CN119242712A - Adeno-associated virus vector carrying miRNA-486 gene specifically targeting skeletal muscle cells and pharmaceutical composition - Google Patents

Adeno-associated virus vector carrying miRNA-486 gene specifically targeting skeletal muscle cells and pharmaceutical composition Download PDF

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CN119242712A
CN119242712A CN202411354648.9A CN202411354648A CN119242712A CN 119242712 A CN119242712 A CN 119242712A CN 202411354648 A CN202411354648 A CN 202411354648A CN 119242712 A CN119242712 A CN 119242712A
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adeno
promoter
skeletal muscle
gene
associated virus
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李方晖
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Nanjing Normal University
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Nanjing Normal University
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Abstract

The invention discloses an adeno-associated virus vector carrying miRNA-486 gene and a pharmaceutical composition for specifically targeting skeletal muscle cells, belonging to the technical field of medicines for treating muscle or neuromuscular system diseases. The adeno-associated virus vector comprises a packaging promoter, an exogenous gene, SV40polyA, a skeletal muscle specific promoter and a miRNA-486 gene coding sequence, wherein the skeletal muscle specific promoter controls the expression of the miRNA-486 gene coding sequence. The invention can specifically target skeletal muscle cells, effectively improve the delivery efficiency of adeno-associated virus and reduce the side effect of adeno-associated virus gene therapy.

Description

Adeno-associated viral vector carrying miRNA-486 gene and specific targeting skeletal muscle cell, and pharmaceutical composition
Technical Field
The invention relates to the technical field of medicaments for treating diseases of muscle or neuromuscular systems, in particular to an adeno-associated virus vector and a pharmaceutical composition carrying miRNA-486 genes, which specifically target skeletal muscle cells.
Background
MiRNA-486 is a kind myomiRs which is mainly derived from skeletal muscle and has a high expression abundance in skeletal muscle (Zhang Wendian. MicroRNAs and sarcopenia [ J ]. Biomedical, 2022,3 (01): 67-69). MicroRNA-486 is a miRNA with stem-loop sequence embedded in ANKYRIN (ANK 1) site, and is strictly conserved in mammals. Research shows that MicroRNA-486 has great potential as a therapeutic target for improving muscular atrophy through exercise intervention (Qiu D,ZhangY,Ni P,et al.Muscle-enriched microRNA-486-mediated regulation ofmuscular atrophy and exercise[J].Journal of Physiology and Biochemistry,2024:1-15.).
However, delivery of therapeutic genes to specific cells in the body has been challenging, particularly in large anatomically distributed tissues (e.g., skeletal muscle). However, lack of muscle cell specificity can lead to unpredictable systemic adverse effects. Therefore, it remains a very important topic to explore targeting strategies to improve drug efficacy, reduce side effects, and how to deliver drugs efficiently to skeletal muscle.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a miRNA-486 gene-carrying adeno-associated virus vector of a specific targeting skeletal muscle cell, wherein the miRNA-486 gene-carrying adeno-associated virus vector of the specific targeting skeletal muscle cell is used for packaging adeno-associated viruses without expressing the miRNA-486 gene, and the packaged adeno-associated viruses can express the miRNA-486 gene under the action of a tissue specific promoter after infecting the skeletal muscle cell.
It is a second object of the present invention to provide a recombinant adeno-associated viral vector system which is capable of successfully packaging adeno-associated viruses whose capsid proteins are AAV1 or AAV7 serotypes and which is capable of selectively targeting skeletal muscle cells.
It is a further object of the present invention to provide a recombinant adeno-associated virus modified with an RGD motif, wherein the RGD motif is linked to a fibrin on the capsid protein of the recombinant adeno-associated virus.
The fourth object of the present invention is to provide a pharmaceutical composition, which can improve the delivery efficiency of adeno-associated virus, reduce the immunogenicity of adeno-associated virus, and reduce the clearance rate of adeno-associated virus in vivo, thereby improving the therapeutic effect and the targeting of liposome.
The invention adopts the following technical scheme:
One of the purposes of the invention is to provide an adeno-associated virus vector carrying miRNA-486 gene which specifically targets skeletal muscle cells, wherein the adeno-associated virus vector comprises a packaging promoter, an exogenous gene, SV40 polyA, a skeletal muscle specific promoter and a miRNA-486 gene coding sequence, and the skeletal muscle specific promoter controls the expression of the miRNA-486 gene coding sequence.
Further, the skeletal muscle specific promoter is one of MCK (creatine kinase) promoter, 3× ENHANCER MCK, MYOG promoter, SM22a promoter, ACTA1 (α -actin) promoter, MHCK7 promoter.
Further, the packaging promoter is one of a CMV (cytomegalovirus) promoter and a PGK (phosphoglycerate kinase) promoter.
The second object of the invention is to provide a recombinant adeno-associated virus vector system, which comprises the adeno-associated virus vector carrying miRNA-486 gene and adenovirus genome plasmid which specifically targets skeletal muscle cells, wherein the adenovirus genome plasmid comprises capsid protein genes of AAV1 or AAV 7.
Further, a fiber gene is arranged near the E4 region of the adenovirus genome plasmid, and a SpyTag sequence is inserted into the fiber gene.
Further, the adenovirus genome of the adenovirus genome plasmid is provided with a fiber gene near the E4 region, and the HI loop region in the fiber gene is inserted with an RGD sequence (arginine-glycine-aspartic acid).
The invention further provides a recombinant adeno-associated virus modified by RGD motif, which is obtained by cotransfecting a packaging cell line with the recombinant adeno-associated virus vector system, and the obtained recombinant adeno-associated virus particles are subjected to covalent modification by RGD motif fused with SpyCatcher, so that the recombinant adeno-associated virus modified by RGD motif is obtained.
The invention also provides a pharmaceutical composition, which comprises the recombinant adeno-associated virus modified by RGD motif, cationic liposome nano-particles and nano-microbubbles, wherein the recombinant adeno-associated virus modified by RGD motif and the nano-microbubbles are coated in the capsules of the cationic liposome nano-particles.
Further, the method also comprises an IL6R specific agonist antibody, wherein the IL6R specific agonist antibody is MT-18, the surface of the cationic liposome nanoparticle is modified by the RGD motif, and the modification method comprises the following steps:
The RGD motif and cysteine are connected to the RGD motif under the enzyme catalysis of transpeptidase, and then react with a maleimide reagent to form a stable thioether bond, and the stable thioether bond is coupled to the surface of the cationic liposome nanoparticle.
Further, the preparation method of the nano-microbubbles comprises the following steps:
S1, microfluidic production of nano microbubbles and micro microbubbles, wherein DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine) and DSPE-PEG2000 (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-n- [ methoxy (polyethylene glycol) -2000 ]) are mixed according to a molar ratio of 95:5 to prepare a mixture with a total lipid concentration of 2 mg/mL;
S2, dissolving lipid in a 1:1 mixture of chloroform and methanol, drying under nitrogen to remove solvent, then re-suspending in Phosphate Buffered Saline (PBS) solution containing 1% (v/v) glycerol, and then combining the lipid solution with C4F10 (PFB) gas in a multiplex micro-spray microfluidic device to generate bubbles;
S3, separating nano microbubbles, namely taking out a nano bubble sample from the bottom of the bottle by using a syringe and a fine needle after 1h, and filtering by using a Polytetrafluoroethylene (PTFE) film with the wavelength of 800nm to remove any large bubbles so as to obtain the nano microbubbles.
Compared with the prior art, the invention has the beneficial effects that:
The invention provides an adeno-associated virus vector carrying miRNA-486 gene of a specific targeting skeletal muscle cell, which comprises a packaging promoter and a skeletal muscle specific promoter, wherein the packaging promoter is used for packaging adeno-associated virus, the adeno-associated virus packaged by the skeletal muscle specific promoter can express the miRNA-486 gene under the action of a tissue specific promoter after infecting the skeletal muscle cell, and exogenous genes such as GFP (green fluorescent protein) are used for observing the transfection efficiency of a recombinant adeno-associated virus vector system under a fluorescent microscope.
The RGD motif modified recombinant adeno-associated virus provided by the invention has the advantages that the fiber protein on the capsid protein of the RGD motif modified recombinant adeno-associated virus is connected with the RGD motif, the RGD motif can be combined with the integrin heterodimer receptor on the surface of skeletal muscle cells, and the capability of infecting skeletal muscle cells of the recombinant adeno-associated virus is improved.
The fourth object of the present invention is to provide a pharmaceutical composition, in which adeno-associated virus is coated in the capsule of cationic liposome nanoparticle, so that the delivery efficiency of adeno-associated virus is improved, and at the same time, the immunogenicity of adeno-associated virus is reduced, and the clearance rate of adeno-associated virus in vivo is reduced, thereby improving the therapeutic effect, and the surface of cationic liposome nanoparticle is modified with the RGD motif, and at the same time, the targeting of cationic liposome nanoparticle is improved.
It is a fourth object of the present invention to provide a pharmaceutical composition wherein the cationic liposome nanoparticle has an intracapsular coating of nanovesicles which under ultrasound, during High Intensity Focused Ultrasound (HIFU), are also found to have a sample temperature above the predictive threshold, such that PFB droplets undergo a phase transition from the liquid to the gaseous state, followed by a 3-fold increase in the diameter of the nanovesicles, resulting in the rupture of the cationic liposome nanoparticles and release of adeno-associated virus, which infect skeletal muscle cells using the surface RGD motif and integrin heterodimer receptor binding, wherein the intracapsular coating of cationic liposome nanoparticles further comprises IL6R specific agonist antibody MT-18, increasing the residence of Treg in muscle by IL6 ra signaling and inducing its phenotypic and functional maturation, recovering miRNA-486 which restores impaired muscle regeneration capacity, and skeletal muscle cell expression in synergy for the treatment of sarcopenia.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings that are required for the embodiments will be briefly described below, and it will be apparent that the drawings in the following description are only embodiments of the present invention, and that other drawings may be obtained from the provided drawings without inventive effort for a person skilled in the art.
Fig. 1 is an image of the white light field (upper) and green fluorescence (lower) of skeletal muscle tissue, heart, lung, spleen, liver and kidney in experimental examples.
Detailed Description
The present invention will be further described with reference to specific embodiments, and it should be noted that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below.
The multi-path micro-spray micro-fluidic device is purchased from micro-nano cubic technology (Beijing) limited company, is equipment developed by utilizing a micro-fluidic technology, can accurately control and operate micro-fluid, generates micro-spray through a plurality of micro-channels, and can realize various applications such as high-throughput experiments, drug screening, tissue engineering, single-cell analysis and the like.
C4F10, also known as perfluorobutane or Perfluorobutane, is a compound that is gaseous at room temperature and is available from Neofluoride Inc. of Hemsl. It is used in medicine as an intravascular ultrasound contrast medium under the trade name Sonazoid. C4F10 has a very low melting point (-129 ℃) and boiling point (-2.1 ℃) with a density of 1.6484g/cu cm (25 ℃) and a vapor pressure of 2010mm Hg at 25 ℃.
DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine) was purchased from shanghai pun biosciences, inc.
DSPE-PEG2000 (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-n- [ methoxy (polyethylene glycol) -2000 ]), CAS number 474922-20-8, available from SiAnruixi Biotechnology Co.
The specific agonist antibody was MT-18 purchased from Abcam China.
Example 1
The embodiment provides an adeno-associated virus vector carrying miRNA-486 gene of specific targeting skeletal muscle cells, wherein the adeno-associated virus vector comprises a packaging promoter, an exogenous gene, SV40 polyA, a skeletal muscle specific promoter and a miRNA-486 gene coding sequence, and the skeletal muscle specific promoter controls the expression of the miRNA-486 gene coding sequence. The miRNA-486 Gene coding sequence is shown in Gene ID 619554, genBank accession number NC_000008.
In this example, the packaging promoter functions to initiate expression of viral replication proteins (Rep) and capsid proteins (Cap), which are typically used to drive expression of these proteins during production of the recombinant AAV vector, thereby enabling efficient replication and packaging of the virus in the host cell, and to initiate expression of the foreign gene, GFP gene, which is expressed during packaging, for observing transfection efficiency of the recombinant adeno-associated viral vector system under a fluorescent microscope. The packaging promoter may be one of a CMV (cytomegalovirus) promoter and a PGK (phosphoglycerate kinase) promoter.
The skeletal muscle specific promoter is one of MCK (creatine kinase) promoter, 3× ENHANCERMCK, MYOG promoter, SM22a promoter, ACTA1 (alpha-actin) promoter, MHCK promoter. The function of the skeletal muscle-specific promoter is to initiate expression of the miRNA-486 gene in skeletal muscle cells.
In this example, the MCK (creatine kinase) promoter is derived from the creatine kinase gene of mice and is capable of driving the specific expression of the gene in skeletal muscle and cardiac muscle.
3× ENHANCERMCK, which is a modified creatine kinase gene promoter, has higher muscle specificity and expression efficiency.
ACTA1 (alpha-actin) promoter ACTA1 promoter is derived from human skeletal muscle alpha-actin gene, and can drive specific expression of gene in skeletal muscle.
MHCK7 promoter MHCK promoter consists of enhancer/promoter region of mouse Muscle Creatine Kinase (MCK) gene and enhancer region of alpha-myosin heavy chain gene, and can drive specific expression of gene in mouse skeletal muscle and cardiac muscle.
In this embodiment, fiber genes are near the E4 region of the adenovirus genome plasmid, spyTag sequences are inserted into the fiber genes, and after packaging, the capsid proteins of the adeno-associated virus particles naturally fuse with SpyTag peptides and then are covalently bonded with RGD peptides fused with Spycatcher, so that redirection of the adeno-associated virus vector is realized, and efficient and specific tissue targeting is ensured.
The SpyTag-SpyCatcher system can also be used for constructing a self-assembled multienzyme complex, and the self-assembly of multienzyme can be realized in cells through the interaction of the SpyTag and the SpyCatcher, so that the catalytic efficiency of enzymatic reaction is improved.
The fiber gene is located near the E4 region of the adenovirus genome, which is not centrally located in the genome, but rather is offset to one side. Fibrin consists of three parts, tail (tail), stem (leaf) and head (knob), where the head (knob) is the part that interacts directly with host cell receptors.
As another embodiment, the adenovirus genome of the adenovirus genome plasmid has fiber gene near E4 region, the HI loop region in the fiber gene is inserted with RGD sequence (arginine-glycine-aspartic acid), and the capsid protein of the adenovirus-associated virus particle is naturally fused with RGD peptide after packaging.
Example 2
This example provides packaging of recombinant adeno-associated virus modified by RGD motif, co-transfecting the adenovirus genome plasmid carrying miRNA-486 gene and constructed in example 1 into packaging cells by liposome transfection reagent, the adenovirus genome plasmid comprising capsid protein gene of AAV7, after 24h transfection, observing transfection efficiency under fluorescence microscope, the adenovirus genome plasmid carrying miRNA-486 gene and specifically targeting skeletal muscle cell, the adenovirus genome plasmid being capsid protein gene of AAV7, after 72h transfection, collecting culture medium supernatant, concentrating by ultrafiltration tube.
Example 3
This example provides a pharmaceutical composition comprising a recombinant adeno-associated virus modified by RGD motif packaged in example 2, and IL 6R-specific agonist antibody MT-18 and nanovesicles coated with RGD motif modified cationic liposome nanoparticles, wherein MT-18 increases the residence of tregs in muscle via IL6Rα signaling and induces their phenotype and functional maturation to regain the ability to restore impaired muscle regeneration, and skeletal muscle cell expressed miRNA-486 synergistically treats sarcopenia. The nanobubbles were also found to be above the predictive threshold during High Intensity Focused Ultrasound (HIFU) under ultrasound, such that the C4F10 (PFB) droplets underwent a phase transition from the liquid to the gaseous state, followed by a 3-fold increase in diameter of the nanobubbles, resulting in rupture of the cationic liposome nanoparticles and release of adeno-associated virus that infected skeletal muscle cells using surface RGD motifs to bind with integrin heterodimer receptors.
In this embodiment, the surface of the cationic liposome nanoparticle is modified by an RGD motif, and the modification method includes the following steps:
under the enzyme catalysis of transpeptidase, the RGD motif and cysteine are connected to the RGD motif, and then react with maleimide reagent to form stable thioether bond, which is coupled to the surface of cationic liposome nano-particle.
In this embodiment, the preparation method of the nano-microbubbles includes the following steps:
S1, microfluidic production of nano microbubbles and micro microbubbles, wherein DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine) and DSPE-PEG2000 (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-n- [ methoxy (polyethylene glycol) -2000 ]) are mixed according to a molar ratio of 95:5 to prepare a mixture with a total lipid concentration of 2 mg/mL;
S2, dissolving lipid in a 1:1 mixture of chloroform and methanol, drying under nitrogen to remove solvent, then re-suspending in Phosphate Buffered Saline (PBS) solution containing 1% (v/v) glycerol, and then combining the lipid solution with C4F10 (PFB) gas in a multiplex micro-spray microfluidic device to generate bubbles;
S3, separating the nano microbubbles, namely taking out a nano bubble sample from the bottom of the bottle by using a syringe and a fine needle after 1h, and filtering by using a Polytetrafluoroethylene (PTFE) film with the wavelength of 800nm to remove any large bubbles so as to obtain the nano microbubbles.
The pharmaceutical composition of this embodiment further comprises a pharmaceutically acceptable carrier or excipient, and the pharmaceutical composition of this embodiment is an injectable formulation.
The adenovirus genome plasmid is plasmid pHelper.
The RGD motif is capable of binding to cell surface receptors of the integrin family, an integrin class of transmembrane receptors that mediate interactions between cells and the extracellular matrix (ECM).
Integrins are transmembrane receptors composed of two subunits, α and β, which play a key role in regulating cell adhesion, signaling, cell migration, and tissue repair. Binding of the RGD motif to integrins can activate intracellular signaling pathways, affecting cell behavior. For example, binding of the RGD motif to integrin αvβ6 may be involved in certain physiological and pathological processes, such as tumor metastasis and tissue regeneration.
Comparative example
Unlike example 3, the pharmaceutical composition provided in the comparative example is a cationic liposome nanoparticle coated adeno-associated virus, the capsid protein of which is not modified by the RGD motif, and the surface of the liposome nanoparticle is not modified by the RGD motif, and the dosage form is an injection preparation.
Experimental example
The pharmaceutical composition prepared in example 3 and the pharmaceutical composition prepared in comparative example were injected intravenously at a dose of 20mg/kg as experimental group and control group, respectively. After 3h of injection, the mice were dissected to obtain images of the white light fields (upper) and green fluorescence (lower) of skeletal muscle tissue, heart, lung, spleen, liver and kidney for targeting. The results in fig. 1 show that the experimental groups 1 and 2 are located in skeletal muscle tissue, indicating that the pharmaceutical composition prepared in example 3 is stable and has good targeting.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.

Claims (10)

1. An adeno-associated viral vector carrying a miRNA-486 gene specifically targeting skeletal muscle cells, wherein the adeno-associated viral vector comprises a packaging promoter, an exogenous gene, SV40 polyA, a skeletal muscle specific promoter, a miRNA-486 gene coding sequence, and wherein the skeletal muscle specific promoter controls expression of the miRNA-486 gene coding sequence.
2. The adeno-associated viral vector carrying the miRNA-486 gene specifically targeting skeletal muscle cells of claim 1, wherein the skeletal muscle-specific promoter is one of MCK promoter, 3 x ENHANCERMCK, MYOG promoter, SM22a promoter, ACTA1 promoter, MHCK7 promoter.
3. The adeno-associated viral vector carrying the miRNA-486 gene specifically targeting skeletal muscle cells of claim 1, wherein the packaging promoter is one of a CMV promoter and a PGK promoter.
4. A recombinant adeno-associated viral vector system comprising an adeno-associated viral vector carrying the miRNA-486 gene specifically targeted to skeletal muscle cells of claim 1 and an adenovirus genome plasmid comprising the capsid protein gene of AAV1 or AAV 7.
5. A recombinant adeno-associated viral vector system according to claim 4, wherein the adenovirus genome of the adenovirus genome plasmid has a fiber gene adjacent to the E4 region, wherein the fiber gene has an SpyTag sequence inserted therein.
6. A recombinant adeno-associated viral vector system according to claim 4, wherein the adenovirus genome of the adenovirus genome plasmid is in the vicinity of the E4 region of the adenovirus genome, and wherein the HI loop region of the fiber gene is inserted into the RGD sequence.
7. The recombinant adeno-associated virus modified by RGD motif is characterized in that the recombinant adeno-associated virus is obtained by cotransfecting a packaging cell line with a recombinant adeno-associated virus vector system as claimed in claim 4, and the obtained recombinant adeno-associated virus particle is subjected to covalent modification by RGD motif fused with Spycatcher to obtain the recombinant adeno-associated virus modified by RGD motif.
8. A pharmaceutical composition comprising an RGD motif-modified recombinant adeno-associated virus of claim 7, a cationic liposome nanoparticle and a nanovesicle, wherein the cationic liposome nanoparticle has an intracapsular coating with the RGD motif-modified recombinant adeno-associated virus and the nanovesicle.
9. The pharmaceutical composition of claim 8, further comprising an IL 6R-specific agonist antibody, wherein the IL 6R-specific agonist antibody is MT-18, wherein the surface of the cationic liposome nanoparticle is modified with the RGD motif, and wherein the modification method comprises the steps of:
The RGD motif and cysteine are connected to the RGD motif under the enzyme catalysis of transpeptidase, and then react with a maleimide reagent to form a stable thioether bond, and the stable thioether bond is coupled to the surface of the cationic liposome nanoparticle.
10. A pharmaceutical composition according to claim 8, wherein the method of preparing the nanomicrobubbles comprises the steps of:
S1, microfluidic production of nano microbubbles and micro microbubbles, wherein 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-n- [ methoxy (polyethylene glycol) -2000] are mixed according to a molar ratio of 95:5 to prepare a mixture with a total lipid concentration of 2 mg/mL;
S2, dissolving lipid in a 1:1 mixture of chloroform and methanol, drying under nitrogen to remove the solvent, then re-suspending in phosphate buffer saline solution containing 1% glycerol by volume, and then combining the lipid solution with C4F10 gas in a multi-path micro-spray microfluidic device to generate bubbles;
s3, separating the nano microbubbles, namely taking out a nano bubble sample from the bottom of the bottle by using a syringe and a fine needle after 1h, and then filtering by using a polytetrafluoroethylene film with the wavelength of 800nm to remove any large bubbles so as to obtain the nano microbubbles.
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