BR122025021657A2 - Scalable methods for producing recombinant adeno-associated viral vector (AAV) in a serum-free cell suspension culture system suitable for clinical use. - Google Patents

Abstract

Methods and compositions for transfecting cells with plasmids are disclosed. In certain embodiments, methods and compositions are described in which transfection efficiency is significantly increased by contacting the cells being transduced with polyethyleneimine (PEI) that is nucleic acid-free during the transfection process. Therapeutically useful adeno-associated viral vectors generated according to the disclosed methods and compositions are also disclosed.

Description

Related Orders

[0001] This patent application claims the benefit of U.S. Patent Application No. 62/261,815, filed December 1, 2015, which is expressly incorporated herein by reference in its entirety.

Field of invention

[0002] This invention relates to the fields of cell transduction (transfection) with nucleic acid, for example, plasmids. More particularly, the invention provides compositions and methods for producing transduced cells, said cells optionally producing Adeno-Associated Viral Vector (AAV).

Introduction

[0003] Several publications and patent documents are cited throughout the descriptive report to describe the state of the art to which this invention belongs. Each of these citations is incorporated herein by reference as if presented in full.

Summary

[0004] The invention provides nucleic acid (plasmid) compositions, such as a nucleic acid encoding a protein or transcribed into a transcript of interest, and polyethyleneimine (PEI), optionally, in combination with cells. In one embodiment, a composition includes a plasmid/PEI mixture, which has a set of components: (a) one or more plasmids comprising nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding auxiliary proteins; (b) a plasmid comprising a nucleic acid encoding a protein or transcribed into a transcript of interest; and (c) a polyethyleneimine (PEI) solution. In particular aspects, the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or are in a molar ratio range of about 100:1 to about 1:0.01, and the mixture of components (a), (b) and (c) was optionally incubated for a period of time of about 10 seconds to about 4 hours.

[0005] In other embodiments, the nucleic acid (plasmids) and polyethyleneimine (PEI) compositions additionally comprise cells. In particular aspects, the cells are in contact with the plasmid/PEI mixture of components (a), (b) and/or (c).

[0006] In additional embodiments, the nucleic acid (plasmid) and polyethyleneimine (PEI) compositions, optionally in combination with cells, further comprise Free PEI. In particular aspects, the cells are in contact with the Free PEI.

[0007] In several other embodiments, the cells were in contact with the mixture of components (a), (b) and/or (c) for at least about 4 hours, or from about 4 hours to about 140 hours, or from about 4 hours to about 96 hours. In particular aspects, the cells were in contact with the mixture of components (a), (b) and/or (c) and, optionally, Free PEI, for at least about 4 hours.

[0008] The compositions of the invention may be present in a container. In particular aspects, a container is a flask, plate, bag or bioreactor, and is optionally sterile, and/or the container is optionally suitable for maintaining cell viability or growth.

[0009] The plasmids of the compositions and methods of the invention include, inter alia, nucleic acids encoding viral proteins, such as AAV capsid proteins. Such plasmids and cells may be in contact with free PEI. In particular aspects, the plasmids and/or cells have been in contact with free PEI for at least about 4 hours, or from about 4 hours to about 140 hours, or from about 4 hours to about 96 hours.

[0010] Methods are also provided for producing transfected cells, which include providing a plasmid; providing a solution comprising polyethyleneimine (PEI); and mixing the nucleic acid (plasmid) with the PEI solution to produce a plasmid/PEI mixture. In particular aspects, such mixtures are incubated for a period ranging from about 10 seconds to about 4 hours. In such methods, cells are then brought into contact with the plasmid/PEI mixture to produce a plasmid/PEI cell culture; then free PEI is added to the nucleic acid/PEI cell culture produced to produce a PEI/plasmid/free PEI cell culture; and then the produced PEI/plasmid/free PEI cell culture is incubated for at least about 4 hours, thereby producing transfected cells. In particular aspects, the plasmid comprises a nucleic acid that encodes a protein or is transcribed into a transcript of interest.

[0011] Methods are further provided for producing transfected cells that produce recombinant AAV vector, which include providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper proteins; providing a plasmid comprising a nucleic acid encoding a protein or transcribed into a transcript of interest; providing a solution comprising polyethyleneimine (PEI); mixing the plasmids mentioned above with the PEI solution, wherein the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or are in a molar ratio range of about 100:1 to about 1:0.01, to produce a plasmid/PEI mixture (and optionally incubating the plasmid/PEI mixture for a period in the range of about 10 seconds to about 4 hours); Contact the cells with the plasmid/PEI mixture to produce a plasmid/PEI cell culture; add free PEI to the plasmid/PEI cell culture produced to produce a free PEI/plasmid/PEI cell culture; and incubate the free PEI/plasmid/PEI cell culture for at least about 4 hours, thereby producing transfected cells that produce recombinant AAV vector comprising a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0012] Methods are additionally provided for producing recombinant AAV vector comprising nucleic acid encoding a protein or transcribed into a transcript of interest, which includes providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper proteins; providing a plasmid comprising a nucleic acid encoding a protein or transcribed into a transcript of interest; providing a solution comprising polyethyleneimine (PEI); Mix the plasmids mentioned above with the PEI solution, wherein the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or are in a molar ratio range of about 100:1 to about 1:0.01, to produce a plasmid/PEI mixture (and optionally incubate the plasmid/PEI mixture for a period of time in the range of about 10 seconds to about 4 hours); contact the cells with the plasmid/PEI mixture produced as described to produce a plasmid/PEI cell culture; add free PEI to the plasmid/PEI cell culture produced to produce a PEI/plasmid/free PEI cell culture; incubate the plasmid/PEI cell culture or the PEI/plasmid/free PEI cell culture produced for at least about 4 hours to produce transfected cells; Harvest the transfected cells produced and/or the culture medium from the transfected cells produced to produce a cell and/or culture medium harvest; and isolate and/or purify recombinant AAV vector from the cell and/or culture medium produced, thereby producing recombinant AAV vector comprising a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0013] Methods are further provided for producing transfected cells that produce recombinant AAV vector with a nucleic acid that encodes a protein or is transcribed into a transcript of interest. In one embodiment, a method includes providing a mixture of components (i) one or more plasmids comprising nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper proteins, (ii) a plasmid comprising a nucleic acid that encodes a protein or is transcribed into a transcript of interest; and (iii) a polyethyleneimine (PEI) solution; Mix plasmids (i) and (ii) with PEI solution (iii) so that the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or in a molar ratio range of about 100:1 to about 1:0.01, to produce a plasmid/PEI mixture (and optionally incubate the plasmid/PEI mixture for a period of time in the range of about 10 seconds to about 4 hours); contact cells with the plasmid/PEI mixture produced to produce a plasmid/PEI cell culture; add Free PEI to the plasmid/PEI cell culture to produce a PEI/plasmid/Free PEI cell culture; and incubate the plasmid/PEI cell culture or the PEI/plasmid/free PEI cell culture for at least about 4 hours to produce transfected cells that produce recombinant AAV vector comprising a nucleic acid that encodes a protein or is transcribed into a transcript of interest.

[0014] The methods and compositions of the invention may include one or more steps or features. An exemplary step or feature includes, but is not limited to, a step of harvesting the transfected cells produced and/or harvesting the culture medium from the transfected cells produced to produce a cell harvest and/or culture medium. A further exemplary step or feature includes, but is not limited to, isolating and/or purifying the recombinant AAV vector from the cell harvest and/or culture medium, thereby producing recombinant AAV vector comprising a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0015] In addition, methods are provided for producing recombinant AAV vector that includes a nucleic acid encoding a protein or transcribed into a transcript of interest. In one embodiment, a method includes providing a mixture of components (i) one or more plasmids comprising nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper proteins, (ii) a plasmid comprising a nucleic acid encoding a protein or transcribed into a transcript of interest; and (iii) a polyethyleneimine (PEI) solution; Mix plasmids (i) and (ii) with PEI solution (iii) so that the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or are in a molar ratio range of about 100:1 to about 1:0.01, to produce a plasmid/PEI mixture (and optionally incubate the plasmid/PEI mixture for a period of time of about 10 seconds to about 4 hours); contact cells with the plasmid/PEI mixture produced to produce a plasmid/PEI cell culture; add Free PEI to the plasmid/PEI cell culture produced to produce a PEI/plasmid/Free PEI cell culture; incubate the plasmid/PEI cell culture or the PEI/plasmid/Free PEI cell culture for at least about 4 hours to produce transfected cells; Harvest the transfected cells produced and/or the culture medium from the transfected cells produced to produce a cell and/or culture medium harvest; and isolate and/or purify recombinant AAV vector from the cell and/or culture medium produced, thereby producing recombinant AAV vector comprising a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0016] In addition, methods are provided for producing recombinant AAV vector that includes a nucleic acid encoding a protein or transcribed into a transcript of interest. In one embodiment, a method includes providing a mixture of components (i) one or more plasmids comprising nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper proteins; (ii) a plasmid comprising a nucleic acid encoding a protein or transcribed into a transcript of interest; and (iii) a polyethyleneimine (PEI) solution; wherein plasmids (i) and (ii) are in a molar ratio range of about 1:0.01 to about 1:100, or are in a molar ratio range of about 100:1 to about 1:0.01, and wherein the mixture of components (i), (ii) and (iii) has optionally been incubated for a period of time of about 10 seconds to about 4 hours; contact the cells with the mixture produced to produce a plasmid/PEI cell culture; add PEI-free to the plasmid/PEI cell culture produced to produce a PEI/plasmid/PEI-free cell culture; incubate the plasmid/PEI cell culture or the PEI/plasmid/PEI-free cell culture for at least about 4 hours to produce transfected cells; Harvest the transfected cells produced and/or the culture medium from the transfected cells produced to produce a cell and/or culture medium harvest; and isolate and/or purify recombinant AAV vector from the cell and/or culture medium produced, thereby producing recombinant AAV vector comprising a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0017] Compositions and methods may also include one or more additional steps or features. Such steps or features include, but are not limited to: where the plasmid/PEI cell culture, or the PEI/plasmid/Free PEI cell culture, or the nucleic acid/PEI cell culture is incubated for a period of time in the range of about 4 hours to about 140 hours, or incubated for a period of time in the range of about 4 hours to about 96 hours. Such steps or features include, but are not limited to: where the plasmid/PEI mixture has a PEI/plasmid weight ratio in the range of about 0.1:1 to about 5:1, or has a PEI/plasmid weight ratio in the range of about 5:1 to about 1:1; or wherein the PEI/plasmid/PEI-free cell culture has a PEI/plasmid weight ratio in the range of about 0.1:1 to about 5:1 or has a PEI/plasmid weight ratio in the range of about 5:1 to about 0.1:1. Such steps or features include, but are not limited to: where the plasmid/PEI mixture has a PEI/plasmid weight ratio in the range of about 1:1 to about 5:1, or has a PEI/plasmid weight ratio in the range of about 5:1 to about 1:1, or wherein the PEI/plasmid/PEI-free cell culture has a PEI/plasmid weight ratio in the range of about 1:1 to about 5:1 or has a PEI/plasmid weight ratio in the range of about 5:1 to about 1:1.

[0018] Forms of PEI (Free PEI, Total PEI, Plasmid/PEI Mixture, or Cells Contacted with Plasmid/PEI Mixture) applicable in the compositions and methods of the invention include a hydrolyzed linear polyethyleneimine. In particular aspects, the PEI (Free PEI, Total PEI, Plasmid/PEI Mixture, or Cells Contacted with Plasmid/PEI Mixture) comprises a hydrolyzed linear polyethyleneimine with a molecular weight in the range of about 4,000 to about 160,000 and/or in the molecular weight range of about 2,500 to about 250,000 in freebase form, or a hydrolyzed linear polyethyleneimine with a molecular weight of about 40,000 and/or about 25,000 in freebase form.

[0019] In several embodiments, the molar ratio between nitrogen (N) in Total PEI and phosphate (P) in the plasmid ranges from about 1:1 to about 50:1 (N:P) in PEI/plasmid/Free PEI cell culture. In several embodiments, the molar ratio between nitrogen (N) in Total PEI and phosphate (P) in the plasmid is about 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 (N:P) in PEI/plasmid/Free PEI cell culture.

[0020] Compositions and methods according to the invention may have plasmid/PEI mixtures incubated for a period of time. In particular aspects, the incubation is in the range of about 30 seconds to about 4 hours. In more particular aspects, the incubation of the plasmid/PEI mixture is in the range of about 1 minute to about 30 minutes.

[0021] Compositions and methods according to the invention may have PEI in various percentage amounts, both by molar ratio and by weight (mass). In particular embodiments, the amount of Free PEI is in the range of about 10% to about 90% of the Total PEI, or the amount of Free PEI is in the range of about 25% to about 75% of the Total PEI, or the amount of free PEI is about 50% of the Total PEI.

[0022] Compositions and methods according to the invention may have PEI added to plasmids and/or cells at various time points. In particular embodiments, free PEI is added to cells before, at the same time as, or after the plasmid/PEI mixture is contacted with the cells.

[0023] The compositions and methods according to the invention include mammalian cells (e.g., HEK 293E or HEK 293F cells). Such cells may be adherent or in suspension culture. In particular aspects, the cells are cultured or maintained in a serum-free culture medium.

[0024] The compositions and methods according to the invention may have cells at particular densities and/or cell growth phases and/or viability. In particular embodiments, the cells are at a density in the range of about 1 x 10⁵ cells/mL to about 1 x 10⁸ cells/mL when contacted with the plasmid/PEI mixture and/or when contacted with free PEI. In further particular embodiments, the viability of the cells when contacted with the plasmid/PEI mixture or with free PEI is about 60% or greater than 60%, or the cells are in log-phase growth when contacted with the plasmid/PEI mixture, or the viability of the cells when contacted with the plasmid/PEI mixture or with free PEI is about 90% or greater than 90%, or the cells are in log-phase growth when contacted with the plasmid/PEI mixture or with free PEI.

[0025] AAV-encoded packaging proteins include, for example, AAV rep and/or AAV cap. Such AAV packaging proteins include, for example, AAV rep and/or AAV cap proteins of any AAV serotype.

[0026] Encoded helper proteins include, for example, adenovirus E2 and/or E4, VARNA proteins and/or non-AAV helper proteins.

[0027] Compositions and methods according to the invention may have nucleic acid (plasmids) in particular amounts or ratios. In particular embodiments, the total amount of plasmid comprising the nucleic acid encoding a protein or transcribed into a transcript of interest and the one or more plasmids comprising nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding auxiliary proteins is in the range of about 0.1 μg to about 15 μg per mL of cells. In further particular embodiments, the molar ratio of the plasmid comprising the nucleic acid encoding a protein or transcribed into a transcript of interest to the one or more plasmids comprising nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding auxiliary proteins is in the range of about 1:5 to about 1:1 or is in the range of about 1:1 to about 5:1.

[0028] Plasmids may contain nucleic acids in different or the same plasmids. In one embodiment, a first plasmid comprises nucleic acids encoding AAV packaging proteins, and a second plasmid comprises nucleic acids encoding auxiliary proteins. In more particular embodiments, the molar ratio of the plasmid comprising the nucleic acid encoding a protein or transcribed into a transcript of interest to a first plasmid comprising nucleic acids encoding AAV packaging proteins to a second plasmid comprising nucleic acids encoding auxiliary proteins is in the range of about 1-5:1:1 or 1:1-5:1 or 1:1:1-5.

[0029] Compositions and method according to the invention include AAV vectors of any serotype, or a variant thereof. In one embodiment, a recombinant AAV vector comprises any of 1-12 AAV serotypes, an AAV VP1, VP2 and/or VP3 capsid protein, or a modified or variant AAV VP1, VP2 and/or VP3 capsid protein, or a wild-type AAV VP1, VP2 and/or VP3 capsid protein. In further particular embodiments, an AAV vector comprises an AAV serotype or an AAV pseudotype, wherein the AAV pseudotype comprises an AAV capsid serotype other than an ITR serotype.

[0030] Compositions and methods according to the invention that provide or include AAV vectors may also include other elements. Examples of such elements include, but are not limited to: an intron, an expression control element, one or more adeno-associated virus (AAV) terminal inverted repeats (ITRs), and/or a filler polynucleotide sequence. Such elements may be within or flank the nucleic acid encoding a protein or transcribed into a transcript of interest, or the expression control element may be operationally linked to the nucleic acid encoding a protein or transcribed into a transcript of interest, or the AAV ITR(s) may flank the 5’ or 3’ terminus of the nucleic acid encoding a protein or transcribed into a transcript of interest, or the filler polynucleotide sequence may flank the 5’ or 3’ terminus of the nucleic acid encoding a protein or transcribed into a transcript of interest.

[0031] Expression control elements include constitutive or regulable control elements, such as a promoter or tissue-specific expression control element (e.g., that provides expression in the liver).

[0032] ITRs can be any of the following: AAV2 or AAV6 serotypes, or a combination thereof. AAV vectors can include any VP1, VP2, and/or VP3 capsid protein having 75% or more sequence identity with any of the VP1, VP2, and/or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, or AAV-2i8, or comprise a modified or variant VP1, VP2, and/or VP3 capsid protein selected from any of the: AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, and AAV-2i8.

[0033] In the compositions and methods of the invention, cells can be subcultured, such as reduced cell density by dilution or removal of cells from the culture. In one embodiment, cells are subcultured to a reduced cell density before contact with the plasmid/PEI mixture.

[0034] In the compositions and methods of the invention, the cells can be used at various densities. In one embodiment, the cells are cultured or subcultured to a cell density in the range of about 0.1 x 106 cells/ml to about 5.0 cells x 106 cells/ml before contact with the plasmid/PEI mixture.

[0035] In the compositions and methods of the invention, cells can be placed in contact with PEI (Free PEI, Total PEI, Plasmid/PEI mixture) for a short or long period of time. In one embodiment, the cells are contacted with the plasmid/PEI mixture between 2 and 5 days after subculture. In another embodiment, the cells are contacted with the plasmid/PEI mixture between 3 and 4 days after subculture.

[0036] The compositions and methods of the invention provide improved cell transfection efficiency and/or recombinant vector production per cell. In one embodiment, the amount of plasmid introduced into transfected cells is at least 50% greater with the step of adding free PEI to the plasmid/PEI cell culture compared to without adding free PEI to the plasmid/PEI cell culture. In another embodiment, the amount of recombinant AAV vector produced is at least 50% or greater with the step of adding free PEI to the plasmid/PEI cell culture compared to without adding free PEI to the plasmid/PEI cell culture. In a further embodiment, the amount of recombinant AAV vector produced is 1-5, 5-10 or 10-20 times greater with the step of adding free PEI to the plasmid/PEI cell culture compared to without adding free PEI to the plasmid/PEI cell culture.

Description of the Drawings

[0037] Figure 1 shows the transfection efficiency of 40 kDa PEI “Max” (A) and 25 kDa PEI (B) dissolved in both TrisHCl and H2O when using plasmid DNA at 2.8, 5.6, and 11.2 μg/mL. The 40 kDa PEI “Max” showed a consistently higher transfection efficiency compared to the 25 kDa PEI.

[0038] Figure 2A-2B shows the effect of free PEI on transfection efficiency and rAAV vector production in 12-well plates. A) Transfection of 293F cells with three plasmids (pAAV-eGFP-WRPE, pAAV-Rep2/Cap2, pAD2-Helper) in serum-free suspension culture in 12-well plates. B) Effect of free PEI on rAAV titer. The PEI/DNA weight ratio was 2:1 or 4:1 with or without the addition of free PEI in the transfection. A DNA quantity of 2.8 μg/mL with a molar ratio of 1:1:1 was used for three plasmids. The diluted PEI was first mixed with diluted DNA at a 1:1 weight ratio to form the complexes. The excess PEI was diluted in 50 μL of culture medium and then added directly to the cells.

[0039] Figure 3A-3B shows the effect of free PEI on transfection efficiency and rAAV titer in shake flasks. A) Transfection efficiency was increased when using free PEI. B) Effect of free PEI on rAAV titer. The PEI/DNA weight ratio was 2:1 and the amount of DNA was 2.8 μg/mL. 1/2 and 1/3 of the amount of PEI was used as free PEI in the transfection.

[0040] Figure 4 shows the growth curve of 293F cells and viability in the bioreactor. Cells were seeded at 0.25x10⁶ cells/mL (Unit 1), 0.35x10⁶ cells/mL (Unit 2), and 0.5x10⁶ cells/mL (Unit 3). Cell density (N) and viability (V) were recorded every 12 hours for 7 days of cell culture in the bioreactor.

[0041] Figure 5A-5B shows cell transfection in the bioreactor. A) 293F cells transfected with three plasmids for up to 72h. GFP-positive cells were detected using an inverted fluorescence microscope. B) Transfection efficiency was measured using flow cytometry. 50%-60% of GFP-positive cells were detected 48h-72h post-transfection. Transfection efficiency was similar between transfection on day 3 and transfection on day 4.

[0042] Figure 6A-6B shows the rAAV titer and vector function assay. A) The vector titer was significantly higher on day 4 of transfection than on day 3 as assessed by qPCR. The highest titer was 1.38E+11 vg/mL under transfection conditions on day 4 and a stirring speed of 150 rpm. B) Vector function was measured by transduction assay. The same volume of cell lysates was added to HEK 293 cells. GFP-positive cells were detected with inverted fluorescence microscopy. The transduction results were consistent with the rAAV titer, with higher titers correlating with a higher transduction rate.

Detailed description

[0043] Compositions and methods for transducing cells with a molecule, such as a nucleic acid (e.g., plasmid), with high efficiency are disclosed herein. These highly efficient transduced cells, when transduced with a nucleic acid encoding a protein or comprising a sequence that is transcribed into a transcript of interest, can produce protein and/or transcript with high efficiency. Additionally, such cells, when transduced with sequences such as plasmids encoding viral packaging proteins and/or helper proteins, can produce recombinant vectors that include the nucleic acid encoding a protein or comprising a sequence that is transcribed into a transcript of interest, which in turn produces recombinant viral vectors with high yield.

[0044] The invention provides a production platform for cell transduction and/or viral vector (e.g., AAV) that includes features that distinguish it from current ‘industry standard’ viral vector (e.g., AAV) production processes. The compositions and methods of the invention are characterized by mixing PEI with nucleic acids under certain conditions. Mixing PEI with nucleic acids results in efficient PEI-induced compaction of nucleic acids to form stable complexes called polyplexes. The method of introducing nucleic acids into cells comprises supplying nucleic acids mixed with PEI under certain conditions and applying the resulting mixture to the cells. Furthermore, the compositions and methods of the invention are characterized by contacting cells with free PEI, or contacting cells with free PEI, in a particular sequence relative to the step of applying the PEI/nucleic acid mixture to the cells. The compositions and methods of the invention are characterized by: 1) high-efficiency cell transduction/transfection of nucleic acids; 3) a unique combination of reagents and process steps that confer unexpected substantial vector yield; and 4) a modular platform that can be used for the production of different AAV serotype/capsid variants.

[0045] The terms “nucleic acid” and “polynucleotide” are used interchangeably to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids and polynucleotides include genomic DNA, cDNA and antisense DNA and processed or unprocessed mRNA, tRNA and inhibitory DNA or RNAi (e.g., small or short hairpin RNA (sh), microRNA (mRNA), small or short interfering RNA (si), trans-splicing RNA or antisense RNA). Nucleic acids and polynucleotides include naturally occurring, synthetic, and intentionally modified or altered sequences (e.g., variant nucleic acid).

[0046] A nucleic acid or plasmid can also refer to a sequence that codes for a protein. Such proteins can be wild-type or a variant, modified, or chimeric protein. A “variant protein” can mean a protein modified in such a way that the modified protein has an amino acid change compared to the wild-type protein.

[0047] Proteins encoded by a nucleic acid or plasmid include therapeutic proteins. Non-limiting examples include a blood clotting factor (e.g., Factor XIII, Factor IX, Factor X, Factor VIII, Factor VIIa, or protein C), CFTR (cystic fibrosis transmembrane regulatory protein), a 65 kDa retinal pigment epithelium-specific antibody (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfonamide, an enzyme involved in lysosomal storage disease (RSD), hypoxanthine guanine phosphoribosyltransferase, β-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, a hormone, a growth factor (e.g., insulin-like growth factors 1 and 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor -3 and -4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factor α and β, etc.), a cytokine (e.g., α-interferon, β-interferon, interferon-Y, interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), a suicide gene product (e.g., herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, etc.), a drug resistance protein (e.g., providing resistance to a drug used in cancer therapy), a tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), a peptide with immunomodulatory properties, a tolerogenic or immunogenic peptide or protein, Tregitopos, or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (Choroideremia), LCA 5 (LCA-Lebercillin), ornithine ketoacid aminotransferase (Gyrate Atrophy), Retinoschisis 1 (X-linked Retinoschisis), USH1C (Usher Syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR forms of RP: retinitis pigmentosa), DFNB1 (Connexin 26 deafness), ACHM 2, 3 and 4 (Achromatopsia), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, gene deficiencies causing lysosomal storage diseases (e.g., sulfatases, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activating proteins, etc.), one or more zinc finger nucleases for genome editing, or donor sequences used as repair templates for genome editing.

[0048] A nucleic acid or plasmid may also refer to a sequence that produces a transcript when transcribed. Such transcripts may be RNA, such as inhibitory RNA (RNAi, for example, small or short hairpin RNA (sh), mRNA (mRNA), small or short interfering RNA (si), transprocessing RNA or antisense RNA).

[0049] Non-limiting examples include inhibitory nucleic acids that inhibit the expression of: huntingtin gene (HTT), a gene associated with dentatorubro-pallidoluysian atrophy (e.g., atrophin 1, ATN1); X-chromosome androgen receptor in spinobulbar muscular atrophy, Human ataxin-1, -2, -3 and -7, voltage-dependent calcium channel Cav2,1P/Q is encoded by TATA-binding protein (CACNA1A), Ataxin 8 opposite-chain, also known as ATXN8OS, Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, 17), FMR1 (fragile X mental retardation 1) in fragile X syndrome, FMR1 (fragile X mental retardation 1) in tremor/ataxia associated with fragile X syndrome, FMR1 (fragile X mental retardation 2) or AF4/FMR2 family member 2 in fragile XE mental retardation; Myotonin-protein kinase (MT-PK) in myotonic dystrophy; Frataxin in Friedreich's ataxia; a mutant of the superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; a gene involved in the pathogenesis of Parkinson's disease and/or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, human immunodeficiency virus transactivator gene transcription, in HIV infection; HIV TAR, human immunodeficiency virus transactivator response element gene, in HIV infection; C-C chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection; liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute kidney injury or delayed renal transplant graft function or acute renal failure from kidney injury; protein kinase N3 (PKN3) preceding recurrent or metastatic solid malignancies; LMP2, also known as proteasome subunit beta type 9 (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta type 8 (PSMB 8), metastatic melanoma; MECL1, also known as proteasome subunit beta type 10 (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; protein kinesin axis in solid tumors, B-cell CLL/lymphoma (BCL-2); apoptosis suppressor in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; Pole-type kinase 1 (PLK1) in liver tumors; diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C; beta-catenin in familial adenomatous polyposis; beta2-adrenergic receptor in glaucoma; RTP801/Redd1, also known as damage-inducible transcriptase 4 (DAN), in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor 1 (VEGFR1) in age-related macular degeneration or choroidal neovascularization; caspase 2 in non-arteritic ischemic optic neuropathy; mutant keratin 6A N17K protein in congenital pachyonychia; influenza A virus gene/genome in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome/gene sequences; respiratory syncytial virus gene/genome sequences in respiratory syncytial virus infection; Ebola filovirus gene/genome sequence in Ebola infection; hepatitis B and C virus gene/genome sequences in hepatitis B and C infection; herpes simplex virus (HSV) genome/gene sequences in HSV infection; coxsackievirus B3 genome/gene sequences in coxsackievirus B3 infection; silencing of a pathogenic allele of a gene (allele-specific silencing) such as torsin A (TOR1A) in primary dystonia, pan-class I and HLA-specific allele in transplantation; mutant rhodopsin (RHO) gene in autosomal dominant hereditary retinitis pigmentosa (adRP); or the inhibitory nucleic acid binds to a transcript of any of the above genes or sequences.

[0050] Nucleic acids (plasmids) can be single, double, or triple, linear or circular, and can be of any length. When discussing nucleic acids (plasmids), a sequence or structure of a particular polynucleotide can be described here according to the convention of giving the sequence in the 5’ to 3’ direction.

[0051] A “plasmid” is a form of nucleic acid or polynucleotide that typically possesses additional elements for expression (e.g., transcription, replication, etc.) or propagation (replication) of the plasmid. A plasmid as used herein may also be used to reference such nucleic acid or polynucleotide sequences. Accordingly, in all respects the compositions and methods of the invention are applicable to nucleic acids and polynucleotides, for example, to introduce nucleic acid or polynucleotide into cells, to transduce (transfect) cells with nucleic acid or polynucleotide, to produce transduced (transfected) cells that have a nucleic acid or polynucleotide, to produce cells that produce viral vectors (e.g., AAV), to produce viral vectors (e.g., AAV), to produce a cell culture medium that has viral vectors (e.g., AAV), etc.

[0052] Compositions and methods of the invention include polyethyleneimine (PEI). PEI is a cationic polymer and is capable of forming a stable complex with nucleic acid, referred to as a polyplex. While not wishing to be limited to any theory, it is believed that the polyplex is introduced into cells via endocytosis.

[0053] PEI can be linear PEI or branched PEI. PEI can be in salt or free base form. In specific embodiments, PEI is linear PEI, such as optionally hydrolyzed linear PEI. Hydrolyzed PEI can be totally or partially hydrolyzed. Hydrolyzed linear PEI has a higher proportion of free (protonable) nitrogens compared to non-hydrolyzed linear PEI, typically having at least 1-5% more free (protonable) nitrogens compared to non-hydrolyzed linear PEI, more typically having 5-10% more free (protonable) nitrogens compared to non-hydrolyzed linear PEI, or more typically having 10-15% more free (protonable) nitrogens compared to non-hydrolyzed linear PEI.

[0054] In specific embodiments, PEI may have a molecular weight in the range of about 4,000 to about 160,000 and/or in the range of about 2,500 to about 250,000 molecular weight in freebase form. In other particular embodiments, PEI may have a molecular weight of about 40,000 and/or about 25,000 molecular weight in freebase form. Specifically, linear PEI with a molecular weight of about 40,000 and/or about 25,000 molecular weight in freebase form. In addition, chemically modified linear PEI or branched PEI may also be used. PEI is commercially available (e.g., Polysciences, Inc., Warrington, PA, USA).

[0055] In the compositions and methods of the invention, a nucleic acid, such as a plasmid, is mixed with PEI to form a PEI mixture or solution. Such a mixture or solution may be referred to as a “plasmid/PEI mixture” or a “nucleic acid/PEI mixture”. The terms “plasmid/PEI mixture” and “nucleic acid/PEI mixture” therefore mean that the PEI has been mixed with the nucleic acid/plasmid. The PEI as presented herein may therefore be mixed with nucleic acid (plasmid) before or substantially simultaneously with cell contact for transduction.

[0056] As used herein, the term “free PEI” means PEI that is substantially or entirely free of nucleic acid (plasmid). PEI as presented herein may therefore also be in the form of free PEI. The “plasmid/PEI mixture” or “nucleic acid/PEI mixture” is therefore distinct from free PEI. If the free PEI is substantially free, the amount of nucleic acid (plasmid) sequences present shall not exceed 5%, as determined by molecular weight or mass. Obviously, the amount may be less than 5%, for example, about 4.5% or less, about 4% or less, about 3.5% or less, about 3% or less, about 2.5% or less, about 2% or less, about 1.5% or less, about 1% or less, or about 0.5% or less.

[0057] As used herein, the term "Total PEI" means the sum of PEI present in the plasmid/PEI mixture and Free PEI. Total PEI therefore includes PEI that is mixed with the plasmid and PEI that is substantially or entirely free of nucleic acid sequences, such as a plasmid.

[0058] The disclosure of the magnitudes, proportions, compositions, solutions, solvents and buffers of PEI, pH, salts and time and duration of cell contact and incubation applies to any one, two or all three: 1) PEI in a plasmid/PEI mixture or in a nucleic acid/PEI mixture; 2) PEI as free PEI (i.e., PEI that is substantially or totally free of nucleic acid or polynucleotide sequences, such as a plasmid); and 3) total PEI (PEI in a plasmid/PEI mixture or in a nucleic acid/PEI mixture + free PEI).

[0059] In particular embodiments, PEI is a solution, such as aqueous solutions (e.g., water). In further particular embodiments, PEI is acidified or neutralized PEI. The term "acidified PEI" means a PEI solution that is prepared by dissolving PEI in an acidic solvent. The acidity of the acidified PEI solution is typically a pH of about 0 to about 3.0, more typically a pH of about 0.5 to about 2.0. The term "neutralized PEI" means a PEI solution that is prepared by dissolving PEI in a neutral solvent or buffer. Neutralized PEI solutions can have a pH in the range of about 6.0 to about 8.0, typically a pH in the range of about 6.5 to about 7.5, more typically a pH in the range of about 6.8 to about 7.2, and most typically a pH in the range of about 7.0 to about 7.2, for example, about 7.1.

[0060] Any solvent or buffer can be used to establish or maintain the pH of a PEI solution within the aforementioned range without destroying the transfection activity of PEI. Examples of acidic solvents include mineral acids such as hydrochloric acid (HCl), and organic acids with pH in the acidic range such as glycine hydrochloric acid solution. Non-limiting examples of neutral solvents/buffers include Tris (trizm base) and HEPES. Buffers can range from about 1 mM to about 100 mM, more typically from about 2 mM to about 50 mM, and most typically from about 5 mM to about 20 mM.

[0061] PEI solutions may optionally include salts. Non-limiting examples of salts include sodium (Na), potassium (K), and magnesium (Mg) salts. In particular aspects, the salt concentrations of a PEI solution range from about 50 mM to about 500 mM, more typically from about 100 mM to about 250 mM, and most typically from about 125 mM to about 175 mM.

[0062] A mixture of nucleic acids (plasmid) and PEI is achieved by mixing nucleic acids (plasmid) and PEI in a solution. The mixture can occur in any solution compatible with PEI-based cell transduction. Non-limiting examples are as defined herein. After mixing, the nucleic acid (plasmid)/PEI mixture can be incubated for a period of time of about 1 minute to about 8 hours; about 10 seconds to about 4 hours; about 1 minute to about 60 minutes; about 1 minute to about 30 minutes; about 10 minutes to about 45 minutes; about 10 minutes to about 30 minutes; and/or about 20 minutes to about 30 minutes. Typically, the times include about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, and about 30 minutes.

[0063] PEI and nucleic acids (plasmid) are mixed in a ratio that is not limited. Typical ratios include a mixture of plasmids in a molar (or weight) ratio range of about 1:0.01 to about 1:100, or in a molar (or weight) ratio range of about 100:1 to about 1:0.01, to produce plasmid/PEI mixture. More typical molar (or weight) ratios include a mixture of plasmids in a molar (or weight) ratio range of about 1:1 to about 1:5, or in a molar (or weight) ratio range of about 1:2 to about 1:4, to produce plasmid/PEI mixture. In further embodiments, the PEI/plasmid weight ratio is in the range of about 0.1:1 to about 5:1, or in the range of about 5:1 to about 0.1:1. In additional embodiments, the PEI/plasmid/Free PEI cell culture has a PEI/plasmid weight ratio in the range of about 0.1:1 to about 5:1, or has a PEI/plasmid weight ratio in the range of about 5:1 to about 0.1:1. In particular embodiments, the plasmid/PEI mixture has a PEI/plasmid weight ratio in the range of about 1:1 to about 5:1, or in the range of about 5:1 to about 1:1. In other particular embodiments, the PEI/plasmid/Free PEI cell culture has a PEI/plasmid weight ratio in the range of about 1:1 to about 5:1, or in the range of about 5:1 to about 1:1.

[0064] The amount of nucleic acids (plasmid) used to produce cell transduction compositions and methods varies. In particular embodiments, the molar ratio between nitrogen (N) in Total PEI and phosphate (P) in the plasmid is in the range of about 1:1 to about 50:1 (N:P) in PEI/plasmid/Free PEI cell culture, or the molar ratio between nitrogen (N) in Total PEI and phosphate (P) in the plasmid is about 1:1 to 10:1 (N:P) in PEI/plasmid/Free PEI cell culture, or the molar ratio between nitrogen (N) in Total PEI and phosphate (P) in the plasmid is about 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1 (N:P) in PEI/plasmid/Free PEI cell culture. In further particular embodiments, the total amount of plasmid comprising the nucleic acid encoding a protein or transcribed into a transcript of interest and the one or more plasmids comprising nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper proteins is in the range of about 0.1 μg to about 15 μg per mL of cells.

[0065] Applying a nucleic acid (plasmid)/PEI mixture to cells is performed by adding the nucleic acid (plasmid)/PEI mixture to the cells in such a way that the nucleic acid (plasmid)/PEI mixture contacts the cells. The cells to which the nucleic acid (plasmid)/PEI solution mixture is added (contacted) can be adherent cells or cells in suspension. These cells may include co-cultures with other cells.

[0066] Cells are contacted for a period of time with a mixture of nucleic acids (plasmid)/PEI that is not limited, in order to achieve cell transduction. Contact of cells with Free PEI typically occurs at the same time as (or immediately after), or after the cells have been contacted with the mixture of nucleic acids (plasmid)/PEI. If there is a time interval between the contact of cells with the mixture of nucleic acids (plasmid)/PEI and the contact of cells with Free PEI, the time interval can be from about 1 second to about 140 hours, typically from about 1 second to about 96 hours, more typically from about 1 second to about 48 or about 72 hours, most typically from about 1 second to about 24 hours, or less, for example, about 16, about 12, about 8 or about 6 hours or less.

[0067] For long-term contact, cells may be affected by PEI cytotoxicity, resulting in an increased number of dead (non-viable) cells, thus reducing transfection efficiency. The incubation time after cells are contacted with Total PEI can vary from seconds to days. Specifically, cells may be contacted with nucleic acids (plasmid)/PEI, or Total PEI, for example, for a period of time from about 1 minute to about 48 hours; from about 1 minute to about 24 hours; from about 1 minute to about 16 hours; from about 1 minute to about 8 hours; from about 1 minute to about 4 hours; from about 1 minute to about 120 minutes; from about 5 minutes to about 60 minutes; from about 10 minutes to about 45 minutes; or from about 10 minutes to about 30 minutes.

[0068] To reduce PEI cytotoxicity, the culture medium can be replaced with fresh culture medium after the cells come into contact with nucleic acids (plasmid)/PEI. Replacing the culture medium after transfection can minimize PEI cytotoxicity without significant loss of cell transfection efficiency.

[0069] Cells for transfection, both before and at the time of contact with the plasmid/PEI mixture or contact with free PEI, have a density in the range of about 1x10⁵ cells/mL to about 1x10⁸ cells/mL when contacted with the plasmid/PEI mixture or when contacted with free PEI. Typically, cells have a density in the range of about 2x10⁵ cells/mL to about 5x10⁶ cells/mL. More typically, cells have a density in the range of about 3x10⁵ cells/mL to about 3x10⁶ cells/mL, for example, about 4x10⁵ cells/mL to about 2x10⁶ cells/mL, or about 3x10⁵ cells/mL to about 1x10⁶ cells/mL.

[0070] Cells for transfection, both before and at the time of contact with the plasmid/PEI mixture and/or contact with Free PEI, may optionally be in the log (exponential) growth phase. Cells for transfection, both before and at the time of contact with the plasmid/PEI mixture and/or contact with Free PEI, may optionally have 60% or more 60% viability, for example, 70%, 80% or 90% or more 90% viability.

[0071] The cells that can be contacted as presented here include mammalian cells, such as human cells. Such cells may be primary cells or cell lines that are capable of growing or maintaining viability in vitro, or have been adapted for in vitro tissue culture. Examples of cell lines include HEK (human embryonic kidney) cells, which include HEK293 cells, such as HEK293F (293F) and HEK293T (293T) cells.

[0072] More generally, such contacted cells as set forth herein may be referred to as “host cells.” A “host cell” denotes, for example, microorganisms, yeast cells, insect cells, and mammalian cells, which may be, or have been, used as recipients of nucleic acid (plasmid) encoding packaging proteins, such as AAV packaging proteins, a nucleic acid (plasmid) encoding helper proteins, a nucleic acid (plasmid) encoding a protein or transcribed into a transcript of interest, or other transfer nucleic acid (plasmid). The term includes the progeny of the original cell, which has been transduced or transfected. Therefore, a “host cell,” as used herein, generally refers to a cell that has been transduced or transfected with an exogenous nucleic acid sequence. It is understood that the offspring of a single parent cell may not necessarily be completely identical in morphology or in genomic or total nucleic acid complement as the original parent, due to natural, accidental, or deliberate mutation.

[0073] Numerous cell growth media suitable for sustaining cell viability or providing cell growth and/or proliferation are commercially available or can be readily produced. Examples of such media include serum-free eukaryotic growth media, such as media for sustaining viability or providing growth of mammalian (e.g., human) cells. Non-limiting examples include Ham's F12 or F12K medium (Sigma-Aldrich), FreeStyle (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific) and mixtures thereof. This media may be supplemented with vitamins and/or minerals and/or salts and/or amino acids, such as amino acids essential for mammalian (e.g., human) cells.

[0074] The terms "transduce" and "transfect" refer to the introduction of a molecule, such as a nucleic acid (plasmid), into a host cell. A cell has been "transduced" or "transfected" when the exogenous nucleic acid has been introduced within the cell membrane. Consequently, a "transduced cell" is a cell into which a "nucleic acid" or "polynucleotide" has been introduced, or a progeny thereof into which an exogenous nucleic acid has been introduced. In particular embodiments, a "transduced" cell (e.g., in a mammal, such as a cell or tissue or organ cell) is a genetic alteration in a cell following the incorporation of an exogenous molecule, e.g., a nucleic acid (e.g., a transgene). A "transduced" cell(s) may be propagated and the introduced nucleic acid transcribed and/or expressed as protein.

[0075] In a “transduced” or “transfected” cell, the nucleic acid (plasmid) may or may not be integrated into the genomic nucleic acid of the recipient cell. If an introduced nucleic acid becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it may be stably maintained in that cell or organism and even transmitted or inherited by progenitor cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist in the recipient cell or host organism extrachromosomally, or only transiently. Several techniques are known (see, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier and Chu et al. (1981) Gene 13:197). Such techniques can be used to introduce one or more halves of exogenous DNA into suitable host cells.

[0076] The term "vector" refers to a small carrier nucleic acid molecule, a plasmid, virus (e.g., AAV vector), or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid. Such vectors can be used for genetic manipulation (i.e., "cloning vectors"), to introduce/transfer polynucleotides into cells, and to transcribe or transform the inserted polynucleotide in cells. An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence with the regulatory regions necessary for expression in a host cell. A vector nucleic acid sequence generally contains at least one origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), intron, ITR(s), selectable marker (e.g., antibiotic resistance), polyadenylation signal. For the purposes of the invention, a "vector," as defined herein, is within the scope of a "plasmid," as that term is used herein.

[0077] A viral vector is derived from or based on one or more nucleic acid elements that comprise a viral genome. Particular viral vectors include lentvirus vectors, pseudo-typed lentviruses, and parvoviruses, such as adeno-associated virus (AAV) vectors.

[0078] The term "recombinant," as a vector modifier, such as recombinant viral vectors, for example, lentiviruses or parvoviruses (e.g., AAV), as well as a sequence modifier such as recombinant polynucleotides and polypeptides, means that the compositions have been manipulated (i.e., engineered) in a way that does not generally occur in nature. A particular example of a recombinant vector, such as an AAV vector, would be when a polynucleotide that is not normally present in the wild-type viral genome (e.g., AAV) is inserted into the viral genome, i.e., it is heterologous. Although the term "recombinant" is not always used here in reference to vectors, such as viral and AAV vectors, as well as sequences such as polynucleotides, recombinant forms including polynucleotides are expressly included despite any omission.

[0079] A recombinant viral “vector” or “AAV vector” is derived from the wild-type genome of a virus, such as AAV, by using molecular methods to remove the wild-type genome of the virus (e.g., AAV) and replace it with a non-native nucleic acid, such as a nucleic acid transcribed into a transcript or encoding a protein. Typically, for AAV, one or both of the inverted terminal repeat (ITR) sequences of the AAV genome are retained in the AAV vector. A “recombinant” viral vector (e.g., AAV) is differentiated from a viral genome (e.g., AAV) since all or part of the viral genome has been replaced by a non-native (i.e., heterologous) sequence relative to the viral genomic nucleic acid genome (e.g., AAV). The incorporation of a non-native sequence, therefore, defines the viral vector (e.g., AAV) as a “recombinant” vector, which in the case of AAV may be referred to as a “rAAV vector”.

[0080] A recombinant vector sequence (e.g., lenti-, parvo-, AAV) can be packaged—referred to herein as a “particle”—for subsequent infection (transduction) of a cell, ex vivo, in vitro, or in vivo. When a recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle may also be referred to as a “rAAV.” Such particles include proteins that encapsulate or package the vector genome. Particular examples include viral envelope proteins and, in the case of AAV, capsid proteins such as AAV VP1, VP2, and VP3.

[0081] A vector “genome” refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsulated to form a viral particle (e.g., AAV). In cases where recombinant plasmids are used to construct or manufacture recombinant vectors, the vector genome does not include the portion of the “plasmid” that does not correspond to the genome sequence of the recombinant plasmid vector. This non-vector portion of the recombinant plasmid genome is referred to as the “plasmid structure,” which is important for plasmid cloning and amplification, a process that is necessary for the propagation and production of recombinant viruses, but is not packaged or encapsulated into virus particles (e.g., AAV). Therefore, a vector “genome” refers to the nucleic acid that is packaged or encapsulated by viruses (e.g., AAV).

[0082] The terms "empty capsid" and "empty particle" refer to an AAV virion that includes an AAV protein shell but does not possess, in whole or in part, a nucleic acid that codes for a protein or is transcribed into a transcript of interest flanked by AAV ITRs. Therefore, the empty capsid does not function to transfer a nucleic acid that codes for a protein or is transcribed into a transcript of interest to the host cell. However, empty capsid formulations have utility in other applications, such as ELISA.

[0083] The term “packaging proteins” refers to viral and/or cellular functions derived from AAV, upon which AAV is dependent for its replication. Therefore, the term captures proteins and RNAs that are necessary in AAV replication, including the halves involved in the activation of AAV gene transcription, the specific splicing stage of AAV mRNA, AAV DNA replication, synthesis of Cap expression products, and AAV capsid assembly. Virus-based accessory functions may originate from any of the known helper viruses, such as adenoviruses, herpesviruses (other than herpes simplex virus type 1), and vaccinia viruses.

[0084] As used herein, "AAV packaging proteins" refers to AAV-derived sequences that function in trans for productive AAV replication. Therefore, AAV packaging proteins are encoded by the AAV main open reading frames (ORFs), rep and cap. Rep proteins have been shown to have many functions, including, but not limited to: recognition, binding, and cleavage of the AAV origin of DNA replication; DNA helicase activity; and modulation of the transcription of AAV (or other heterologs) promoters. Cap (capsid) proteins provide the necessary packaging functions. AAV packaging proteins are used herein to supplement the trans AAV functions that are absent from AAV vectors.

[0085] “Nucleic acids encoding AAV packaging proteins” generally refer to a nucleic acid molecule that includes nucleotide sequences that provide suppressed AAV functions of an AAV vector that must be used to produce a recombinant transduction AAV vector. Nucleic acids encoding AAV packaging proteins are commonly used to provide transient expression of AAV rep and/or cap genes to supplement missing AAV functions that are necessary for AAV replication; however, the nucleic acid constructs do not possess AAV ITRs and cannot replicate or package themselves. Nucleic acids encoding AAV packaging proteins may be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. Several nucleic acid constructs have been described, such as the commonly used pAAV/Ad and pIM29+45 plasmids that encode both Rep and Cap expression products. See, for example, Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. Several vectors have been described that encode Rep and/or Cap expression products (e.g., U.S. Patent Nos. 5,139,941 and 6,376,237).

[0086] The term "nucleic acids encoding helper proteins" generally refers to a nucleic acid molecule(s) that includes nucleotide sequences encoding proteins that provide helper function(s). A vector with nucleic acid(s) encoding helper protein(s) can be transfected into a suitable host cell, where the vector is then able to support the AAV virion product in the host cell. Infectious viral particles, such as those existing in nature, such as adenovirus, herpesvirus, or vaccinia virus particles, are expressly excluded from the term.

[0087] Therefore, auxiliary protein vectors can be in the form of a plasmid, phage, transposon, or cosmid. In particular, it has been shown that the complete complement of adenovirus genes is not required for auxiliary functions. For example, adenovirus mutants incapable of DNA replication and late gene synthesis have been shown to be permissive for AAV replication. Ito et al., (1970) J. Gen. Virol 9:243; Ishibashi et al, (1971) Virology 45:317.

[0088] It has been shown that mutants in the E2B and E3 regions support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing ancillary function. Carter et al., (1983) Virology 126:505. However, adenoviruses defective in the E1 region, or having a deleted E4 region, are unable to support AAV replication. Thus, for adenoviral ancillary proteins, it is likely that the E1A and E4 regions are required for AAV replication, directly or indirectly. Laughlin et al., (1982) J. Virol. 41:868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78:1925; Carter et al., (1983) Virology 126:505. Other characterized Ad mutants include: EIB (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., (1980) Virology 104: 502); E2A (Handa et al., (1975) J. Gen. Virol 29:239; Strauss et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol. 35:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:2927; Myers et al. al., (1981) J. Biol. E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)).

[0089] Studies of helper proteins provided by adenoviruses having mutations in E1B have reported that E1B 55k is required for AAV virion production, while E1B 19k is not. Additionally, International Publication WO 97/17458 and Matshushita et al., (1998) Gene Therapy 5: 938-945, describe helper function vectors encoding various Ad genes. An example of a helper vector comprises an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirus E2A 72 kD coding region, an adenovirus E1A coding region, and an adenovirus E1B region without an intact E1B BS5k coding region (see, for example, International Publication No. WO 01/83797).

[0090] A "transgene" is used here to refer appropriately to a nucleic acid that is intended for, or has been, introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene that is transcribed into a transcript or that codes for a polypeptide or protein.

[0091] An “Expression control element” refers to nucleic acid sequence(s) that influence the expression of an operationally linked nucleic acid. Control elements, including expression control elements as presented here, such as promoters and enhancers, vector sequences that include AAV vectors may include one or more “expression control elements”. Typically, these elements are included to facilitate proper heterologous polynucleotide transcription and, if appropriate, transformation (e.g., a promoter, enhancer, splicing signal for introns, maintenance of the correct gene reading frame to allow transformation into mRNA frame and stop codons, etc.). Such elements typically act in cis, referred to as a “cis-acting” element, but may also act in trans.

[0092] Expression control can be at the level of transcription, translation, splicing, message stability, etc. Typically, an expression control element that modulates transcription is juxtaposed near the 5’ end (i.e., “upstream”) of a transcribed nucleic acid. Expression control elements can also be located at the 3’ end (i.e., “downstream”) of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements can be located adjacent to or distant from the transcribed sequence (e.g., 1-10, 10-25, 25-50, 50-100, 100 to 500 or more nucleotides of the polynucleotide), even at considerable distances. However, due to the length limitations of certain vectors, such as AAV vectors, expression control elements will typically be within 1 to 1000 nucleotides of the transcribed nucleic acid.

[0093] Functionally, the expression of operationally bound nucleic acid is at least partly controllable by the element (e.g., promoter) such that the element modulates nucleic acid transcription and, as appropriate, transcript transformation. A specific example of an expression control element is a promoter, which is usually located 5’ from the transcribed sequence. A promoter typically increases an expressed amount of operationally bound nucleic acid compared to an amount expressed when no promoter is present.

[0094] An “enhancer” as used herein may refer to a sequence that is located adjacent to the heterologous polynucleotide. Enhancer elements are typically located upstream of a promoter element, but they also function and may be located downstream or within a nucleic acid sequence. Therefore, an enhancer element may be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a nucleic acid. Enhancer elements typically increase the expression of an operationally linked nucleic acid, above the expression provided by a promoter element.

[0095] An expression construct may comprise regulatory elements that serve to direct expression in a particular cell or tissue type. Expression control elements (e.g., promoters) include those active in a given tissue or cell type, referred to herein as “tissue-specific expression control elements/promoters.” Tissue-specific expression control elements are typically active in specific cells or tissues (e.g., liver). Expression control elements are typically active in particular cells, tissues, or organs because they are recognized by transcriptional activator proteins, or other transcriptional regulators, that are unique to a specific cell, tissue, or organ type. Such regulatory elements are known to those skilled in the art (see, for example, Sambrook et al. (1989) and Ausubel et al. (1992)).

[0096] The incorporation of tissue-specific regulatory elements into the plasmids of the present invention provides at least partial tissue tropism for nucleic acid expression. Examples of promoters that are active in the liver are the TTR promoter, the human alpha 1-antitrypsin (hAAT) promoter; albumin, Miyatake, et al. J. Virol. 71:5124-32 (1997); hepatitis B virus core promoter, Sandig, et al., Gene Ther. 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot, et al., Hum. Gene. Ther., 7:1503-14 (1996)], among others. An example of an active enhancer in the liver is apolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0097] Expression control elements also include ubiquitous or promiscuous promoters/enhancers that are capable of directing the expression of a polynucleotide in many different cell types. Such elements include, but are not limited to, the immediate cytomegalovirus (CMV) promoter/enhancer sequences, the Rous sarcoma virus (RSV) promoter/enhancer sequences, and other viral promoters/enhancers active in a variety of mammalian cell types or synthetic elements that are not present in nature (see, for example, Boshart et al., Cell, 41: 521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter, and the phosphoglycerol kinase (PGK) promoter.

[0098] Expression control elements can also confer expression in a regulable manner, i.e., a signal or stimulus increases or decreases the expression of the operationally linked heterologous polynucleotide. A regulable element that increases the expression of the operationally linked polynucleotide in response to a signal or stimulus is also referred to as an "inducible element" (i.e., it is induced by a signal). Particular examples include, but are not limited to, an inducible hormone promoter (e.g., steroid). Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimulus present; the greater the amount of signal or stimulus, the greater the increase or decrease in expression. Particular, non-limiting examples include the zinc-inducible sheep metallothionein promoter (MT); the steroid hormone-inducible mouse mammary tumor virus promoter (MMTV); the T7 polymerase promoter system (WO 98/10088); the tetracycline repressible system (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline-inducible system (Gossen, et al., Science. 268:1766-1769 (1995); see also Harvey, et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); the RU486-inducible system (Wang, et al., Nat. Biotech. 15:239-243 (1997) and Wang, et al., Gene Ther. 4:432-441 (1997)); and the rapamycin-inducible system (Magari, et al., J. Clin. Invest. 100:2865-2872 (1997); Rivera et al. Nat. Medicine. 2:1028-1032 (1996)). Other regulable control elements that may be useful in this context are those that are regulated by a specific physiological state, for example, temperature, acute phase, development.

[0099] Expression control elements also include elements native to the nucleic acid. A native control element (e.g., promoter) can be used when it is desired that the expression of the heterologous polynucleotide mimic native expression. The native element can be used when the expression of the heterologous polynucleotide needs to be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. Other native expression control elements, such as introns, polyadenylation sites, or Kozak consensus sequences, can also be used.

[0100] The term "operationally linked" means that the regulatory sequences necessary for the expression of a coding sequence are placed in the appropriate positions relative to the coding sequence in order to effect the expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and second nucleic acid molecule in which a hybrid nucleic acid molecule is generated.

[0101] In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates the expression of the nucleic acid. More specifically, for example, two operationally linked DNA sequences mean that the two DNAs are arranged (cis or trans) in a relationship such that at least one of the DNA sequences is able to exert a physiological effect on the other sequence.

[0102] Consequently, additional elements for vectors include, without limitation, an expression control element (e.g., promoter/intensifier), a transcription termination signal or stop codon, untransformed 3’ or 5’ regions (e.g., polyadenylation (polyA) sequences) flanking a sequence, such as one or more copies of an AAV ITR sequence, or an intron.

[0103] Other elements include, for example, polynucleotide filler sequences, for example, to improve packaging and reduce the presence of contaminating nucleic acid. AAV vectors typically accept DNA inserts having a size range that is generally from about 4 kb to about 5.2 kb, or slightly more. Therefore, for shorter sequences, the inclusion of a filler in order to adjust the length to near or to the normal size of the virus genomic sequence acceptable for packaging the AAV vector into virus particles. In several embodiments, a nucleic acid filler sequence is a segment of untranslated nucleic acid (non-protein coding). For a nucleic acid sequence smaller than 4.7 Kb, the filler polynucleotide sequence has a length that, when combined (e.g., inserted into a vector) with the sequence, has a total length between approximately 3.0-5.5 Kb, or between approximately 4.0-5.0 Kb, or between approximately 4.3-4.8 Kb.

[0104] An intron can also function as a filler polynucleotide sequence, so as to achieve a length for packaging the AAV vector into a virus particle. Introns and intron fragments that function as a filler polynucleotide sequence can also enhance expression.

[0105] The “polypeptides”, “proteins”, and “peptides” encoded by “nucleic acid” or “plasmids” include full-length native sequences, as with naturally occurring wild-type proteins, as well as functional subsequences, modified forms, or sequence variations provided that the subsequence, modified form, or variant retains some degree of functionality of the entire native protein. For example, a protein may have a deletion, substitution, or addition and retain at least some partial function or activity.

[0106] The terms "modify" or "variant" and grammatical variations thereof mean that a nucleic acid or polypeptide deviates from a reference sequence. Modified and variant sequences may therefore have substantially the same, greater or lesser expression, activity or function than a reference sequence, but at least retain partial activity or function of the reference sequence.

[0107] Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (e.g., 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100, 100-150, 150-200, 200-250, 250-500, 500-750, 750-850 or more nucleotides or residues).

[0108] An example of an amino acid modification is a conservative amino acid substitution or a deletion (e.g., subsequences or fragments) of a reference sequence. In particular embodiments, a modified or variant sequence retains at least part of a function or activity of the unmodified sequence.

[0109] All forms of nucleic acids from mammals and non-mammals that are transcribed and nucleic acids that encode proteins are included. Thus, the invention includes genes and proteins from non-mammals, non-human mammals, and humans, which genes and proteins function in a substantially similar manner to human genes and proteins.

[0110] After the production of recombinant virus vectors (e.g., AAV) as presented here, if desired, viral virions (e.g., rAAV) can be purified and/or isolated from host cells using a variety of conventional methods. Such methods include column chromatography, CsCl gradients, and the like. For example, a plurality of column purification steps, such as purification on an anion exchange column, an affinity column, and/or a cation exchange column, can be used. (See, for example, International Publication No. WO 02/12455 and U.S. Patent Application No. 20030207439). Alternatively, or in addition, CsCl gradient steps can be used. (See, for example, U.S. Patent Applications No. 20120135515; and 20130072548). Furthermore, if infectious virus is used to express packaging and/or helper proteins, residual virus can be inactivated using various methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60 °C for, for example, 20 minutes or more. This treatment effectively inactivates the helper virus since AAV is heat-stable while adenovirus helper is thermally sensitive to heat.

[0111] The term "isolate," when used as a modifier of a composition, means that the compositions are made by human hands or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they are normally associated in nature, for example, one or more contaminants such as protein, nucleic acid, lipid, carbohydrate, cell membrane.

[0112] With regard to RNA molecules, the term "isolate" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues) such that it exists in a "substantially pure" form (the term "substantially pure" is defined below).

[0113] With regard to protein, the term "isolated protein" or "isolated and purified protein" is sometimes used herein. This term refers primarily to a protein produced by the expression of an isolated nucleic acid molecule. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in a "substantially pure" form.

[0114] The term “isolate” does not exclude man-made combinations, for example, a recombinant vector sequence (e.g., rAAV), or virus particles that encapsulate or encapsulate a vector genome and a pharmaceutical formulation. The term “isolate” also does not exclude alternative physical forms of the composition, such as hybrids/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivative forms, or man-made forms expressed in host cells.

[0115] The term "substantially pure" refers to a preparation comprising at least 50-60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation may comprise at least 75% by weight, or about 90-99% by weight, of the compound of interest. Purity measured by methods appropriate for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC anise and the like).

[0116] Nucleic acid molecules, expression vectors (e.g., vector genomes), plasmids, can be prepared using recombinant DNA technology methods. The availability of nucleotide sequence information allows the preparation of isolated nucleic acid molecules by a variety of means. For example, nucleic acids (e.g., plasmids) can be produced using various conventional recombinant DNA cloning technologies, through cellular expression or in vitro transformation, and chemical synthesis techniques. Purity can be determined by sequencing, gel electrophoresis, and the like. For example, nucleic acids can be isolated using hybridization techniques or computer-based database screening. Such techniques include, but are not limited to: (1) hybridization of DNA or cDNA genomic libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides having shared structural features, for example, using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of pairing a nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of a subtracted nucleic acid library.

[0117] Nucleic acids can be maintained as DNA in any convenient cloning vector. In one embodiment, nucleic acids are maintained in a plasmid. Alternatively, nucleic acids can be maintained in a vector suitable for expression in mammalian cells.

[0118] The nucleic acids of the invention, vectors, expression vectors (e.g., rAAV), and recombinant virus particles, methods, and uses enable the treatment of genetic diseases. For deficiency state diseases, gene transfer can be used to bring a normal gene into affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations. For imbalance disease states, gene transfer could be used to create a disease state in a model system, which could then be used in efforts to neutralize the disease state. The use of site-specific integration of nucleic acid sequences to correct defects is also possible.

[0119] Viral vectors, such as lenti- and parvovirus vectors, including AAV serotypes and their variants, provide a means for delivering nucleic acid into cells ex vivo, in vitro, and in vivo, encoding proteins such that the cells express the encoded protein. AAVs are useful viruses as gene therapy vectors, as they can penetrate cells and introduce nucleic acid/genetic material so that the nucleic acid/genetic material can be stably maintained in the cells. Furthermore, these viruses can produce nucleic acid/genetic material at specific locations, for example. Because AAVs are not associated with pathogenic disease in humans, AAV vectors are able to deliver heterologous polynucleotide sequences (e.g., proteins and therapeutic agents) to human patients without causing substantial AAV pathogenesis or disease.

[0120] Viral vectors that may be used include, but are not limited to, adeno-associated virus (AAV) vectors of multiple serotypes (e.g., AAV-1 to AAV-12 and others) and hybrid/chimeric AAV vectors, lentivirus vectors and pseudo-type lentivirus vectors (e.g., Ebola virus, vesicular stomatitis virus (VSV), and feline immunodeficiency virus (FIV)), herpes simplex virus vectors, adenoviral vectors (with or without tissue-specific promoters/enhancers), vaccinia virus vectors, retroviral vectors, lentiviral vectors, nonviral vectors, and others.

[0121] AAV and lentiviral particles can be used advantageously as vehicles for effective gene delivery. Such virions possess a number of desirable characteristics for such applications, including tropism for both cell division and non-division. Initial clinical experience with these vectors also demonstrated no sustained toxicity and minimal or undetectable immune responses. AAVs are known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells. Non-viral vectors, for example, those based on plasmid DNA or minicircles, are also suitable gene transfer vectors.

[0122] Consequently, in various embodiments of the invention, a vector includes a lenti- or parvoviral vector, such as an adenoviral vector. In particular embodiments, a recombinant vector is a parvovirus vector. Parvoviruses are small viruses with a single-stranded DNA genome. “Adeno-associated viruses” (AAVs) are in the parvovirus family.

[0123] AAV vectors and lentiviral vectors typically do not include viral genes associated with pathogenesis. Such vectors typically have one or more of the wild-type AAV genes, eliminated in whole or in part, for example, rep and/or cap genes, but retain at least one functional flanking ITR sequence, as required for salvage, replication, and packaging of the recombinant vector into an AAV vector particle. For example, only the essential parts of the vector, for example, the ITR and LTR elements, respectively, are included. An AAV vector genome would therefore include sequences required in cis for replication and packaging (e.g., functional ITR sequences).

[0124] Recombinant AAV vectors, as well as their methods and uses, include any viral strain or serotype. As a non-limiting example, a recombinant AAV vector can be based on any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, or AAV-2i8, for example. Such vectors may be based on the same lineage or serotype (or subgroup or variant), or be different from each other. As a non-limiting example, a recombinant AAV vector based on a serotype genome may be identical to one or more of the capsid proteins that package the vector. Additionally, a recombinant AAV vector genome may be based on an AAV serotype genome (e.g., AAV2) distinct from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome may be based on AAV2, while at least one of the three capsid proteins may be AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV-2i8, or a variant thereof. AAV variants include capsid variants and chimeras of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV-2i8.

[0125] In particular embodiments, adeno-associated virus (AAV) vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV-2i8, as well as variants (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions), for example, as set forth in WO 2013/158879 (International Application PCT/US2013/037170), WO 2015/013313 (International Application PCT/US2014/047670) and US 2013/0059732 (U.S. Application No. 13/594,773, discloses LK01, LK02, LK03, etc.).

[0126] AAV and AAV variants (e.g., capsid variants) (e.g., VP0, VP1, VP2, and/or VP3) may or may not be distinct from other AAV serotypes, including, for example, AAV1-AAV12 (e.g., distinct from VP1, VP2, and/or VP3 sequences of any of the AAV1-AAV12 serotypes).

[0127] As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological distinction is determined based on the lack of cross-reactivity between antibodies to one AAV compared to another AAV. These cross-reactivity differences are generally due to differences in capsid protein sequences/antigenic determinants (e.g., due to differences in VP1, VP2, and/or VP3 sequences of AAV serotypes). Although AAV variants that include capsid variants may not be serologically distinct from a reference AAV or another AAV serotype, they differ in at least one nucleotide or amino acid residue compared to the reference or other AAV serotype.

[0128] According to the traditional definition, a serotype means that the virus of interest has been tested against specific serum for all existing serotypes and characterized for neutralizing activity, and no antibodies have been found that neutralize the virus of interest. As more isolates of naturally occurring viruses are discovered and/or capsid mutants are generated, there may or may not be serological differences with any of the currently existing serotypes. Therefore, in cases where the new virus (e.g., AAV) has no serological difference, this new virus (e.g., AAV) would be a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are of another serotype according to the traditional definition of serotype. Therefore, for convenience and to avoid repetition, the term "serotype" refers broadly to both serologically distinct viruses (e.g., AAV) as well as viruses (e.g., AAV) that are not serologically distinct but may be within a subgroup or variant of a given serotype.

[0129] In several exemplary embodiments, an AAV vector related to a reference serotype has a polynucleotide, polypeptide or subsequence thereof that includes or consists of a sequence that is at least 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV-2i8 (e.g., such as an ITR, or a VP1, VP2 and/or VP3 sequence).

[0130] Compositions, methods and uses of the invention include AAV sequences (polypeptides and nucleotides) and their subsequences that exhibit less than 100% sequence identity with a reference AAV serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV-2i8, but are distinct from and not identical to known AAV genes or proteins, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV-2i8 genes or proteins, etc. In one embodiment, an AAV polypeptide or its subsequence includes or consists of a sequence that is at least 75% or more identical, for example, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to any reference sequence of AAV or its subsequence, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV-2i8 (e.g., capsid VP1, VP2). and/or VP3 or ITR). In particular aspects, an AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions.

[0131] Recombinant AAV vectors, including AAV1, AAV2, AAV3, AAV4, AAV10, AAV11, AAV12 or AAV-2i8 and related variant, hybrid and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art, to include one or more nucleic acid sequences (transgenes) flanked with one or more functional AAV ITR sequences.

[0132] Nucleic acids (plasmids), vectors, recombinant vectors (e.g., rAAV), and recombinant virus particles can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful for, among other things, administration and delivery to an individual in vivo or ex vivo. In particular embodiments, the pharmaceutical compositions contain a pharmaceutically acceptable vehicle or excipient. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition and that can be administered without undue toxicity.

[0133] As used herein, the term “pharmaceutically acceptable” and “physiologically acceptable” means a biologically acceptable gaseous, liquid or solid formulation, or a mixture thereof, that is suitable for one or more routes of administration, delivery or in vivo contact. A “pharmaceutically acceptable” or “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, for example, the material can be administered to a subject without causing substantial undesirable biological effects. Thus, such a pharmaceutical composition can be used, for example, in the administration of a nucleic acid, vector, viral particle or protein to an individual.

[0134] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline solution, glycerol, sugars and ethanol. Pharmaceutically acceptable salts may also include, for example, salts of mineral acids such as hydrochlorides, hydrobromides, phosphates, sulfates and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents and the like, may be present in such vehicles.

[0135] The pharmaceutical composition may be provided as a salt and may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than the corresponding free base forms. In other cases, a preparation may be a lyophilized powder that may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose and 2-7% mannitol, in a pH range of 4.5 to 5.5, which is combined with buffer before use.

[0136] Pharmaceutical compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersing and suspending media, coatings, isotonics, and absorption-promoting or retarding agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), micropearls, powder, granules, and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral, and antifungal agents) may also be incorporated into the compositions.

[0137] Pharmaceutical compositions may be formulated to be compatible with a particular route of administration or distribution. Thus, pharmaceutical compositions include vehicles, diluents or excipients suitable for administration by various routes.

[0138] Compositions and methods can be sterile. Compositions can be made and methods can be carried out in containers suitable for such processes. Such containers include dishes, flasks, rotary bottles, bags, bioreactors, vessels, tubes, vials, etc. The containers can be made of materials that include, but are not limited to, glass, plastic, and polymers such as polystyrene, polybutylene, polypropylene, etc.

[0139] The compositions and steps of the method can be performed in a designated order or rearranged order. The steps of the method can be performed in stages or at intervals with intervening time periods. In other words, a method step can be performed, and then a time interval can occur between the next step, such intervals varying, for example, from about 1 second to about 60 seconds; from about 1 minute to about 60 minutes; from about 1 hour to about 24 hours; from about 1 day to about 7 days; or from about 1 week to about 48 weeks.

[0140] Protocols for the generation of adenoviral vectors have been described in U.S. Patents Nos. 5,998,205; 6,228,646; 6,093,699; and 6,100,242; and international patent applications WO 94/17810 and WO 94/23744, which are incorporated herein by reference in their entirety.

[0141] The invention is useful in the production of cells and vectors for human and veterinary medical applications. Therefore, suitable subjects include mammals, such as humans, as well as non-human mammals. The term “subject” refers to an animal, typically a mammal, such as humans, non-human primates (monkeys, gibbons, gorillas, chimpanzees, orangutans, apes), a domestic animal (dogs and cats), a farm animal (domestic fowl, such as chickens and ducks, horses, cows, goats, sheep, pigs) and experimental animals (mouse, rat, rabbit, guinea pig). Human subjects include fetal, neonatal, infant, juvenile and adult subjects. Subjects include animal disease models, for example, mice and other animal models of blood clotting diseases such as HemA and others known to those skilled in the art.

[0142] A “unit dosage form” as used herein refers to physically discrete units suitable as unit dosages for the individual to be treated; each unit containing a predetermined amount optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filler) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect). Unit dosage forms may be contained within, for example, ampoules and vials, which may contain a liquid composition, or a composition in a lyophilized or freeze-dried state; a sterile liquid vehicle, for example, may be added prior to administration or in vivo delivery. Individual unit dosage forms may be included in multi-dose kits or containers. Recombinant vector sequences (e.g., rAAV), recombinant virus particles and their pharmaceutical compositions may be packaged in a unit or multiple dosage form for ease of administration and dosage uniformity.

[0143] The invention provides kits with packaging material and one or more components. A kit typically includes a label or packaging insert including a description of the components or instructions for use of the components. A kit may contain a collection of such components, for example, a nucleic acid (plasmid), PEI, cells.

[0144] A kit refers to a physical structure that houses one or more kit components. The packaging material may keep the components sterile and may be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0145] Labels or inserts may include identifying information for one or more components. Labels or inserts may include information identifying the manufacturer, batch numbers, place and date of manufacture, expiration dates. Labels or inserts may include information identifying the manufacturer, batch numbers, manufacturer location, and date. Labels or inserts may include instructions for using one or more components of the kit in a manufacturing method, use, or protocol. Instructions may include instructions for producing the compositions or practicing any of the methods described herein.

[0146] Labels or inserts include “printed material”, for example, paper or cardboard, either separate from or affixed to a component, kit or packaging material (for example, a box) or connected to an ampoule, tube or vial containing a kit component. Labels or inserts may additionally include computer-readable media such as a printed barcode label, a disc, an optical disc such as a CD or DVD-ROM/RAM, DVD, MP3, magnetic tape or an electrical storage medium such as RAM and ROM or hybrids thereof, such as magnetic/optical storage media, FLASH media or memory cards.

[0147] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that normally understood by a person skilled in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0148] All patents, patent applications, publications and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, shall prevail.

[0149] Several terms related to the biological molecules of the invention are used above and also throughout the specification and claims.

[0150] All features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving the same purpose, equivalent or similar. Thus, unless expressly indicated otherwise, the features disclosed (e.g., PEI, plasmid, vector (e.g., rAAV, or recombinant virus particle)) are an example of a genus of equivalent or similar features.

[0151] As used herein, the singular forms "a", "and" and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a plasmid" or "a nucleic acid" includes a plurality of such plasmids or nucleic acids, reference to "a vector" includes a plurality of such vectors, and reference to "a virus" or "particle" includes a plurality of such viruses/particles.

[0152] As used herein, all numerical values or numerical ranges include whole numbers within such ranges and fractions of the values or whole numbers within ranges, unless the context clearly indicates otherwise. Thus, to illustrate, reference to 80% or more of identity includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so forth.

[0153] Reference to an integer that is greater than or less than any number greater than or less than the reference number, respectively. Thus, for example, a reference to less than 100 includes 99, 98, 97, etc. down to the number one (1); and less than 10 includes 9, 8, 7, etc. down to the number one (1).

[0154] As used herein, all numerical values or intervals include fractions of the values and integers within such intervals and fractions of the integers within such intervals, unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical interval, such as 1-10, includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. The reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so on.

[0155] A reference to a series of intervals includes intervals that combine the limit values of different intervals within the series. Thus, to illustrate the reference to a series of ranges, for example, from 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-850, includes intervals of 120, 1-30, 1-40, 1-50, 1-60, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 50-75, 50-100, 50-150, 50-200, 50-250, 100200, 100-250, 100-300, 100-350, 100-400, 100-500, 150-250, 150-300, 150350, 150-400, 150-450, 150-500, etc.

[0156] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments and aspects. The invention also specifically includes embodiments in which the particular subject matter is excluded, wholly or partially, such as substances or materials, steps and conditions of the method, protocols or procedures. For example, in certain embodiments or aspects of the invention, materials and/or steps of the method are excluded. Thus, although the invention is not generally expressed herein in terms of what the invention does not include, aspects that are not expressly excluded from the invention are nevertheless disclosed herein.

[0157] A number of embodiments of the invention have been described. However, a person skilled in the art, without departing from the spirit and scope of the invention, may make various alterations and modifications to the invention to adapt it to various uses and conditions. Therefore, the following examples are intended to illustrate, but not limit, the scope of the claimed invention in any form.

EXAMPLE 1

[0158] This example includes a description of various materials and methods.

[0159] Cell culture: FreeStyle™293F cells (293F) acquired from Thermo Fisher Scientific (Invitrogen™ by Thermo Fisher Scientific, R790-07) were cultured in FreeStyle TMF17 (F17) expression medium (Gibco Cat. No. A13835-01) or FreeStyle™293 (FS) expression medium (Gibco Cat. No. 12338-018) supplemented with 4 mM GlutaMAX (Life Technologies, Cat. No. 35050-061). Cells were cultured in various cell culture apparatus, including spin flasks and bioreactors. For rotary flask culture (Bellco Glass, cat. 1965-83100 or Corning, cat. 3152), cells were cultured at 37°C with 70 rpm agitation and a humidified atmosphere of 8% CO2; for bioreactors (DASGIP Parallel Bioreactor system, Eppendorf), cell culture was controlled by programmed parameters (OD 30%, pH 7.2, 170 or 150 rpm). Typically, cells were seeded at 0.25–0.5 x 10⁶/mL, subcultured every 2–3 days when cell density reached approximately 1.6–2 x 10⁶/mL by adding fresh cell culture medium. Cell density and viability were determined using a hemocytometer after staining with Trypan Blue.

[0160] Plasmids: Three plasmids were used to produce recombinant adeno-associated viral vectors (rAAVs): 1) a transgene plasmid containing eGFP flanked by ITRs; 2) a packaging plasmid containing rep and cap genes from AAV serotype 2; and 3) an adenoviral helper plasmid containing the E2, E4, and VARNA genes from adenovirus. All plasmids were purchased and manufactured by Aldevron. P

[0161] Preparation of PEI solutions: Linear polyethyleneimine (PEI) 25 kDa (Polysciences, Cat. No. 23966-2), PEI “Max” 40 kDa (Polysciences, Cat. 24765-2, hydrochloride salt of linear PEI 25 kDa) and PEIpro (Polyplus, cat. 115-010) were used as transfection reagents. For most transfection studies, PEI “Max” was dissolved in 5 mM Tris to make a 0.5 mg/mL solution and the pH was adjusted to 7.10. PEI 25 kDa was first dissolved in hot water at 80 °C; after cooling, Tris buffer was added to produce 0.5 mg/mL of PEI in 5 mM Tris buffer solution, pH 7.10. For some studies, PEI “Max” 40 kDa was compared with PEI 25KDa in transfection, PEI 40KDa and 25KDa were dissolved in 5mM Tris buffer with or without 150mM NaCl or in water with or without 150mM NaCl, adjust the pH to 7.10.

[0162] Transfection: 293F cells were grown in FS medium or F17 medium plus 4mM GlutaMAX™ supplement in spin flasks. One day before transfection, the cells were subcultured by adding fresh medium. On the day of transfection, cell density was determined using a hemocytometer and further diluted with fresh FS medium or F17 medium to a final density of 0.35-1 x 10⁶ cells/mL. Transfection was performed in cell suspension culture wells or spin flasks.

[0163] Three plasmids as described above were used in transfection at a molar ratio of 1:1:1. The total amount of DNA used for transfection was 0.7 to 11.2 μg per ml of cell culture. The PEI/DNA complex was prepared with different proportions of PEI and DNA, incubated at room temperature for 1 min to 30 min, then the DNA/PEI complexes were added dropwise to the cell culture. To evaluate the effect of free PEI on transfection efficiency and rAAV productivity, PEI molecules were prepared in the same way as described above without incubation with DNA and added to cell culture immediately after the DNA/PEI complexes were added. Samples, including cells and cell culture media, were taken at 24, 48, and 72 hours after transfection for transfection efficiency and other assays, and cell culture was harvested at 72 hours after transfection.

[0164] Transfection efficiency was evaluated using an inverted fluorescence microscope (Leica) or a flow cytometer (Becton Dickinson Biosciences). eGFP-positive cells were detected using a fluorescence microscope. The percentage of GFP-positive cells was evaluated using a BD FACS Canto flow cytometer.

[0165] Production of rAAV vectors in bioreactors: A DASGIP 2G Parallel Bioreactor system (Eppendorf) equipped with two inclined blade rotors was used to scale up the vector production process. The final working volume was set to 400 mL in the studies. Agitation was set to 150 rpm or 170 rpm, the temperature was maintained at 37 °C and pH 7.2 during cell culture. Dissolved oxygen was maintained at 30% by supplementation with a gas mixture of oxygen, carbon dioxide, and air. All these parameters were monitored and controlled by the DASGIP Control System with DASGIP Control 4.0 software. 293F cells cultured in F17 medium were inoculated at a cell density of 0.4 x 10⁶ cells/mL with viability greater than 95%. Cells were subcultured on day 2 or day 3 after seeding by adding fresh medium; cell density was approximately 0.4–0.7 cells x 10⁶/mL after subculture. Twenty-four hours after subculture, cells were transfected with the PEI/DNA complex as described in the legends, and cell density was approximately 1 x 10⁶ cells/mL at transfection. PEI/DNA weight ratio 2:1 with 1/2 PEI as free PEI at transfection. Samples were collected every 24h for up to 72h.

[0166] Quantification of rAAV vectors: rAAV vectors were released from 293F cell collections transfected by three freeze/thaw cycles or Microfluidizer™ (Microfluidics) three times. Cell debris was pelleted by centrifugation and supernatants were collected for PCR and real-time transduction assays.

[0167] The number of copies of the AAV vector genome was determined by real-time polymerase chain reaction (Q-PCR) (QuanStudio 7, Life Technologies) using TaqMan Master Mix (cat. 4304437, Life Technologies). 10 μl of cell lysate were treated with 7.6 U of DNase I (Cat # 79254, Qiagen) to digest contamination from unpackaged DNA, and then treated with 0.2% SDS/5mM EDTA/0.2M NaCl at 95°C for 10 min to inactivate DNase I and release vector DNA. The primers and probe detected the eGFP transgene sequence: Forward primer: 5'-GCACAAGCTGGAGTACAACTA-3', reverse primer 5'-TGTTGTGGCGGATCTTGAA-3', and probe 5'-/56-FAM/AGCAGAAGA/ZEN/ACGGCATCAAGGTGA/3IABkFQ/-3'. To generate a standard curve, a pAAV-eGFP-WRPE plasmid was linearized by HindIII-HF digestion and used in 1:5 serial dilutions of 1 x 10⁸ to 1.28 x 10³ copies of the gene. All samples were performed in triplicate.

[0168] Transduction assays were performed by adding 50 μL of cell lysates to HEK 293 cells seeded in 48-well plates. Etoposide was added to each well at the time of transduction to a final concentration of 3 μM. After 48 h of incubation, GFP-positive cells were evaluated using an inverted fluorescence microscope.

EXAMPLE 2

[0169] This example includes a description of several results.

[0170] Effect of transfection medium on transfection efficiency and rAAV production: To develop a scalable serum-free suspension culture system for large-scale production of the rAAV vector, several suitable media for cell suspension culture, including FS medium, F17 medium, SFM4Transfx-293 and others, were evaluated for growing 293F cells. Studies reveal that FS medium 293 and F17 medium support good cell growth – cell density reached 2.5 ~ 3 x 10⁶ cells/mL in a spinning flask. These two media also support efficient gene transfection in 293F cells.

[0171] Effect of free PEI on transfection efficiency and rAAV production: High transfection efficiency is a step towards achieving high rAAV production. Polyethyleneimine (PEI) was used as a transfection reagent to transfect 293F cells in suspension culture.

[0172] Among the numerous parameters evaluated, the molar ratio of nitrogen (N) in the PEI molecule to DNA phosphate (P) (N:P ratio) drastically affects transfection efficiency, from very poor transfection to highly efficient transfection when the N:P ratio was changed from low to high. Cytotoxicity was also found at high N:P ratio, such as N:P ratio 30, when used for transfection.

[0173] Several different PEI molecules were studied including PEI “Max” 40KDa, PEI 25KDa, PEIPro and others. PEI molecules were prepared using Tris buffer or DI water with or without 150 mM NaCl, at pH 7.1, an appropriate amount of plasmid DNA was mixed with PEI molecules in a fixed N:P ratio, incubated at room temperature for different time periods such as 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes and up to 30 minutes, and then used to transfect cells.

[0174] The data show that both PEI “Max” 40KDa and PEI 25KDa provided high-efficiency cell transfection, with PEI “Max” 40KDa consistently providing high transfection efficiency (Fig. 1). Thus, most of the data were generated using PEI “Max” 40KDa as the transfection reagent.

[0175] RAAV vectors were produced by transfecting 293F cells using three plasmids as described in the materials and methods. A different cell culture apparatus, including a 12-well plate, cell culture spin flask, and bioreactor, was used to culture 293F cells and produce VAAr vectors. The PEI/DNA mixture was prepared at a weight ratio of 2:1 and 4:1 for transfecting the cells. Different amounts of DNA per ml of cell culture were tested for transfection efficiency, and a molar ratio of 1:1:1 between the three plasmids was typically used for transfection. 293F cells were cultured in serum-free suspension culture using FS medium and F17 medium.

[0176] PEI-mediated gene transfer is a very complicated cellular biological process, involving binding to cellular receptors, endocytosis, intracellular trafficking, entry into nuclei, and gene expression are just some of the main steps. While not wishing to be limited by any theory, an appropriate amount of free PEI molecules can increase transfection efficiency and, in turn, increase rAAV production.

[0177] As indicated in Fig. 2A, cell transfection efficiency was significantly improved when free PEI molecules were added to cell culture after the addition of the DNA/PEI complex, compared to transfection efficiency with only the DNA/PEI complex. This phenomenon, transfection efficiency of free PEI, was observed in 12-pot cell culture plates (Fig. 2A), cell culture spinner flasks, and bioreactor. Furthermore, rAAV vector production increased correspondingly when free PEI molecules were added to the transfection, with 2 to 3 times more rAAV vectors produced (Fig. 2B).

[0178] The finding that Free PEI increases transfection efficiency and rAAV production was tested on larger cell culture scales, spinner flasks, and bioreactors. Transfection results in spinner flasks with or without Free PEI (Fig. 3) are consistent with what was found in 12-well plates, namely, that transfection efficiency was significantly increased when Free PEI was used in transfection and rAAV productivity increased 2 to 3 times, from vector genome 1,3E+10 (VG) per mL of cell lysate without free PEI to approximately 2,4E+10 VG/mL with free PEI.

[0179] The cell growth state at transfection also had a significant impact on rAAV vector production in the bioreactor. To better evaluate ways to improve rAAV production, the cell growth state was tested to determine the impact on rAAV vector yield. In cell suspension culture, cells were seeded at 0.25 x 10⁶ cells/mL, 0.35 x 10⁶ cells/mL, and 0.5 x 10⁶ cells/mL, and a cell growth curve was established over 7 days of cell culture (Fig. 4) in a 2L bioreactor. Cell growth phases were roughly defined and the exponential growth phase was determined between 48h and 84h. The peak cell density was 4.8 to 5.8 x 10⁶ cells/mL on day 6, and then the cell density began to fall. Cell viability was above 90% during culture.

[0180] For transfection and rAAV production, 293F cells were inoculated at a cell density of 0.4 x 10⁶ cells/ml with viability greater than 95% in a 2-liter bioreactor with working volumes of approximately 400 ml. While attempting to identify the best cell culture window(s) for plasmid transfection and rAAV production, it was discovered that transfecting cells at different cell culture states also has a significant effect on rAAV yield.

[0181] The cells were then subcultured on either day or two of three post-seeding by adding fresh cell culture media to reduce cell density; cell densities are in the range of approximately 0.4–0.7 x 10⁶ cells/mL. The cells were then transfected 24 hours after subculture using the PEI-mediated transfection method as described. Typically, cell densities at transfection will reach 7E+05 cells/mL to 1.3E+06 cells/mL of medium. The conditions of two independent experiments and the corresponding results are shown in Table 1. Table 1 Production of rAAV vectors in bioreactors

[0182] It appears that subcultured cells on day 3 post-seeding and transfected cells on day 4 post-seeding resulted in significantly higher rAAV productivity compared to subcultured cells on day 2 post-seeding and transfected on day 3 post-seeding.

[0183] It is interesting to note that transfection efficiency does not appear to differ between these two compared conditions, as indicated by the data in Figure 5. Shown in Fig. 5A is a comparison of transfection efficiency as indicated by eGFP gene expression, and Fig. 5B is further quantitative FACS data, suggesting approximately 50% of cells transfected in all tested conditions; however, the vector titer was significantly higher on day 4 of transfection as assessed by qPCR (Fig. 6A), 2 times higher, 5 times higher, at some point even 7 to 8 times higher on day 4 transfection compared to day 3 transfection. The highest titer was 1.38E+11 vg/mL under the day 4 transfection condition and agitation speed at 150 rpm.

[0184] The rAAV-GFP transduction function of these experiments was evaluated by transduction of HEK 293 cells, consistent with qPCR analysis, when the same volume of cell lysate from each production condition was added to HEK 293 cells, a higher transduction was observed from the day 4 transfection samples compared to the day 3 transfection sample (Fig. 6B). These data indicate that the cell growth stage and metabolic state may play an important role in rAAV production, and that there may be a cellular metabolic window that is more permeable to rAAV biosynthesis, in addition to transfection efficiency.

[0185] The new PEI-mediated rAAV production system recently developed is a versatile and fully scalable cGMP-compliant rAAV production platform that can be used to produce any serotypes of rAAV vectors in serum-free suspension cell culture. In combination with PEI as a transfection reagent, such as 40kDa PEI™ Max™ Efficient molecules, addition of free PEI in the transfection process and a cell growth step initiated for transfection, a very high rAAV yield was obtained under suspension cell culture conditions, approximately 10 times higher than in similar serum-free suspension culture systems reported in the literature (summarized in Table 2). Table 2 Comparison of vector yield from serum-free suspension cell culture methods with that reported in the literature (reference).

[0186] Although some embodiments of the invention have been described and specifically exemplified above, the invention is not intended to be limited to such embodiments. Various modifications may be made without departing from the scope and spirit of the invention, as set forth in the following claims.

Claims (14)

1. Composition, characterized by comprising: (a) a plasmid/PEI mixture comprising: (i) one or more plasmids comprising nucleic acids encoding adeno-associated virus (AAV) packaging proteins, comprising AAV rep or AAV cap, and nucleic acids encoding adenovirus E2, E4, VA RNA or non-AAV auxiliary functions; (ii) a plasmid comprising a nucleic acid encoding a protein or being transcribed into a transcript of interest; and (iii) a polyethyleneimine (PEI) solution, wherein said plasmids of (i) and (ii) are in a molar ratio range of 1:0.01 to 1:100, or in a molar ratio range of 100:1 to 1:0.01 in the plasmid/PEI mixture; and (b) mammalian cells; wherein the cells are in contact with the plasmid/PEI mixture; and wherein the cells and the plasmid/PEI mixture comprise a plasmid/PEI cell culture; and (c) Free PEI in an amount between 25% and 65% of the Total PEI; wherein said Free PEI and said plasmid/PEI cell culture constitute a Free PEI/plasmid/PEI cell culture. 2. Composition according to claim 1, characterized by further comprising a plasmid that includes nucleic acids encoding the capsid proteins of AAV. 3. Composition, according to claim 1 or claim 2, characterized in that the cells produce a recombinant AAV vector that includes a nucleic acid encoding a protein or is transcribed into a transcript of interest. 4. Composition, according to any one of claims 1 to 3, characterized in that the composition is contained in a container, which optionally includes a flask, a plate, a bag or a bioreactor, which is optionally sterile and/or which is optionally suitable for maintaining cell viability or growth. 5. Composition, according to any one of claims 1 to 4, characterized by the plasmid/PEI cell culture, or said free PEI/plasmid/PEI cell culture being incubated for a period of time in the range of 4 hours to 140 hours. 6. Composition according to any one of claims 1 to 5, characterized in that (a) the plasmid/PEI mixture having a PEI:plasmid ratio between 0.1:1 and 5:1, or a PEI:plasmid ratio in the range of 5:1 to 0.1:1, or (b) the PEI/plasmid/PEI-free cell culture having a PEI:plasmid ratio in the range of 0.1:1 to 5:1, or having a PEI:plasmid ratio in the range of 5:1 to 0.1:1. 7. Composition, according to any one of claims 1 to 6, characterized in that the PEI of said plasmid/PEI mixture and/or the free PEI comprise a hydrolyzed linear polyethyleneimine. 8. Composition, according to any one of claims 1 to 7, characterized by the plasmid/PEI mixture PEI and/or the free PEI comprising a hydrolyzed linear polyethyleneimine with a molecular weight in the range of 4,000 to 160,000 and/or in the range of 2,500 to 250,000 in free base form. 9. Composition, according to any one of claims 1 to 8, characterized in that the molar ratio between nitrogen (N) in the total PEI and phosphate (P) in the plasmid is in the range of 1:1 to 50:1 (N:P) in the PEI Free/plasmid/PEI cell culture. 10. Composition, according to any one of claims 1 to 9, characterized in that the plasmid/PEI mixture has been incubated for a period of time between 30 seconds and 4 hours. 11. Composition, according to any one of claims 1 to 10, characterized in that the amount of free PEI is 50% of the total PEI. 12. Composition, according to any one of claims 1 to 11, characterized in that Free PEI is added to the cells before, at the same time as, or after the plasmid/PEI mixture comes into contact with the cells. 13. Composition, according to any one of claims 1 to 12, characterized in that the cells are in culture suspension. 14. Composition, according to any one of claims 1 to 13, characterized in that the mammalian cells are HEK 293E cells (human embryonic kidney) or HEK 293F cells.