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

Abstract

The invention discloses an adeno-associated virus vector carrying a miRNA-486 gene and specifically targeting skeletal muscle cells and a pharmaceutical composition, and belongs to the technical field of drugs 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 is used for controlling the expression of the miRNA-486 gene coding sequence. According to the present invention, the skeletal muscle cells can be specifically targeted, the adeno-associated virus delivery efficiency can be effectively improved, and the side effect of adeno-associated virus gene therapy can be reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for treating muscle or neuromuscular system diseases, and in particular to an adeno-associated virus vector carrying a miRNA-486 gene that specifically targets skeletal muscle cells and a drug composition. Background Art

[0002] MiRNA-486 is a type of myomiRs that is mainly derived from skeletal muscle and is expressed at a high abundance in skeletal muscle (Zhang Wendian. microRNAs and sarcopenia [J]. Biomedicalization, 2022, 3(01): 67-69). MicroRNA-486 is a miRNA with a stem-loop sequence embedded in the ANKYRIN1 (ANK1) site, which is strictly conserved in mammals. Studies have shown that microRNA-486 has great potential to become a therapeutic target for improving muscle atrophy through exercise intervention (Qiu D, Zhang Y, Ni P, et al. Muscle-enriched microRNA-486-mediated regulation of muscular atrophy and exercise [J]. Journal of Physiology and Biochemistry, 2024: 1-15.).

[0003] However, delivering therapeutic genes to specific cells in the body has always been challenging, especially in tissues with large anatomical distributions such as skeletal muscle. However, the lack of muscle cell specificity can lead to unpredictable systemic adverse reactions. Therefore, it is necessary to explore targeted strategies to improve drug efficacy and reduce side effects, and how to effectively deliver drugs to skeletal muscle remains a very important topic. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, one of the objects of the present invention is to provide an adeno-associated virus vector carrying the miRNA-486 gene that specifically targets skeletal muscle cells. The adeno-associated virus vector carrying the miRNA-486 gene that specifically targets skeletal muscle cells is used to package adeno-associated viruses that do not express the miRNA-486 gene. After the packaged adeno-associated viruses infect skeletal muscle cells, they can express the miRNA-486 gene under the action of a tissue-specific promoter.

[0005] The second object of the present invention is to provide a recombinant adeno-associated virus vector system, which can successfully package adeno-associated viruses, the capsid protein of which is AAV1 or AAV7 serotype, and can selectively target skeletal muscle cells.

[0006] The third object of the present invention is to provide a recombinant adeno-associated virus modified with an RGD motif, wherein the fiber protein on the capsid protein of the recombinant adeno-associated virus modified with an RGD motif is connected to the RGD motif.

[0007] The fourth object of the present invention is to provide a pharmaceutical composition that can improve the delivery efficiency of adeno-associated virus while reducing the immunogenicity of adeno-associated virus and lowering its clearance rate in the body, thereby improving the therapeutic effect and improving the targeting of liposomes.

[0008] The purpose of the present invention is achieved by the following technical solutions:

[0009] One of the purposes of the present invention is to provide an adeno-associated virus vector carrying the miRNA-486 gene that specifically targets skeletal muscle cells. 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. The skeletal muscle-specific promoter controls the expression of the miRNA-486 gene coding sequence.

[0010] Furthermore, the skeletal muscle-specific promoter is one of the following: MCK (creatine kinase) promoter, 3×enhancer MCK, MYOG promoter, SM22a promoter, ACTA1 (α-actin) promoter, and MHCK7 promoter.

[0011] Furthermore, the packaging promoter is one of a CMV (cytomegalovirus) promoter and a PGK (phosphoglycerate kinase) promoter.

[0012] The second object of the present invention is to provide a recombinant adeno-associated virus vector system, including an adeno-associated virus vector carrying the miRNA-486 gene that specifically targets skeletal muscle cells and an adenovirus genome plasmid, wherein the adenovirus genome plasmid includes the capsid protein gene of AAV1 or AAV7.

[0013] Furthermore, the fiber gene is located near the E4 region of the adenovirus genome of the adenovirus genome plasmid, and a SpyTag sequence is inserted into the fiber gene.

[0014] Furthermore, the fiber gene is located near the E4 region of the adenovirus genome of the adenovirus genome plasmid, and an RGD sequence (arginine-glycine-aspartic acid) is inserted into the HI loop region of the fiber gene.

[0015] The third object of the present invention is to provide a recombinant adeno-associated virus modified with an RGD motif, which is obtained by co-transfecting a packaging cell line with a recombinant adeno-associated virus vector system, and the obtained recombinant adeno-associated virus particles are covalently modified with the RGD motif fused with SpyCatcher to obtain the recombinant adeno-associated virus modified with the RGD motif.

[0016] The fourth object of the present invention is to provide a pharmaceutical composition, comprising the recombinant adeno-associated virus modified with an RGD motif, cationic liposome nanoparticles and nanobubbles, wherein the recombinant adeno-associated virus modified with an RGD motif and the nanobubbles are encapsulated in the capsules of the cationic liposome nanoparticles.

[0017] Furthermore, it also includes an IL6R specific agonist antibody, wherein the IL6R specific agonist antibody is MT-18; the surface of the cationic liposome nanoparticle is modified with the RGD motif, and the modification method includes the following steps:

[0018] The RGD motif and cysteine ​​are connected to the RGD motif under the enzymatic catalysis of transpeptidase, and then reacted with a maleimide reagent to form a stable thioether bond, which is then coupled to the surface of the cationic liposome nanoparticle.

[0019] Furthermore, the method for preparing the nanobubbles comprises the following steps:

[0020] S1. Microfluidic production of nanobubbles and microbubbles: DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000]) were mixed at a molar ratio of 95:5 to prepare a mixture with a total lipid concentration of 2 mg / mL;

[0021] S2, lipids were dissolved in a 1:1 mixture of chloroform and methanol, dried under nitrogen to remove the solvent, and then resuspended in a phosphate-buffered saline (PBS) solution containing 1% (v / v) glycerol. The lipid solution was then combined with C4F10 (PFB) gas in a multichannel microspray microfluidic device to generate bubbles;

[0022] S3. Isolation of nanobubbles: After 1 hour, the nanobubble sample was taken out from the bottom of the bottle using a syringe and a fine needle, and then filtered using an 800 nm polytetrafluoroethylene (PTFE) membrane to remove any large bubbles to obtain the nanobubbles.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides an adeno-associated virus vector carrying the miRNA-486 gene that specifically targets skeletal muscle cells. The vector comprises a packaging promoter and a skeletal muscle-specific promoter. The packaging promoter is used to package adeno-associated virus. After the adeno-associated virus packaged by the skeletal muscle-specific promoter infects skeletal muscle cells, the miRNA-486 gene can be expressed under the action of the tissue-specific promoter. Exogenous genes such as GFP (green fluorescent protein) are used to observe the transfection efficiency of the recombinant adeno-associated virus vector system under a fluorescence microscope.

[0025] The present invention provides a recombinant adeno-associated virus modified with an RGD motif. The fibrous protein on the capsid protein of the recombinant adeno-associated virus modified with the RGD motif is connected to the RGD motif. The RGD motif can bind to the integrin heterodimer receptor on the surface of skeletal muscle cells, thereby improving the ability of the recombinant adeno-associated virus to infect skeletal muscle cells.

[0026] The fourth object of the present invention is to provide a pharmaceutical composition, in which adeno-associated virus is encapsulated in the capsule of cationic liposome nanoparticles, which can improve the delivery efficiency of adeno-associated virus while reducing the immunogenicity of adeno-associated virus and reducing its clearance rate in the body, thereby improving the therapeutic effect. The surface of the cationic liposome nanoparticles is modified with the RGD motif, and the targeting of the cationic liposome nanoparticles is improved.

[0027] The fourth object of the present invention is to provide a pharmaceutical composition, wherein the cationic liposome nanoparticles are coated with nanobubbles in the capsule, and the nanobubbles are under the action of ultrasound. During the high-intensity focused ultrasound (HIFU) process, the sample temperature is also found to be higher than the predicted threshold, so that the PFB droplets undergo a phase change from liquid to gas state, and then the diameter of the nanobubbles increases by 3 times, resulting in the rupture of the cationic liposome nanoparticles and the release of adeno-associated viruses. The adeno-associated viruses utilize the RGD motif on the surface to bind to the integrin heterodimer receptor to infect skeletal muscle cells; the cationic liposome nanoparticles are coated with nanobubbles in the capsule, and the nanobubbles also include an IL6R-specific agonist antibody, MT-18, which increases the residence of Treg in the muscle through IL6Rα signal transduction, and induces its phenotypic and functional maturation, regains the ability to restore damaged muscle regeneration, and synergizes with miRNA-486 expressed by skeletal muscle cells to treat sarcopenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0029] Figure 1These are white light field (upper) and green fluorescence (lower) images of skeletal muscle tissue, heart, lung, spleen, liver, and kidney in the experimental example. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific embodiments. It should be noted that, under the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0031] The multi-channel microspray microfluidic device was purchased from Micro-Nano Cube Technology (Beijing) Co., Ltd. The multi-channel microspray microfluidic device is a device developed using microfluidic technology. It can accurately control and manipulate tiny fluids. This device generates microsprays through multiple microchannels and can achieve a variety of applications such as high-throughput experiments, drug screening, tissue engineering, and single-cell analysis.

[0032] C4F10, also known as perfluorobutane or Perfluorobutane, is a compound that is a gas at room temperature and is purchased from Changzhou New Fluoro Chemical Co., Ltd. It is used as an intravascular ultrasound contrast agent in medicine under the trade name Sonazoid. C4F10 has a very low melting point (-129°C) and boiling point (-2.1°C), a density of 1.6484 g / cu cm (25°C), and a vapor pressure of 2010 mm Hg at 25°C.

[0033] DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) was purchased from Shanghai Pengshuo Biotechnology Co., Ltd.

[0034] DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000]), CAS No. 474922-20-8, was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0035] The specific agonist antibody was MT-18 purchased from Abcam China.

[0036] Example 1

[0037] This embodiment provides an adeno-associated virus vector carrying a miRNA-486 gene that specifically targets skeletal muscle cells. 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. 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.

[0038] In this embodiment, the role of the packaging promoter is to start the expression of viral replication protein (Rep) and capsid protein (Cap) which are usually used to drive the expression of viral replication protein (Rep). In the production process of recombinant AAV vector, the packaging promoter controls the expression of these proteins, so that the virus can be effectively replicated and packaged in the host cell, and starts the expression of foreign genes. The foreign gene is the GFP gene. The GFP protein is expressed during the packaging process, which is used to observe the transfection efficiency of the recombinant adeno-associated virus vector system under a fluorescence microscope. The packaging promoter can be one of the CMV (cytomegalovirus) promoter and the PGK (phosphoglycerate kinase) promoter.

[0039] The skeletal muscle specific promoter is one of the MCK (creatine kinase) promoter, 3×enhancerMCK, MYOG promoter, SM22a promoter, ACTA1 (α-actin) promoter, and MHCK7 promoter. The function of the skeletal muscle specific promoter is to activate the expression of the miRNA-486 gene in skeletal muscle cells.

[0040] In this embodiment, MCK (creatine kinase) promoter: MCK promoter is derived from the mouse creatine kinase gene and can drive the specific expression of the gene in skeletal muscle and cardiac muscle.

[0041] 3×enhancerMCK: This is a modified creatine kinase gene promoter with higher muscle specificity and expression efficiency.

[0042] ACTA1 (α-actin) promoter: The ACTA1 promoter is derived from the human skeletal muscle α-actin gene and can drive the specific expression of the gene in skeletal muscle.

[0043] MHCK7 promoter: The MHCK7 promoter consists of the enhancer / promoter region of the mouse muscle creatine kinase (MCK) gene and the enhancer region of the α-myosin heavy chain gene, and can drive the specific expression of the gene in mouse skeletal muscle and cardiac muscle.

[0044] In this embodiment, the fiber gene is located near the E4 region of the adenovirus genome of the adenovirus genome plasmid. The SpyTag sequence is inserted into the fiber gene. After packaging, the capsid protein of the adeno-associated virus particle will naturally fuse with the SpyTag peptide, and then covalently bind to the RGD peptide fused with SpyCatcher to achieve redirection of the adeno-associated virus vector and ensure efficient and specific tissue targeting.

[0045] The SpyTag-SpyCatcher system can also be used to construct self-assembling multi-enzyme complexes. Through the interaction between SpyTag and SpyCatcher, multi-enzyme self-assembly can be achieved in cells, thereby improving the catalytic efficiency of enzymatic reactions.

[0046] The fiber gene is located near the E4 region of the adenovirus genome, which is not located in the center of the genome but rather to one side. The fiber protein consists of three parts: the tail, the shaft, and the knob, of which the knob is the part that directly interacts with the host cell receptor.

[0047] As another embodiment, the fiber gene is located near the E4 region of the adenovirus genome of the adenovirus genome plasmid, and the RGD sequence (arginine-glycine-aspartic acid) is inserted into the HI loop region of the fiber gene. After packaging, the RGD peptide will naturally fuse to the capsid protein of the adeno-associated virus particle.

[0048] Example 2

[0049] This embodiment provides the packaging of a recombinant adeno-associated virus modified with an RGD motif. The adeno-associated virus vector carrying the miRNA-486 gene and the adenovirus genome plasmid specifically targeting skeletal muscle cells constructed in Example 1 are co-transfected into packaging cells by a liposome transfection reagent. The adenovirus genome plasmid includes the capsid protein gene of AAV7. After transfection for 24 hours, the transfection efficiency is observed under a fluorescence microscope. The adeno-associated virus vector carrying the miRNA-486 gene and the adenovirus genome plasmid specifically targeting skeletal muscle cells, the adenovirus genome plasmid includes the capsid protein gene of AAV7. After transfection for 72 hours, the culture medium supernatant is collected and concentrated using an ultrafiltration tube.

[0050] Example 3

[0051] This embodiment provides a pharmaceutical composition, wherein a recombinant adeno-associated virus modified with an RGD motif, an IL6R-specific agonist antibody MT-18, and nano-microbubbles packaged in Example 2 are coated with cationic liposome nanoparticles modified with an RGD motif, and MT-18 increases the residence of Treg in muscle through IL6Rα signal transduction, and induces its phenotypic and functional maturation, regains the ability to restore damaged muscle regeneration, and synergistically treats sarcopenia with miRNA-486 expressed by skeletal muscle cells. Under the action of ultrasound, during high-intensity focused ultrasound (HIFU), the sample temperature of the nanobubbles was also found to be higher than the predicted threshold, causing the C4F10 (PFB) droplets to undergo a phase transition from a liquid to a gas state, and then the diameter of the nanobubbles increased by 3 times, resulting in the rupture of the cationic liposome nanoparticles and the release of the adeno-associated virus, which infects skeletal muscle cells by binding to the integrin heterodimer receptor using the surface RGD motif.

[0052] In this embodiment, the surface of the cationic liposome nanoparticles is modified with the RGD motif, and the modification method comprises the following steps:

[0053] The RGD motif and cysteine ​​are connected to the RGD motif under the enzymatic catalysis of transpeptidase, and then reacted with a maleimide reagent to form a stable thioether bond, which is then coupled to the surface of the cationic liposome nanoparticles through the thioether bond.

[0054] In this embodiment, the method for preparing nanobubbles comprises the following steps:

[0055] S1. Microfluidic production of nanobubbles and microbubbles: DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000]) were mixed at a molar ratio of 95:5 to prepare a mixture with a total lipid concentration of 2 mg / mL;

[0056] S2, lipids were dissolved in a 1:1 mixture of chloroform and methanol, dried under nitrogen to remove the solvent, and then resuspended in a phosphate-buffered saline (PBS) solution containing 1% (v / v) glycerol. The lipid solution was then combined with C4F10 (PFB) gas in a multichannel microspray microfluidic device to generate bubbles;

[0057] S3. Isolation of nanobubbles: After 1 hour, the nanobubble sample was taken out from the bottom of the bottle using a syringe and a fine needle, and then filtered using an 800 nm polytetrafluoroethylene (PTFE) membrane to remove any large bubbles and obtain nanobubbles.

[0058] The pharmaceutical composition of this embodiment further includes a pharmaceutically acceptable carrier or excipient, and the pharmaceutical composition of this embodiment is an injection preparation.

[0059] The adenovirus genome plasmid is plasmid pHelper.

[0060] The RGD motif is able to bind to cell surface receptors of the integrin family, a class of transmembrane receptors that mediate the interaction between cells and the extracellular matrix (ECM).

[0061] Integrins are transmembrane receptors composed of two subunits, α and β, which play a key role in regulating cell adhesion, signal transduction, cell migration and tissue repair. The binding of RGD motif to integrin can activate intracellular signaling pathways and affect cell behavior. For example, the binding of RGD motif to integrin αvβ6 may be involved in certain physiological and pathological processes, such as tumor metastasis and tissue regeneration.

[0062] Comparative Example

[0063] Different from Example 3, the pharmaceutical composition provided in the comparative example is cationic liposome nanoparticles encapsulating adeno-associated virus, the capsid protein of the adeno-associated virus is not modified by the RGD motif, and the surface of the liposome nanoparticles is not modified by the RGD motif, and the dosage form is an injection preparation.

[0064] Experimental example

[0065] The mice were injected with the pharmaceutical composition prepared in Example 3 and the pharmaceutical composition prepared in the comparative example into the tail vein, respectively, as the experimental group and the control group, with an injection dose of 20 mg / kg. 3 hours after the injection, in order to determine the targeting, the mice were dissected to obtain white light field (upper part) and green fluorescence (lower part) images of skeletal muscle tissue, heart, lung, spleen, liver and kidney. Figure 1 The results showed that experimental group 1 and experimental group 2 were localized in skeletal muscle tissue, indicating that the pharmaceutical composition prepared in Example 3 was stable and had good targeting properties.

[0066] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. An adeno-associated virus vector carrying the miRNA-486 gene that specifically targets skeletal muscle cells, characterized in that: The adeno-associated virus vector comprises a packaging promoter, a foreign gene, SV40 polyA, 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.

2. An adeno-associated virus vector carrying the miRNA-486 gene that specifically targets skeletal muscle cells according to claim 1, characterized in that: The skeletal muscle specific promoter is one of MCK promoter, 3×enhancerMCK, MYOG promoter, SM22a promoter, ACTA1 promoter and MHCK7 promoter.

3. The adeno-associated virus vector carrying the miRNA-486 gene that specifically targets skeletal muscle cells according to claim 1, characterized in that: The packaging promoter is one of a CMV promoter and a PGK promoter.

4. A recombinant adeno-associated virus vector system, characterized in that: It comprises an adeno-associated virus vector carrying the miRNA-486 gene and an adenovirus genome plasmid that specifically targets skeletal muscle cells as described in claim 1, wherein the adenovirus genome plasmid comprises the capsid protein gene of AAV1 or AAV7.

5. A recombinant adeno-associated virus vector system according to claim 4, characterized in that: The fiber gene is located near the E4 region of the adenovirus genome of the adenovirus genome plasmid, and a SpyTag sequence is inserted into the fiber gene.

6. A recombinant adeno-associated virus vector system according to claim 4, characterized in that: The fiber gene is located near the E4 region of the adenovirus genome of the adenovirus genome plasmid, and the RGD sequence is inserted into the HI loop region of the fiber gene.

7. A recombinant adeno-associated virus modified with an RGD motif, characterized in that: The recombinant adeno-associated virus particles are covalently modified with the RGD motif fused with SpyCatcher to obtain the RGD motif-modified recombinant adeno-associated virus obtained by co-transfecting a packaging cell line with a recombinant adeno-associated virus vector system as described in claim 4.

8. A pharmaceutical composition, characterized in that It comprises a recombinant adeno-associated virus modified with an RGD motif, cationic liposome nanoparticles and nanobubbles as described in claim 7, wherein the recombinant adeno-associated virus modified with an RGD motif and the nanobubbles are encapsulated in the capsules of the cationic liposome nanoparticles.

9. A pharmaceutical composition according to claim 8, characterized in that Also included is an IL6R specific agonist antibody, wherein the IL6R specific agonist antibody is MT-18; the surface of the cationic liposome nanoparticle is modified with the RGD motif, and the modification method comprises the following steps: The RGD motif and cysteine ​​are connected to the RGD motif under the enzymatic catalysis of transpeptidase, and then reacted with a maleimide reagent to form a stable thioether bond, which is then coupled to the surface of the cationic liposome nanoparticle.

10. A pharmaceutical composition according to claim 8, characterized in that The method for preparing the nanobubbles comprises the following steps: S1. Microfluidic production of nanobubbles and microbubbles: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000] were mixed at a molar ratio of 95:5 to prepare a mixture with a total lipid concentration of 2 mg / mL; S2, the lipids were dissolved in a 1:1 mixture of chloroform and methanol, dried under nitrogen to remove the solvent, and then resuspended in a phosphate-buffered saline solution containing 1% glycerol by volume, and then the lipid solution was combined with C4F10 gas in a multi-channel microspray microfluidic device to generate bubbles; S3. Isolation of nanobubbles: After 1 hour, the nanobubble sample was taken out from the bottom of the bottle using a syringe and a fine needle, and then filtered using an 800 nm polytetrafluoroethylene membrane to remove any large bubbles to obtain the nanobubbles.

Images