viral RNA vector, a non-viral vector, and a combination thereof.

219. The method of any one of claims 1-218, wherein the TNA is a single-stranded nucleic acid or a double-stranded nucleic acid.

220. The method of any one of claims 1-219, wherein the TNA is siRNA.

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221. The method of claim 220, wherein the siRNA comprises a sense strand and an antisense strand, wherein the antisense strand has a sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi).

222. The method of claim 221, wherein the siRNA is sufficiently complementary to a target mRNA that specifies the amino acid sequence of a cellular protein involved or predicted to be involved in an autoimmune disease or disorder.

223. The method of claim 221 or claim 222, wherein the siRNA is sufficiently complementary to a target selected from Table 27.

224. The method of any one of claims 211-223, wherein the sense strand or the antisense strand is modified by the substitution of at least one nucleotide with a modified nucleotide, such that in vivo stability and/or target efficiency is enhanced as compared to a corresponding unmodified siRNA.

225. The method of claim 224, wherein the modified nucleotide is an internal nucleotide.

226. The method of claim 224 or claim 225, wherein substantially all of the nucleotides of the sense strand comprise a modification; substantially all of the nucleotides of the antisense strand comprise a modification; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise a modification.

227. The method of claim 224 or claim 225, wherein all of the nucleotides of the sense strand comprise a modification; all of the nucleotides of the antisense strand comprise a modification; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

228. The method of any one of claims 224-227, wherein at least one of the nucleotide modifications is selected from the group consisting of a deoxy-nucleotide modification, a 3 ’-terminal deoxythimidine (dT) nucleotide modification, a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxy- nucleotide modification, a locked nucleotide modification, an unlocked nucleotide modification, a conformationally restricted nucleotide modification, a constrained ethyl nucleotide modification, an abasic nucleotide modification, a 2’ -amino nucleotide modification, a 2’-O-allyl- nucleotide modification, 2’-C -alkyl- nucleotide modification, a 2’- methoxyethyl nucleotide modification, a 2’-O-alkyl- nucleotide modification, a morpholino nucleotide modification, a phosphoramidate nucleotide modification, a non-natural base comprising

240

MEl\57916143.vl 131698-30520 nucleotide modification, a tetrahydropyran nucleotide modification, a 1,5-anhydrohexitol nucleotide modification, a cyclohexenyl nucleotide modification, a nucleotide comprising a phosphorothioate group nucleotide modification, a nucleotide comprising a methylphosphonate group nucleotide modification, a nucleotide comprising a 2 ’-phosphate nucleotide modification, a nucleotide comprising a 5 ’-phosphate nucleotide modification, a nucleotide comprising a 5 ’-phosphate mimic nucleotide modification, a thermally destabilizing nucleotide modification, a glycol modified nucleotide (GNA) nucleotide modification, an unlocked nucleic acid (UNA) modification, a iAB modification, and a 2-O-(N-methylacetamide) nucleotide modification; and combinations thereof.

229. The method of any one of claims 224-228, wherein the modified nucleotide is: a sugar-modified nucleotide selected from the group consisting of 2'-fluoro-cytidine, 2'-fluoro- uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino- adenosine, 2'-amino-guanosine and 2'-amino-butyryl-pyrene-uridine; a nucleobase-modified nucleotide selected from the group consisting of 5 -bromo-uridine, 5 -iodouridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 5 -fluoro-cytidine, and 5 -fluoro-uridine, 2,6-diaminopurine, 4-thio-uridine; and 5 -amino-allyl -uridine; a 2'-deoxy ribonucleotide and is present within the sense strand; a 2'-fluoro modified ribonucleotide; and/or selected from the group consisting of a 2'-fluoro, 2'-amino and 2'-thio modified ribonucleotide.

230. The method of any one of claims 224-229, wherein the modified nucleotide is a nucleotide analogue or artificial nucleotide base.

231. The method of claim 230 wherein the nucleotide analogue or artificial nucleotide base comprises a 5'-vinylphosphonate modified nucleotide with a modification at a 5' hydroxyl group of the ribose moiety (5'-(E)-vinylphosphonate (5'-EVP) modified nucleotide).

232. The method of any one of claims 1-231, wherein the TNA is an siRNA; wherein the ionizable lipid is Lipid 87 and is present in the LNP in an amount of about 47.5 mol% of the total lipid present in the LNP; wherein the helper lipid is DSPC and is present in the LNP in an amount of about 10 mol% of the total lipid present in the LNP; wherein the sterol is cholesterol and is present in the LNP in an amount of about 39.5 mol% of the total lipid present in the LNP; wherein the first lipid-anchored polymer is DODA-PG39 and is present in an amount of about 2.8 mol% of the total lipid present in the LNP; and

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MEl\57916143.vl 131698-30520 wherein the second lipid-anchored polymer is DODA-PG68 and is present in an amount of about 0.2 mol% of the total lipid present in the LNP.

233. The method of any one of claims 1-232, wherein the TNA is mRNA.

234. The method of any one of claims 1-233, wherein the LNP does not comprise polyethylene glycol (PEG).

235. The method of any one of claims 1-234, wherein the LNP does not induce or minimally induces antibody-mediated clearance in the subject’s blood.

236. The method of any one of claims 1-235, wherein the LNP has a half-life (ti/2) in blood in vivo of about 3 hours to about 72 hours.

237. The method of any one of claims 1-236, wherein the LNP has a half-life (ti/2) in blood in vivo of greater than 3 hours.

238. The method of any one of claims 1-237, wherein the LNP has a half-life (ti/2) in blood in vivo of greater than 4 hours.

239. The method of any one of claims 1-238, wherein the LNP has a half-life (ti/2) in blood in vivo of greater than 3 hours, 4, hours, 5 hours, 6, hours, 8, hours, 10 hours, 12 hours, 14 hours, 15 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 25 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 35 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 45 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 55 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 65 hours, 66 hours, 68 hours, 70 hours, or 72 hours.

240. The method of any one of claims 1-239, wherein the LNP has an in vivo half-life (ti/2) that is prolonged in the subject’s blood as compared to the in vivo half-life of an LNP that does not comprise PG or a PG derivative.

241. The method of any one of claims 1-240, wherein the LNP has an in vivo half-life (ti/2) that is prolonged in the subject’s blood as compared to the in vivo half-life of an LNP that comprises polyethylene glycol (PEG).

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242. The method of any one of claims 1-241, wherein the in vivo half-life of the LNP is increased by at least a factor of about two or more as compared to the in vivo half-life of an LNP that does not comprise PG or a PG derivative.

243. The method of any one of claims 1-242, wherein the in vivo half-life of the LNP is increased by at least a factor of about two or more as compared to the in vivo half-life of an LNP that comprises PEG.

244. The method of any one of claims 1-243, wherein the in vivo half-life of the LNP is increased by at least a factor of about three or more as compared to the in vivo half-life of an LNP that does not comprise PG or a PG derivative.

245. The method of any one of claims 1-244, wherein the in vivo half-life of the LNP is increased by at least a factor of about three or more as compared to the in vivo half-life of an LNP that comprises PEG.

246. The method of any one of claims 1-245, wherein the LNP and/or the TNA persists in the subject’s blood at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 or more hours after the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or subsequent dose.

247. The method of any one of claims 1-246, wherein each dose is separated by a time period of at least about 6 hours, at least about 1 day, at least about 7 days, at least about 2 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 4 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 5 years, or at least about 10 years.

248. The method of any one of claims 1-247, wherein each dose is separated by a time period of about 6-24 hours, about 1-500 days, about 1-100 weeks, about 1-24 months, or about 1-10 years.

249. The method of any one of claims 1-248, wherein each dose is separated by a time period of about 7 days.

250. The method of any one of claims 1-249, wherein each dose is administered on a periodic schedule.

251. The method of any one of claims 1-250, wherein each dose is administered on a variable schedule.

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252. The method of any one of claims 1-251, wherein each dose is about 0.01-10.0 mg/kg.

253. The method of any one of claims 1-252, wherein each dose is at least 0.01 mg/kg, at least 0.05 mg/kg, at least 0.1 mg/kg, at least 0.5 mg/kg, at least 1.0 mg/kg, at least 1.5 mg/kg, at least 2.0 mg/kg, at least 2.5 mg/kg, at least 3.0 mg/kg, at least 4.0 mg/k, or at least 5.0 mg/kg.

254. The method of any one of claims 1-253, wherein each dose is about 0. 1 mg/kg.

255. The method of any one of claims 1-253, wherein each dose is about 0.25 mg/kg.

256. The method of any one of claims 1-253, wherein each dose is about 0.5 mg/kg.

257. The method of any one of claims 1-253, wherein each dose is about 1.0 mg/kg.

258. The method of any one of claims 1-253, wherein each dose is about 1.5 mg/kg.

259. The method of any one of claims 1-253, wherein each dose is about 2.0 mg/kg.

260. The method of any one of claims 1-253, wherein each dose is about 2.5 mg/kg.

261. The method of any one of claims 1-253, wherein each dose is about 3.0 mg/kg.

262. The method of any one of claims 1-253, wherein each dose is about 4.0 mg/kg.

263. The method of any one of claims 1-253, wherein each dose is about 5.0 mg/kg.

264. The method of any one of claims 1-263, wherein each dose comprises the same amount of the LNP.

265. The method of any one of claims 1-264, wherein each dose comprises a different amount of the LNP.

266. The method of any one of claims 1-265, wherein the subject is a human.

267. The method of any one of claims 1-266, wherein the disease, disorder or condition is related to abnormal expression of a gene product.

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268. The method of any one of claims 1-267, wherein the disease, disorder, or condition is a blood disease, disorder, or condition.

269. The method of any one of claims 1-268 wherein the disease, disorder, or condition is an autoimmune disease, disorder, or condition.

270. The method of claim 269, wherein the autoimmune disease, disorder, or condition is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, type I diabetes, systemic lupus erythematosus, psoriasis, psoriatic arthritis, Sjogren’s syndrome, Crohn’s disease, Celiac disease, ulcerative colitis, Graves’ disease, Hashimoto’s thyroiditis, Addison’s disease, dermatomyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome, myasthenia gravis, autoimmune vasculitis, pernicious anemia, vitiligo, systemic sclerosis, scleroderma, diffuse scleroderma, limited scleroderma, linear scleroderma, localized scleroderma, hemolytic anemia, inflammatory bowel disease (IBD), achantholysis, acute disseminated encephalomyelitis, adult-onset Still disease, allergic glomerulonephritis, ANCA associated vasculitis, ankylosing spondylitis, anti- glomerular basement membrane disease, anti-N-methyl-D-aspartate receptor encephalitis, antiphospholipid syndrome, antisynthetase syndrome, autoimmune gastritis, autoimmune hypophysitis, autoimmune liver disease, autoimmune lung disease, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, autoimmune skin disease, autoimmune thyroiditis, autoimmune uveitis, Behcet diseae, bullous pemphigoid, Churg Strauss syndrome, dermatitis herpetiformis, endocrine ophthalmopathy, erythematous pemphigus, Felty syndrome, Giant cell arteritis, IgA glomerulonephritis, membranous glomerulonephritis, Goodpasture syndrome, Granulomatosis with polyangiitis, Graves’ ophthalmopathy, Hailey Hailey syndrome, heparin induced thrombocytopenia, autoimmune hepatitis, idiopathic hypoparathryroidism, idiopathic thrombocytopenic purpura, IgA pemphigus, immune complex nephritis, immunoglobulin A nephropathy juvenile rheumatoid arthritis, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, linear IgA bullous dermatosis, lupus nephritis, lupus vasculitis, microscopic polyangiitis, morphea, mucous membrane phemphigoid, myasthenia gravis, nonarticular rheumatism, sympathetic ophthalmia, opsoclonous myoclonus syndrome, paraneoplastic pemphigus, pemphigoid, pemphigoid gestationis, pemphigus, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, autoimmune polyendocrinopathies, polyradiculoneuropathy, postpartum thyroiditis, primary biliary cirrhosis, rheumatic disease, rheumatic heart disease, rheumatoid polymyalgia, rheumatoid nodule, rheumatoid vasculitis, stiff-person syndrome, CREST syndrome, systemic juvenile idiopathic arthritis, sarcoidosis, and uveomeningoencephalitic syndrome.

261. The method of any one of claims 1-270, wherein the disease, disorder, or condition is a genetic disease or disorder.

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272. The method of claim 271, wherein the genetic disease or disorder is selected from the group consisting of sickle cell disease, melanoma; hemophilia A (clotting factor VIII (FVIII) deficiency), hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR defect), familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson’s disease, phenylketonuria (PKU), congenital hepatic porphyria, an inherited disorder of hepatic metabolism; Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, a mucopolysaccharide storage disease, Niemann- Pick disease, Fabry disease, Schindler disease, GM2 -gangliosidosis Type II (Sandhoff Disease), Tay- Sachs disease, Metachromatic Leukodystrophy, Krabbe disease, mucolipidosis (ML), Sialidosis Type II, a glycogen storage disease (GSD), Gaucher disease, cystinosis, Batten disease, Aspartylglucosaminuria, Salla disease, Danon disease (LAMP -2 deficiency), Lysosomal Acid Lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (NCL), sphingolipidoses, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, spinocerebellar ataxia, spinal muscular atrophy (SMA), Friedreich’s ataxia, Duchenne muscular dystrophy (DMD), a Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber Congenital Amaurosis, Stargardt disease, wet macular degeneration (wet AMD), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha- 1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC), and Cathepsin A deficiency.

273. The method of any one of claims 1-16, wherein the method provides anti -tumor immunity to a subject in need thereof.

74. The method of any one of claims 1-158 or 273, wherein the disease, disorder or condition is associated with an elevated expression of a tumor antigen.

275. The method of any one of claims 1-274, wherein the LNP further comprises a third lipid- anchored polymer, wherein the third lipid-anchored polymer comprises:

(i) a third lipid moiety comprising at least one hydrophobic tail;

(ii) a third polymer;

(iii) an optional third linker, wherein the third polymer is conjugated to the third lipid moiety via the third linker; and

(iv) a reactive species conjugated to the third polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety.

276. The method of claim 275, wherein the third polymer is polyglycerol (PG) or a PG derivative.

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277. The method of claims 258 or claim 276, further comprising a targeting moiety conjugated to the third polymer via the reactive moiety.

278. The method of any one of claims 275-277, wherein the targeting moiety conjugated to the third polymer is different from the targeting moiety conjugated to the second polymer.

279. The method of any one of claims 275-278, wherein the third lipid-anchored polymer is different from the second lipid-anchored polymer.

280. The method of any one of claims 275-279, wherein the targeting moiety conjugated to the third polymer and the targeting moiety conjugated to the second polymer bind to different antigens on the same tissue or cell type.

281. A method of synthesizing a linker-conjugated dioctadecylamine (DODA-1) of the following structure:

(DODA-1), said method comprising:

(DODA-A1);

(DODA-A2); and

(c) reacting DODA-A2 with a reducing agent to produce DODA-1.

282. The method of claim 281, wherein in (a) the base is N,N-Diisopropylethylamine (DIPEA).

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283. The method of claim 264 or claim 265, wherein in (b) the coupling reagent is l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI) .

284. The method of any one of claims 281-283, wherein in (b) the catalyst is 4- (dimethylamino)pyridine (DMAP).

285. The method of any one of claims 281-284, wherein in (b) the base is diisopropylethylamine (DIPEA).

286. The method of any one of claims 281-285, wherein in (c) the reducing agent is sodium borohydride.

287. The method of any one of claims 281-286, further comprising:

(dl) reacting DODA-1 with 2,3-epoxy-l-(l-ethoxyethoxy)propane (EEGE) in the presence of a base or an organocatalyst under argon atmosphere to produce DODA conjugated to a linker and polymerized EEGE (intermediate DODA-PG-OH) of the following structure:

(DODA-PG-OH), wherein n is an integer from 8 to 100; and

(DODA-PG).

288. The method of claim 287, wherein in (dl) the base is a phosphazene base.

289. The method of claim 288, wherein the base is P4-t-Bu.

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290. The method of claim 287, wherein in (dl) the organocatalyst is N-heterocyclic carbene (NHC) or an N-heterocyclic olefin (NHO).

291. The method of any one of claims 287-290, wherein in (el) the acidic conditions comprise a strong acid.

292. The method of claim 291, wherein the strong acid is selected from the group consisting of hydrochloric acid (HC1), hydrobromic acid (HBr), hydroiodic acid (HI), perchloric acid (HCIO4), chloric acid (HCIO3), sulfuric acid (H2SO4), and nitric acid (HNO3).

293. The method of claim 292, wherein the strong acid is HC1.

294. The method of any one of claims 281-293, further comprising:

(DODA-PG-OH), wherein n is an integer from 8 to 100; and

(DODA-PG-L1);

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(DODA-PG-L2); and

(DODA-PG-Maleimide) .

295. The method of claim 294, wherein in (d2) the base is a phosphazene base.

296. The method of claim 295, wherein the base is P4-t-Bu.

297. The method of claim 294, wherein in (d2) the organocatalyst is N-heterocyclic carbene

(NHC) or an N-heterocyclic olefin (NHO).

298. The method of any one of claims 294-297, wherein in (e2) the reducing agent is sodium hydride.

299. The method of any one of claims 294-298, wherein in (f2) the acidic conditions comprise a strong acid.

290. The method of claim 299, wherein the strong acid is selected from the group consisting of hydrochloric acid (HC1), hydrobromic acid (HBr), hydroiodic acid (HI), perchloric acid (HCIO4), chloric acid (HCIO3), sulfuric acid (H2SO4), and nitric acid (HNO3).

301. The method of claim 290, wherein the strong acid is HC1.

302. The method of any one of claims 294-301, wherein in (g2) the base is triethylamine.

250

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