AAV carrier with high skin and muscle affinity and application thereof

Abstract

The invention relates to an AAV carrier with high skin and muscle affinity and an application of the AAV carrier. Specifically, the invention finds that the AAV59 capsid protein has high affinity to a skin muscle layer, so that the AAV vector of the invention is constructed, the AAV vector comprises the AAV59 capsid protein and a viral genome packaged in the AAV59 capsid protein, the genome contains one or more exogenous genes, and the AAV vector can be used for diagnosing, preventing and / or treating skin diseases or is used for medical cosmetology. The AAV vector provided by the invention has improved skin muscle layer infection efficiency and expression level, and can deliver therapeutic genes into cells of the skin muscle layer, thereby realizing accurate treatment of skin diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a skin-muscle layer high-affinity AAV carrier and its applications. Background Technology

[0002] Skin diseases can be classified in many ways, including but not limited to hereditary skin diseases such as epidermolysis bullosa (EB), keratosis, hirsutism, albinism, and ichthyosis; autoimmune skin diseases such as cutaneous lupus erythematosus (CLE), pemphigus, psoriasis, vitiligo, and dermatomyositis (DM); allergic skin diseases such as eczema, urticaria, and atopic dermatitis (AD); and infectious skin diseases such as bacterial infections, fungal infections, and viral infections.

[0003] As the largest organ in the human body, the complex structure and function of the skin make it susceptible to various internal and external factors, leading to a range of skin diseases. These diseases typically involve multiple factors, including genetics, immunity, hormones, and the environment. These diseases not only affect a patient's appearance but can also impair bodily functions and reduce quality of life. Skin diseases sometimes present with similar symptom patterns, making clinical diagnosis difficult. Traditional drug treatments for most skin diseases primarily involve topical hormones or immunomodulators, which have limited efficacy, require long-term maintenance, and are prone to drug resistance and side effects. Therefore, seeking safe and effective treatments has always been a key focus in the treatment of skin diseases.

[0004] Among existing treatment methods, gene therapy is a type of therapy that packages effective DNA into a vector, delivers it to target tissues, and expresses the therapeutic molecules. Its good safety and efficacy have been demonstrated in numerous clinical trials. The key to gene therapy lies in how to accurately deliver the therapeutic gene to the target tissue and ensure its effective expression. Adeno-associated virus (AAV), as a commonly used gene delivery vector, has broad application prospects in the field of gene therapy due to its low immunogenicity, high safety, high infection efficiency, and diverse serotypes.

[0005] The dermal muscle layer is a crucial structure for maintaining skin shape and function. Improving the affinity of AAV carriers for this layer can advance the application of gene therapy in skin diseases. Furthermore, AAV's affinity for the dermal muscle layer can also be applied in the aesthetic medicine industry. The dermal muscle layer connects to hair follicles (arrector pili muscles), allowing AAV to express proteins related to hair follicle regeneration, thus treating hair loss. With age, the dermal muscle layer gradually loosens, leading to wrinkles and sagging skin; therefore, AAV can be used as a carrier for anti-aging treatments. However, the targeting specificity of existing traditional AAV carriers in skin tissue is not ideal, especially in the muscle tissue and cells of the skin.

[0006] Therefore, there is an urgent need in this field to develop AAV carriers with high affinity for the skin and muscle layers and their application in the treatment of skin diseases. Summary of the Invention

[0007] The purpose of this invention is to provide an AAV carrier with high affinity for the skin and muscle layers and its applications.

[0008] In a first aspect of the present invention, a recombinant AAV vector is provided, comprising:

[0009] (1) AAV59 capsid protein; and

[0010] (2) A viral genome packaged in the AAV59 capsid protein, the genome containing one or more exogenous genes for the diagnosis, prevention and / or treatment of skin diseases, or for medical aesthetic purposes.

[0011] In another preferred embodiment, the AAV59 capsid protein is modified from the AAV5 capsid protein, the amino acid sequence of which is shown in SEQ ID NO:1.

[0012] In another preferred embodiment, the amino acid sequence of the AAV59 capsid protein is the amino acid sequence shown in SEQ ID NO:2 or a derivative thereof.

[0013] In another preferred embodiment, the derived sequence is a sequence that has at least 95% identity with the sequence shown in SEQ ID NO:2, preferably at least 97%, 98% or 99% identity, and still has high skin affinity.

[0014] In another preferred embodiment, the derived sequence is the sequence shown in SEQ ID NO:2 that still possesses high skin affinity after the deletion, insertion, and / or substitution of one or more (e.g., typically 1-10, preferably 1-6, more preferably 1-5, even more preferably 1-3, and most preferably 1) amino acid residues.

[0015] In another preferred embodiment, the sequence of the AAV59 capsid protein is shown in SEQ ID NO:2.

[0016] In another preferred embodiment, the nucleotide sequence encoding the AAV59 capsid protein is shown in SEQ ID NO:3.

[0017] In another preferred embodiment, the viral genome is an artificially recombinant viral genome.

[0018] In another preferred embodiment, the exogenous gene includes: (1) a reporter gene; (2) a skin disease-related positive regulatory therapeutic gene; (3) a nucleotide sequence that inhibits the expression of a skin disease-related negative regulatory gene; and (4) a nucleotide sequence that encodes a gene editing system for treating skin diseases.

[0019] In another preferred embodiment, the reporter gene is selected from the group consisting of: green fluorescent protein gene (GFP), human growth hormone gene (hGH), secretory alkaline phosphatase gene (SEAP), β-galactosidase gene (LacZ), chloramphenicol acetyltransferase gene (CAT), luciferase gene, or combinations thereof.

[0020] In another preferred embodiment, the reporter gene is the green fluorescent protein gene (GFP).

[0021] In another preferred embodiment, the skin disease-related positive regulatory therapeutic genes include: genes encoding functional proteins for treating skin diseases, or genes that, when expressed, can benefit the treatment and / or prevention of skin diseases.

[0022] In another preferred embodiment, the positively regulated therapeutic gene is selected from the group consisting of: KRT14, KRT5, DSP, FLG, COL7A1, VEGF, PTCH1, TP53, EGFR, MST1R, ALOX5, ADAR1, STAT3, SOD2, XP, NOTCH3, TNXB, MVK, MC1R, OCA2, TYR, POMC, SLC24A5, DSG1, CTSC, FLG2, TGM1, Shh, or combinations thereof.

[0023] In another preferred embodiment, the negatively regulated gene includes: a gene that is highly expressed in skin diseases (relative to a healthy state), or a gene whose expression is suppressed, which can be beneficial for the treatment and / or prevention of skin diseases.

[0024] In another preferred embodiment, the nucleotide sequence capable of inhibiting the expression of the negatively regulated gene includes: microRNA, siRNA, or shRNA sequences specifically targeting the negatively regulated gene, and / or coding sequences for antibodies specifically targeting the negatively regulated gene.

[0025] In another preferred embodiment, the negatively regulated genes include, but are not limited to: SMO, EGFR, ICAM-1, MMPs, HLA-C, ADRB2, KLK5, STAT1, IFIH1, NOS2, CCR2, FGFR2, CTLA4, or combinations thereof.

[0026] In another preferred embodiment, the gene editing system for treating skin diseases includes a Cas protein reaction system.

[0027] In another preferred embodiment, the Cas protein reaction system includes: Cas protein, guide RNA and / or target gene homologous sequence, wherein the target gene homologous sequence is used for homologous recombination to repair target gene mutation sites.

[0028] In another preferred embodiment, the Cas protein reaction system is used to repair target gene mutation sites in the cell genome of the skin and restore the normal function of the target gene; wherein the target gene is a positively regulated therapeutic gene.

[0029] In another preferred embodiment, the Cas protein reaction system is used to knock out or knock down the expression of a target gene in skin cells; wherein the target gene is a negatively regulated gene.

[0030] In another preferred embodiment, the medical aesthetic treatment includes: anti-aging, wrinkle reduction, scar improvement, and skin pigmentation management.

[0031] In another preferred embodiment, the genes used for medical aesthetics include: COL1A1, COL3A1, EGF, FGF, SOD, CAT, Klotho, SIRT1, HMOX1, or combinations thereof.

[0032] In another preferred embodiment, the viral genome also includes regulatory elements, including a promoter, an enhancer, a polyA, and two ITRs located at both ends.

[0033] In another preferred embodiment, the promoter includes, but is not limited to: CB promoter, CMV promoter, CAG promoter, UBC promoter, tetracycline promoter TRE, Synapsin I promoter, CamKIIa promoter, c-fos promoter, Mecp2 promoter, NSE promoter, SST promoter, TH promoter, GFAP promoter, GFAP104 promoter, GfaABC1D promoter, ALDH1L1 promoter, MBP promoter, Rpe65 promoter, and VMD2 promoter.

[0034] In another preferred embodiment, the promoter is a CB promoter.

[0035] In another preferred embodiment, the recombinant AAV vector has high affinity for and targeting of skin tissue and / or skin cells.

[0036] In another preferred embodiment, the skin tissue is selected from the group consisting of the epidermis, dermis, subcutaneous tissue, or a combination thereof.

[0037] In another preferred embodiment, the skin tissue is subcutaneous tissue.

[0038] In another preferred embodiment, the skin cells include all cell types in the epidermis, dermis, and subcutaneous tissue.

[0039] In another preferred embodiment, the skin cells are selected from the group consisting of keratinocytes, melanocytes, Langerhans cells, Merkel cells, dermal fibroblasts, vascular endothelial cells, cutaneous nerve cells, epithelial cells, hair follicle cells, mast cells, leukocytes, astrocytes, or combinations thereof.

[0040] In a second aspect of the invention, a pharmaceutical composition comprising the following components is provided:

[0041] (i) the recombinant AAV vector as described in the first aspect of the invention; and

[0042] (ii) Pharmaceutically acceptable excipients;

[0043] The pharmaceutical composition is used to treat skin diseases and / or for medical aesthetic purposes.

[0044] In another preferred embodiment, component (i) accounts for 0.1-99.9 wt% of the total weight of the pharmaceutical composition, preferably 10-80 wt%, more preferably 30-60 wt%.

[0045] In another preferred embodiment, the excipients include, but are not limited to, salts, organic compounds, and surfactants, or combinations thereof.

[0046] In another preferred embodiment, the excipients include, but are not limited to, solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, and absorption delay agents, or combinations thereof.

[0047] In another preferred embodiment, the excipient comprises a saline solution, including but not limited to: buffered saline, physiological saline, phosphate buffer, citrate buffer, acetate buffer, bicarbonate buffer, sucrose solution, salt solution, polysorbate solution, or combinations thereof.

[0048] In another preferred embodiment, the excipient includes, but is not limited to, stabilizers, preservatives, transfection promoters that facilitate cell uptake, or combinations thereof.

[0049] In another preferred embodiment, the stabilizer includes, but is not limited to, monosodium glutamate, glycine, EDTA, albumin (e.g., human serum albumin), or combinations thereof.

[0050] In another preferred embodiment, the preservative includes, but is not limited to, 2-phenoxyethanol, sodium benzoate, potassium sorbate, methylparaben, phenol, thimerosal, antibiotics, or combinations thereof.

[0051] In another preferred embodiment, the transfection promoter comprises calcium ions.

[0052] In another preferred embodiment, the pharmaceutical composition is liquid.

[0053] In another preferred embodiment, the pharmaceutical composition is an injectable preparation, including intramuscular injection, intradermal injection, subcutaneous injection, and intravenous injection.

[0054] In another preferred embodiment, the pharmaceutical composition is an intradermal injection.

[0055] In another preferred embodiment, the pharmaceutical composition is administered by intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, transdermal administration, or a combination thereof.

[0056] In another preferred embodiment, the pharmaceutical composition is administered by intradermal injection.

[0057] In another preferred embodiment, the pharmaceutical composition may be in the form of ointment, cream, lotion, paste, gel, spray, aerosol or oil.

[0058] In another preferred embodiment, the carrier includes, but is not limited to, petrolatum, lanolin, polyethylene glycol, alcohol, transdermal enhancer, or combinations thereof.

[0059] In another preferred embodiment, the skin disease is selected from the group consisting of: hereditary skin diseases, autoimmune skin diseases, allergic skin diseases, infectious skin diseases, or combinations thereof.

[0060] In another preferred embodiment, the hereditary skin diseases include, but are not limited to: epidermolysis herpes (EB), keratosis, hirsutism, albinism, and ichthyosis.

[0061] In another preferred embodiment, the autoimmune skin disease includes, but is not limited to, cutaneous lupus erythematosus (CLE), pemphigus, psoriasis, vitiligo, and dermatomyositis (DM).

[0062] In another preferred embodiment, the allergic skin disease includes, but is not limited to, eczema, urticaria, and atopic dermatitis (AD).

[0063] In another preferred embodiment, the infectious skin disease includes, but is not limited to, bacterial infections, fungal infections, and viral infections.

[0064] In a third aspect of the invention, a genetically engineered cell is provided, the genetically engineered cell being packaged to produce a recombinant AAV vector as described in the first aspect of the invention, the genetically engineered cell comprising:

[0065] (i) A first nucleic acid construct containing an exogenous therapeutic gene for skin diseases;

[0066] (ii) a second nucleic acid construct containing the rep and cap genes, wherein the cap gene encodes the AAV59 capsid protein; and

[0067] (iii) A third nucleic acid construct, wherein the third nucleic acid construct is an auxiliary plasmid.

[0068] In another preferred embodiment, the genetically engineered cell is a eukaryotic cell.

[0069] In another preferred embodiment, the genetically engineered cells are selected from the group consisting of 293T cells, HEK293 cells, Sf9 cells, BHK cells, or combinations thereof.

[0070] In another preferred embodiment, the helper plasmid is derived from adenovirus (Ad), herpes simplex virus (HSV), or other helper plasmids with helper functions.

[0071] In another preferred embodiment, the first nucleic acid construct is a plasmid.

[0072] In another preferred embodiment, the first nucleic acid construct also contains a foreign reporter gene.

[0073] In another preferred embodiment, the exogenous reporter gene is selected from the group consisting of GFP, hGH, SEAP, LacZ, CAT, luciferase gene, or combinations thereof.

[0074] In another preferred embodiment, the second nucleic acid construct is a plasmid.

[0075] In another preferred embodiment, the first nucleic acid construct, the second nucleic acid construct, and / or the third nucleic acid construct may exist temporarily in the genetically engineered cell or be stably integrated into the genome of the genetically engineered cell.

[0076] In a fourth aspect of the invention, there is provided the use of the AAV59 capsid protein for preparing a recombinant AAV vector as described in the first aspect of the invention.

[0077] In another preferred embodiment, the AAV59 capsid protein is modified from the AAV5 capsid protein, the amino acid sequence of which is shown in SEQ ID NO:1.

[0078] In another preferred embodiment, the amino acid sequence of the AAV59 capsid protein is the amino acid sequence shown in SEQ ID NO:2 or a derivative thereof.

[0079] In another preferred embodiment, the derived sequence is a sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:2, preferably at least 97%, 98% or 99% identity.

[0080] In another preferred embodiment, the sequence of the AAV59 capsid protein is shown in SEQ ID NO:2.

[0081] In another preferred embodiment, the nucleotide sequence encoding the AAV59 capsid protein is shown in SEQ ID NO:3.

[0082] In a fifth aspect of the invention, there is provided the use of the recombinant AAV vector as described in the first aspect of the invention for preparing a pharmaceutical composition as described in the second aspect of the invention.

[0083] In a sixth aspect of the invention, a method for delivering a target gene into tissues or cells of the skin is provided, comprising:

[0084] (1) The target gene was packaged into the AAV59 capsid protein to form a recombinant AAV vector; and

[0085] (2) Contact the tissue or cells of the skin with the recombinant AAV carrier.

[0086] In another preferred embodiment, the skin tissue includes, but is not limited to, the epidermis, the dermis, the subcutaneous tissue, or a combination thereof.

[0087] In another preferred embodiment, the cells of the skin include, but are not limited to, all cell types in the epidermis, dermis, and subcutaneous tissue, such as keratinocytes, melanocytes, Langerhans cells, Merkel cells, dermal fibroblasts, vascular endothelial cells, cutaneous nerve cells, epithelial cells, hair follicle cells, mast cells, leukocytes, and astrocytes.

[0088] In another preferred embodiment, the target gene includes: (1) a reporter gene; (2) a skin disease-related positive regulatory therapeutic gene; (3) a nucleotide sequence that inhibits the expression of a skin disease-related negative regulatory gene; (4) a nucleotide sequence encoding a gene editing system for treating skin diseases; and (5) a gene for medical aesthetics.

[0089] In another preferred embodiment, the method is a non-diagnostic / non-therapeutic method.

[0090] In a seventh aspect of the invention, a method for preventing and / or treating skin diseases is provided, comprising the steps of: administering an effective amount of a recombinant AAV carrier as described in the first aspect of the invention or a pharmaceutical composition as described in the second aspect of the invention to a subject in need.

[0091] In another preferred embodiment, the pharmaceutical composition is administered alone or in combination with other drugs for treating the skin disease in a method of treating the skin disease.

[0092] In another preferred embodiment, the application includes in vivo injection.

[0093] In another preferred embodiment, the in vivo injection method includes, but is not limited to: intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, or a combination thereof.

[0094] In another preferred embodiment, the administration includes transdermal delivery.

[0095] In another preferred embodiment, the transdermal administration method includes, but is not limited to, ointments, creams, lotions, pastes, gels, sprays, aerosols, oils, or combinations thereof.

[0096] In another preferred embodiment, the object includes a human or a non-human mammal.

[0097] In another preferred embodiment, the non-human mammals include, but are not limited to: non-human primates, sheep, dogs, cats, horses, cattle, chickens, rats, and mice.

[0098] In an eighth aspect of the invention, a method is provided for identifying the skin affinity of a recombinant AAV vector to a subject, the recombinant AAV vector comprising an artificially recombinant viral genome, the viral genome comprising a reporter gene.

[0099] In another preferred embodiment, the reporter gene is selected from the group consisting of GFP, hGH, SEAP, LacZ, CAT, and Luciferase.

[0100] In another preferred embodiment, the reporter gene is GFP.

[0101] In another preferred embodiment, the method for identifying the skin affinity of the recombinant AAV vector to a subject includes:

[0102] (1) In vivo injection of the recombinant AAV carrier into the skin of the subject to administer an effective dose of the recombinant AAV carrier to the subject, the injection method including but not limited to intramuscular injection, intradermal injection, subcutaneous injection, and intravenous injection, preferably intradermal injection; and

[0103] (2) Collect skin samples from the subjects and use biochemical methods to identify the affinity of the recombinant AAV vector for the subject's skin. The biochemical methods include immunohistochemistry, Western blotting, ELISA and quantitative PCR, with immunohistochemistry being preferred.

[0104] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0105] Figure 1 A schematic diagram of the CB-GFP vector structure is shown, which contains the following elements in sequence: 5'ITR, CB promoter, GFP gene coding sequence, bGH poly(A) sequence, and 3'ITR.

[0106] Figure 2 The results of silver staining used to calculate the gray values ​​of silver staining are shown. Each well is numbered: M is the marker; 2 is the blank control; 3 is standard-1; 4 is standard-2; 5 is standard-3; 6 is standard-4; 7 is AAV5-GFP; 8 is T64-GFP; and 9 is AAV59-GFP.

[0107] Figure 3 The standard curves used to measure the titers of serotypes AAV5 and AAV59 viral particles (named AAV5-GFP and AAV59-GFP, respectively) produced and collected using the CB-GFP vector are shown.

[0108] Figure 4 The image shows a demonstration of injecting the virus into the skin on the back of a mouse via intradermal injection.

[0109] Figure 5 Immunohistochemical results of GFP staining on the dorsal skin of mice after injection with AAV5-GFP (left column) and AAV59-GFP (right column) viruses are shown. The scale bar is 200 μm and the magnification is 10×.

[0110] Figure 6The quantitative statistical graphs of GFP fluorescence signal intensity after AAV5-GFP and AAV59-GFP virus injection into the dorsal skin of mice are shown, where “****” indicates P<0.0001. Detailed Implementation

[0111] Through extensive and in-depth research, the inventors unexpectedly discovered that the AAV59 capsid protein from patent WO2022226294A1 has a high affinity for the skin-muscle layer. Specifically, after packaging the AAV59 capsid protein of this invention with a recombinant genome containing the exogenous reporter gene GFP into AAV59-GFP viral particles, and administering them to subjects via intradermal injection, and collecting corresponding tissue samples, immunohistochemistry and imaging techniques revealed that the AAV59-GFP viral particles exhibit a strong affinity for the skin-muscle layer, significantly higher than that of AAV5-GFP viral particles packaged from traditional AAV serotypes (e.g., AAV5) that are currently widely used in clinical trials and even pharmaceuticals. Based on this, the present invention was completed.

[0112] the term

[0113] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0114] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0115] As used herein, the term "treatment" refers to administering to a patient an oral or topical therapeutic agent comprising the AAV viral vector of the present invention and pharmaceutical compositions thereof. The patient suffers from one or more diseases, and the therapeutic agent has a therapeutic effect on these diseases. Typically, the patient is given the AAV viral vector of the present invention and pharmaceutical compositions thereof in an amount that effectively relieves the symptoms of one or more diseases (therapeutic effective amount).

[0116] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0117] As used in this article, the term "adeno-associated virus (AAV)" refers to a virus belonging to the genus Parvovirus of the family Parvoviridae that can infect humans and other mammals.

[0118] As used herein, the term "ITR" refers to a DNA sequence of approximately 145 nucleotides that mediates the biological functions of AAV viruses, including replication, packaging, and integration. ITRs can originate from any AAV, including but not limited to AAV serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and any other now-known or later-discovered AAVs. The 5' ITR and 3' ITR flanking the ITR do not necessarily originate from the same AAV serotype, as long as they function as intended. The nucleotide sequence of the ITR region is known.

[0119] As used herein, the term "artificially recombinant viral genome" or "recombinant genome" refers to an artificially designed or synthesized exogenous DNA sequence that replaces the natural AAV genome between ITRs. An AAV containing an artificially recombinant viral genome is called a "recombinant AAV." Recombinant AAVs can achieve different functions based on the expression of the recombinant genome they contain.

[0120] As used in this article, the term "affinity" refers to the property of a virus to prefer infecting and / or entering certain types of cells or tissues.

[0121] As used in this article, the term "vector" refers to a molecular tool for transporting, transducing, and expressing a foreign target gene contained therein in target cells.

[0122] As used in this article, the term "treatment" refers to both therapeutic and preventative measures. Those requiring treatment may include individuals who already have a skin condition and those who may eventually develop it.

[0123] As used in this article, the term "skin disease" refers to a range of diseases that occur on the skin, mucous membranes and their appendages (such as hair, nails, sweat glands and sebaceous glands), including but not limited to hereditary skin diseases, autoimmune skin diseases, allergic skin diseases, infectious skin diseases, etc.

[0124] As used herein, the term "excipient" refers to a natural or synthetic substance attached to the active ingredient in a drug, such as a solvent, dispersion medium, coating, antibacterial or antifungal agent, isotonic agent, and absorption delay agent. These excipients can aid in the storage of viral particles and administration to subjects. Excipients may include any suitable component, such as, but not limited to, saline.

[0125] As used herein, the terms “subject” and “object” are used interchangeably and include any human or non-human mammal, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, rats, mice, etc.

[0126] As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in humans and / or animals and is acceptable to them. It refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or that exhibits a detectable therapeutic or preventative effect. Therapeutic effects also include a reduction in physiological symptoms. The precise effective amount for a given subject depends on the subject's body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. For a given condition, the effective amount can be determined using routine laboratory methods.

[0127] Adeno-associated virus (AAV)

[0128] Adeno-associated virus (AAV) is a virus belonging to the genus *Devicynis* in the family Parvoviridae, and is one of the smallest known viral species. AAV viral particles are non-vesicular icosahedral structures, with a diameter between 20 and 26 nm, composed of 60 copies of three capsid proteins, VP1, VP2, and VP3, in a 1:1:10 ratio. As used herein, the term "AAV" includes naturally occurring adeno-associated virus and recombinant forms of adeno-associated virus (rAAV), as well as mutant forms of AAV. Numerous AAV serotypes exist, including AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV10, AAV-DJ, and AAV-DJ / 8.

[0129] The AAV genome contains two main genes: the `rep` gene, which encodes the Rep proteins (Rep 76, Rep 68, Rep 52, and Rep 40), and the `cap` gene, which encodes the AAV capsid proteins (VP1, VP2, and VP3). Those skilled in the art know that the AAV capsid proteins contain VP1, VP2, and VP3 proteins, and that VP2 and VP3 proteins undergo transcription and translation at the start codon within the VP1 protein; that is, the VP1 sequence contains the VP2 and VP3 sequences. As the most commonly used viral vector in gene therapy, AAV is characterized by non-pathogenicity, low immunogenicity, serotype diversity, and persistent expression of exogenous genes.

[0130] The AAV59 capsid protein is derived from a mutation of the AAV5 capsid protein, the amino acid sequence of which is shown in SEQ ID NO:1. The amino acid sequence of the AAV59 capsid protein is shown in SEQ ID NO:2 or a derived sequence thereof, wherein the derived sequence refers to a sequence shown in SEQ ID NO:2 that still possesses high skin affinity after the deletion, insertion, and / or substitution of one or more amino acid residues (e.g., typically 1-10, preferably 1-6, more preferably 1-5, even more preferably 1-3, and most preferably 1). In a preferred embodiment, the nucleotide sequence encoding the AAV59 capsid protein is shown in SEQ ID NO:3.

[0131] This invention is the first to discover that the AAV59 capsid protein has high affinity for skin tissue, thereby developing a recombinant AAV vector that specifically targets skin combinations or cells.

[0132] The recombinant AAV vector of the present invention

[0133] As used herein, the terms “recombinant AAV vector,” “AAV virus particle,” and “recombinant AAV” are used interchangeably and all refer to the recombinant AAV vector of the present invention containing the AAV59 capsid protein and the artificial recombinant viral genome packaged in the AAV59 capsid protein.

[0134] In this invention, the artificial recombinant viral genome contains one or more exogenous genes for the diagnosis, prevention, and / or treatment of skin diseases, or for use in medical aesthetics. The exogenous genes include: (1) reporter genes; (2) positively regulated therapeutic genes related to skin diseases; (3) nucleotide sequences that inhibit the expression of negatively regulated genes related to skin diseases; (4) nucleotide sequences encoding gene editing systems for the treatment of skin diseases; and (5) genes related to medical aesthetics, etc.

[0135] In one embodiment of the present invention, the exogenous gene is a reporter gene, such as the green fluorescent protein (GFP) gene.

[0136] In one embodiment of the present invention, the exogenous gene encodes a biological molecule with a specific function, including but not limited to proteins with therapeutic functions for skin diseases, microRNA (miRNA), siRNA, shRNA, antibodies, and Cas9 guide RNA. The genes encoding proteins with therapeutic functions for skin diseases include, but are not limited to, KRT14, KRT5, DSP, FLG, COL7A1, VEGF, PTCH1, TP53, EGFR, MST1R, ALOX5, ADAR1, STAT3, SOD2, XP, NOTCH3, TNXB, MVK, MC1R, OCA2, TYR, POMC, SLC24A5, and DSG. 1. CTSC, FLG2, TGM1, Shh; target genes of miRNA or antibody include, but are not limited to, SMO, EGFR, ICAM-1, MMPs, HLA-C, ADRB2, KLK5, STAT1, IFIH1, NOS2, CCR2, FGFR2, CTLA4, etc.; target genes of Cas9 guide RNA include, but are not limited to, KRT14, KRT5, DSP, FLG, COL7A1, VEGF, PTCH1, TP53, EGFR, etc. FR, MST1R, ALOX5, ADAR1, STAT3, SOD2, XP, NOTCH3, TNXB, MVK, MC1R, OCA2, TYR, POMC, SLC24A5, DSG1, CTSC , FLG2, TGM1, Shh, SMO, EGFR, ICAM-1, MMPs, HLA-C, ADRB2, KLK5, STAT1, IFIH1, NOS2, CCR2, FGFR2, CTLA4, etc.

[0137] In one embodiment of the present invention, the AAV viral vector of the present invention comprises an artificially recombinant viral genome, the artificially recombinant viral genome comprising a transcriptional regulatory sequence (promoter), a gene coding sequence (CDS), and a poly(A) sequence for maintaining the activity and stability of messenger RNA.

[0138] In one embodiment of the present invention, the strength of AAV affinity can be measured by the amount of exogenous reporter genes carried by different serotypes of AAV expressed in a specific type of cell or tissue under the same conditions. For example, GFP can be used as an exogenous reporter gene, and the fluorescence intensity of GFP can be used as an indicator of the level of AAV skin affinity.

[0139] In one embodiment of the invention, an AAV viral vector is produced using a DNA plasmid comprising a 5' ITR, a recombinant genome, and a 3' ITR, wherein the 5' ITR and 3' ITR are located flanking the recombinant genome, respectively. The AAV viral vector can be produced by simultaneously introducing the aforementioned DNA plasmid, a plasmid encoding the AAV cap / rep gene, and a helper plasmid provided by adenovirus or herpesvirus into a suitable host cell using known techniques, such as transfection. The DNA plasmid can be expressed in the host cell and packaged into viral particles.

[0140] Pharmaceutical Composition

[0141] In one embodiment, the pharmaceutical composition comprises viral particles with AAV59 as a capsid and excipients.

[0142] In one embodiment of the present invention, the pharmaceutical composition can efficiently deliver a recombinant genome into the skin to express biological molecules with specific functions, including but not limited to proteins with therapeutic functions for skin diseases, microRNA (miRNA), siRNA, shRNA, antibodies, and Cas9 guide RNA.

[0143] In one embodiment of the present invention, the present invention further provides a method for injecting AAV virus particles into the skin of a subject, including but not limited to administering an effective dose of AAV virus particles to the subject via intramuscular injection, intradermal injection, subcutaneous injection, or intravenous injection. The above methods should be selected based on the pathogenesis of skin diseases and can be used in combination.

[0144] In one embodiment of the present invention, the present invention further provides a method for administering AAV virus particles to a subject transdermally in vivo, including but not limited to: ointments, creams, lotions, pastes, gels, sprays, aerosols, oils, etc., to deliver an effective dose of AAV virus particles to the subject. The above methods should be selected based on the pathogenesis of skin diseases and can be used in combination.

[0145] The pharmaceutical compositions of the present invention may also include other drugs for treating skin diseases, or be used in combination with other drugs for treating skin diseases.

[0146] Other medications used to treat skin diseases include, but are not limited to: Aureobasidium polyglycoside tablets, cyclosporine, fluticasone propionate cream, vitamin A, vitamin E, retinoic acid, zinc oxide boric acid, calcipotriol, tocilizumab, glucocorticoids, calmodulin regulators, azathioprine, leflunomide, ebastine, epinastine hydrochloride, compound glycyrrhizin, tacrolimus, loratadine, calamine lotion, cetirizine hydrochloride, diphenhydramine hydrochloride, erythromycin, mupirocin, penciclovir, acyclovir, bifonazole, itraconazole, terbinafine hydrochloride, mometasone furoate, etc.

[0147] The pharmaceutical compositions of this invention can be used to treat the following skin diseases: hereditary skin diseases, autoimmune skin diseases, allergic skin diseases, and infectious skin diseases. Hereditary skin diseases include, but are not limited to: epidermolysis bullosa (EB), keratosis, hirsutism, albinism, and ichthyosis. Autoimmune skin diseases include, but are not limited to: cutaneous lupus erythematosus (CLE), pemphigus, psoriasis, vitiligo, and dermatomyositis (DM). Allergic skin diseases include, but are not limited to: eczema, urticaria, and atopic dermatitis (AD). Infectious skin diseases include, but are not limited to: bacterial infections, fungal infections, and viral infections.

[0148] The effective amount of a pharmaceutical composition can vary depending on the mode of administration and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the drug, such as tissue distribution, bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

[0149] application

[0150] This invention also provides the application of the AAV59 capsid protein in preparing the recombinant AAV vector of this invention. Utilizing the high affinity of the AAV59 capsid protein for skin, it can be used to prepare a vector for delivering exogenous genes to the skin, namely the recombinant AAV vector of this invention.

[0151] In one embodiment of the present invention, the AAV59 capsid protein of the present invention can carry a reporter gene to prepare a recombinant AAV vector for identifying the affinity of the recombinant AAV vector for the skin of a subject. For example, the reporter gene can be GFP, hGH, SEAP, LacZ, CAT, and Luciferase; preferably, the reporter gene is GFP. The recombinant AAV vector is injected in vivo into the skin of the subject to administer an effective dose of the recombinant AAV vector to the subject. Skin samples are collected from the subject, and the affinity of the recombinant AAV vector for the subject's skin can be identified using biochemical methods.

[0152] In one embodiment of the present invention, the AAV59 capsid protein of the present invention can carry positively regulatory therapeutic genes related to skin diseases for the preparation of recombinant AAV vectors for treating skin diseases. Positively regulatory therapeutic genes include genes encoding functional proteins for treating skin diseases, or genes that, when expressed, can benefit the treatment and / or prevention of skin diseases, such as genes selected from the group consisting of: KRT14, KRT5, DSP, FLG, COL7A1, VEGF, PTCH1, TP53, EGFR, MST1R, ALOX5, ADAR1, STAT3, SOD2, XP, NOTCH3, TNXB, MVK, MC1R, OCA2, TYR, POMC, SLC24A5, DSG1, CTSC, FLG2, TGM1, Shh, etc.

[0153] In one embodiment of the present invention, the AAV59 capsid protein of the present invention can carry a nucleotide sequence that inhibits the expression of negatively regulatory genes associated with skin diseases, for use in preparing a recombinant AAV vector for treating skin diseases. The nucleotide sequence of the negatively regulatory gene expression is a gene that is highly expressed in skin diseases (relative to a healthy state), or a gene whose expression, when suppressed, can be beneficial for the treatment and / or prevention of skin diseases, including but not limited to: SMO, EGFR, ICAM-1, MMPs, HLA-C, ADRB2, KLK5, STAT1, IFIH1, NOS2, CCR2, FGFR2, CTLA4, or combinations thereof. The nucleotide sequence capable of inhibiting the expression of the negatively regulatory gene includes: microRNA, siRNA, or shRNA sequences specifically targeting the negatively regulatory gene, and / or coding sequences for antibodies specifically targeting the negatively regulatory gene.

[0154] In one embodiment of the present invention, the AAV59 capsid protein of the present invention may carry genes related to gene editing, such as genes encoding the Cas protein reaction system, for editing target genes related to skin diseases, such as restoring the normal function of positively regulated therapeutic genes, or knocking out or knocking down the expression of negatively regulated genes in skin cells.

[0155] In one embodiment of the present invention, the AAV59 capsid protein of the present invention can also carry genes or sequences related to medical aesthetics for the preparation of recombinant AAV vectors for medical aesthetics, such as recombinant AAV vectors for anti-aging, wrinkle reduction, scar improvement, and skin pigmentation management. Genes related to medical aesthetics include, but are not limited to: COL1A1, COL3A1, EGF, FGF, SOD, CAT, Klotho, SIRT1, HMOX1, etc.

[0156] The main advantages of this invention include:

[0157] (1) This invention is the first to discover that AAV59 has a high affinity for the skin and muscle layer, and that the effect of using AAV59 to deliver exogenous genes to the skin of subjects is significantly better than that of traditional AAV capsids (e.g., AAV5).

[0158] (2) In this invention, AAV59 is used as a delivery vector for gene therapy drugs, which can be applied to the field of skin disease treatment and to develop a new method for treating skin diseases with gene therapy.

[0159] (3) Since AAV59 is not a naturally occurring AAV capsid, it is less common for subjects to have been infected with AAV and have AAV59 neutralizing antibodies in their bodies compared to traditional AAV capsids (such as AAV5), thus it can be applied to a wider range of subjects.

[0160] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0161] Example 1: Construction of CMV-GFP plasmid

[0162] GFP was selected as the exogenous reporter gene. The expression of the GFP gene coding sequence carried by AAV serotypes AAV59 and AAV5 in the skin was quantitatively analyzed to compare the skin affinity of AAV59 and AAV5. Therefore, the inventors constructed the CB-GFP plasmid to provide the genomic sequence of recombinant AAV.

[0163] The construction method is as follows: The plasmid backbone (B07-pscAAV plasmid backbone) and the GFP gene coding sequence obtained by PCR were digested with nucleotide endonucleases NcoI and HindIII at 37℃ for 1 hour to obtain the corresponding sticky ends. After gel extraction and recovery of the target fragment, ligation was performed overnight at 16℃ using T4 ligase. After transformation, single clones were selected for culture, and plasmids were extracted. The plasmid was verified to be correct by Sanger sequencing.

[0164] A schematic diagram of plasmid construction is shown below. Figure 1As shown, the expression cassette of the CB-GFP plasmid contains the following elements: 5' ITR, CB promoter, GFP gene coding sequence, bGH poly(A) sequence, and 3' ITR. Using a three-plasmid transfection method, the CB-GFP plasmid, the plasmid encoding the AAV cap / rep gene, and the helper plasmid were simultaneously introduced into host cells 293 to produce the AAV viral vector.

[0165] Example 2: Construction of CMV-GFP plasmid

[0166] Silver staining is used to detect proteins in polyacrylamide gels. It works by reducing silver ions to metallic silver in an alkaline pH environment, causing them to precipitate on the protein surface and display a color. The steps for determining the viral titers of AAV5-GFP and AAV59-GFP using silver staining are as follows:

[0167] Mix AAV standard, test virus, and 5× Loading buffer and denature at 100℃ for 5 minutes. Prepare SDS-PAGE gel; after complete solidification, add sample and marker to the wells of the stacking gel, and run the gel at 100V for 20 minutes. After the sample reaches the separating gel, run the gel at 120V for 1 hour. Prepare a fixative solution with ethanol, acetic acid, and double-distilled water in a 5:1:4 volume ratio. Fix the gel in the fixative solution for 40 minutes, then wash with 30% ethanol and double-distilled water. Incubate with 1× silver staining sensitizer for 2 minutes, then wash twice with double-distilled water for 1 minute each time. Add 1× silver solution and incubate for 10 minutes, then wash with double-distilled water for 1 minute. Add 100ml of silver staining developer and incubate for 3-10 minutes. When ideal protein bands appear, add stop solution and let stand for 10 minutes.

[0168] After the reaction, the gray value of the alkaline gel was calculated. A standard curve was constructed by setting the gray value of the standard on the x-axis and the titer (Vp / mL) of the standard on the y-axis. Then, the corresponding viral titer (Vp / mL) was calculated from the gray value of the AAV sample to be determined. The standard curve for viral titer quantification was plotted as follows: Figure 3 As shown, R 2 =0.9997.

[0169] Based on the calculation results of the standard curve and the silver staining gray value of the virus, the viral titers of AAV5-GFP and AAV59-GFP differ by approximately two times (as shown in Table 1, AAV5-GFP: 4.07 × 10⁻⁶). 13 Vp / mL; AAV59-GFP: 2.36 × 10⁻⁶ 13 Vp / mL).

[0170] Table 1 Virus titer

[0171]

[0172] Example 3: Skin-muscle layer affinity of AAV5-GFP and AAV59-GFP

[0173] The skin-muscle affinity of AAV5-GFP and AAV59-GFP was compared by intradermal injection of equal doses of AAV5-GFP or AAV59-GFP into the dorsal skin of mice.

[0174] The specific method for intradermal injection is as follows: Eight-week-old SPF-grade male C57BL / 6JGpt mice were selected for intradermal injection into the skin on their backs. A schematic diagram is shown below. Figure 4 As shown. Mice were anesthetized, their back hair was shaved, and 20 μL of AAV virus (AAV5-GFP or AAV59-GFP, both at a titer of ~2 × 10⁻⁶) was injected intradermally (into the dermis). 13 Vp / mL).

[0175] Frozen sectioning and immunohistochemical analysis of mouse skin tissue were performed as follows: Mice were sacrificed two weeks after viral injection and perfused through the heart successively with 1×PBS and 4% PFA solution. Skin tissue within 1 cm of the injection site was harvested and fixed in 4% PFA solution for 24 h. Subsequently, it was dehydrated using gradients of 15% and 30% sucrose solutions. Longitudinal sections of skin tissue with a thickness of 20 μm were obtained using the frozen section method. The sections were incubated with DAPI (1:1000) at room temperature for 10 min. The skin sections were imaged using a confocal microscope (Leica SP8), and quantitative GFP fluorescence analysis was performed under consistent imaging parameters and without overexposure.

[0176] The results are as follows Figure 5 and Figure 6 As shown. The results showed that, under the same skin region and the same imaging parameters, the GFP fluorescence signal in the skin-muscle layer of the AAV59-GFP group was significantly stronger than that of the AAV5-GFP group (as shown). Figure 5 (Scale bar: 200 μm, magnification: 10×), statistical results show that the above differences are significant. Figure 6 (P<0.0001).

[0177] The above results indicate that intradermal injection of AAV59 into the dermis of mice can induce viral expression in the muscle layer of the skin, and its expression can extend to the non-injection area (the diameter of the injection area is about 0.5 cm, and the diameter of the fluorescent expression area is about 1 cm two weeks after injection). Its affinity for the skin muscle layer is superior to that of the traditional serotype AAV5.

[0178] It can be seen that AAV59 has a better affinity for the skin and muscle layer than traditional AAV serotypes such as AA5, indicating that AAV59 has a promising application prospect in gene therapy for skin diseases.

[0179] sequence

[0180] The amino acid sequence of AAV5 (SEQ ID NO: 1)

[0181] MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIP AQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFT LPQYGYATLNRDNTENPTERSSFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYL EGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0182] The amino acid sequence of AAV59 (SEQ ID NO: 2)

[0183] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDRSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPTGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0184] Nucleotide sequence encoding AAV59 (SEQ ID NO: 3)

[0185]

[0186] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A recombinant AAV vector, characterized in that, The recombinant AAV vector comprises: (1) AAV59 capsid protein; and (2) A viral genome packaged in the AAV59 capsid protein, the genome containing one or more exogenous genes for the diagnosis, prevention and / or treatment of skin diseases, or for medical aesthetic purposes.

2. The recombinant AAV vector as described in claim 1, characterized in that, The amino acid sequence of the AAV59 capsid protein is the amino acid sequence shown in SEQ ID NO:2 or a derivative thereof.

3. The recombinant AAV vector as described in claim 1, characterized in that, The exogenous genes include: (1) reporter genes; (2) skin disease-related positive regulatory therapeutic genes; (3) nucleotide sequences that inhibit the expression of skin disease-related negative regulatory genes; (4) nucleotide sequences that encode gene editing systems for treating skin diseases; and (5) genes related to medical aesthetics.

4. The recombinant AAV vector as described in claim 1, characterized in that, The recombinant AAV vector has high affinity and targeting properties for skin tissue and / or skin cells.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the following components: (i) the recombinant AAV vector as described in claim 1; and (ii) Pharmaceutically acceptable excipients; The pharmaceutical composition is used to treat skin diseases and / or for medical aesthetic purposes.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is administered via intradermal injection.

7. The pharmaceutical composition according to claim 5, characterized in that, The skin diseases mentioned are selected from the following group: hereditary skin diseases, autoimmune skin diseases, allergic skin diseases, infectious skin diseases, or combinations thereof.

8. A genetically engineered cell, characterized in that, The genetically engineered cells are packaged to produce the recombinant AAV vector as described in claim 1, wherein the genetically engineered cells contain: (i) A first nucleic acid construct containing an exogenous therapeutic gene for skin diseases; (ii) A second nucleic acid construct containing the rep and cap genes, wherein the cap gene encodes the AAV59 capsid protein; and (iii) A third nucleic acid construct, wherein the third nucleic acid construct is an auxiliary plasmid.

9. The use of an AAV59 capsid protein, characterized in that, Used to prepare the recombinant AAV vector as described in claim 1.

10. A method for identifying the skin affinity of a recombinant AAV vector to a subject, the recombinant AAV vector comprising an artificially recombinant viral genome, the viral genome comprising a reporter gene.

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