Gene introduction method, gene therapy method, and tissue regeneration method

Abstract

[Problem] To do not have a gene introduction method that has high gene introduction efficiency and high specificity to skin, subcutaneous tissue, skin ulcer surfaces and constituent cells thereof. More generally, it is difficult to achieve high gene introduction efficiency in gene introduction into a living body. The skin appendages are difficult to regenerate in form and function; when the tip tissues (upper arms, forearms, hands, fingers, thighs, shanks, feet, toes) of the upper limbs and the lower limbs are lost, regeneration is difficult. The solution is that: a novel AAV (AAV is an abbreviation of an adeno-associated virus) capsid having high gene introduction efficiency and high specificity to cells of skin / subcutaneous tissues and skin ulcer surfaces, and a novel AAV capsid having high gene introduction efficiency and high specificity to epidermal tissues and cells constituting the epidermal tissues, namely keratinocytes; disclosed is a novel tissue-specific promoter having high gene expression and high specificity against epidermal tissue and keratinocytes constituting the epidermal tissue, and a pharmaceutical product having improved gene introduction efficiency by using a steroid together with gene introduction in a living body. The present invention relates to a pharmaceutical product for inducing regeneration, regeneration and function improvement of a skin appendage by introducing a gene into a living body in order to achieve sufficient skin appendage functions, a research and development platform for developing a regeneration method for peripheral tissues and defective tissues of upper and lower limbs, and a pharmaceutical product for regeneration, and more particularly, to a pharmaceutical product for regeneration for inducing regeneration, regeneration and function improvement of the skin appendage by introducing a gene into the living body in a state where the function of the skin appendage is reduced. Thus, a method for solving the above problem is provided.

Description

Technical Field

[0001] The present invention relates to gene therapy, gene transfer, tissue regeneration induction, restoration induction, and genome editing methods. In addition, the present invention relates to using the gene therapy, gene transfer, tissue regeneration induction, tissue restoration induction, and genome editing methods as therapeutic methods by applying them to clinical symptoms and diseases, and using them as a research and development platform for therapeutic methods and biological research. Background Art

[0002] Gene transfer technology, which introduces nucleic acids such as DNA and RNA into cells inside and outside a living organism, can be used as a method for treating diseases that have been difficult to treat with conventional therapies, treating genetic diseases, and regenerative medicine to achieve tissue regeneration. It can also be widely used as a method for drug innovation and biological research.

[0003] In order to use gene transfer as a gene therapy method, a method for its development, or a method for drug innovation / biological research, it is often required to obtain stronger gene expression in a larger number of cells, that is, to achieve high gene transfer efficiency.

[0004] As methods for gene introduction into animal cells, methods based on viral vectors such as adeno-associated virus vectors (AAV vector), adenovirus vectors, retrovirus vectors, lentivirus vectors, and Sendai virus vectors, methods based on administration of DNA itself (naked) such as plasmids and minicircle DNA, calcium phosphate methods, liposome transfection methods, electroporation methods, etc. can be used. In addition, lipid nanoparticles (LNP) can be used for RNA delivery.

[0005] In particular, in gene transfer into living organisms, it is often difficult to obtain high gene transfer efficiency into target organs, tissues, and cells, and thus, in the utilization and development of gene transfer as a therapeutic method, there is a demand for achieving higher gene transfer efficiency.

[0006] In particular, there are no excellent gene introduction methods for the skin and skin ulcer areas. Therefore, in the promotion of research and development of treatments for skin genetic diseases (see non-patent document 1) and tissue regeneration treatments for skin ulcers (see non-patent documents 2 and 3), methods that can achieve high gene introduction efficiency and tissue-specific gene expression are required.

[0007] Skin appendages are composed of hair follicles, sebaceous glands, sweat glands, etc., and play the role of protection from mechanical obstacles, heat preservation, moisture retention, temperature regulation, etc. Skin appendages are formed during the fetal period and organogenesis through the interaction of epithelial tissue and mesenchymal tissue (see Non-Patent Document 4).

[0008] Representative diseases caused by the loss, disorder, or dysfunction of skin appendages include alopecia and sebum deficiency. In addition to temperature regulation disorders and itching caused by dry skin, they also cause cosmetic surgery disorders.

[0009] It is difficult to regenerate lost skin appendages clinically, and it is usually necessary to transplant existing appendages from other parts of the body through skin grafting, skin flap formation, etc.

[0010] In this regard, there are the following methods: a method of producing cells with skin appendage inducing ability as a cell supply source for the purpose of treating defects, disorders, and dysfunctions of skin appendages by introducing an introduced gene containing at least one gene that is relatively strongly expressed in cells with skin appendage inducing ability into somatic cells that do not have skin appendage inducing ability (see Patent Document 1); and a method of inducing skin without skin appendages by introducing genes into somatic cells present in a living body (Non-Patent Document 1). However, there are no reports on a method of inducing skin tissue with skin appendages from somatic cells in a living body by introducing genes into somatic cells present in a living body.

[0011] The upper limbs consisting of the upper arm, forearm, hand, and fingers, and the lower limbs consisting of the thigh, calf, foot, and toes are complex tissues composed of bones, cartilage, muscles, tendons, fat, peripheral nerves, blood vessels, skin, etc.

[0012] In mammals, represented by humans, it is difficult to regenerate any type of higher-order tissue defect clinically, and there is no good treatment except for replacing it functionally or cosmetically by installing artificial limbs, prosthetic feet, prostheses, etc.

[0013] Furthermore, when there is a loss of bone, cartilage, muscle, or fat, especially in the tissues that make up the upper and lower limbs, it is difficult to regenerate the tissue clinically even if it is a single tissue, and therefore tissue transplantation from other parts of the body is performed.

[0014] Prior art literature

[0015] Patent Literature

[0016] Patent Document 1: International Publication No. 2022 / 244502

[0017] Non-patent literature

[0018] Non-patent literature 1: Ain QU, Campos EVR, Huynh A, Witzigmann D, Hedtrich S. Gene Delivery to the Skin-How Far Have We Come? "Trends Biotechnol", 2021, 39(5), p474-487.

[0019] Non-patent literature 2: Kurita M, Araoka T, Hishida T, O'Keefe DD, Takahashi Y, Sakamoto A, Sakurai M, Suzuki K, Wu J, Yamamoto M, Hernandez-Benitez R, Ocampo A, Reddy P, Shokhirev MN, Magistretti P, Nunez Delicado E, Eto H, Harii K, IzpisuaBelmonte JC,“Nature”,2018,561(7722),p243-247

[0020] Non-patent document 3: Kato M, Ishikawa S, Shen Q, Du Z, Katashima T, Naito M, Numahata T, Okazaki M, Sakai T, Kurita M. "Commun Biol", 2023, 6(1), p508.

[0021] Non-patent document 4: Lee J, B■scke R, Tang PC, Hartman BH, Heller S, Koehler KR. "Cell Research", 2018, 22(1), p242-254 Summary of the invention

[0022] The present invention includes: a gene transfer method with high gene transfer efficiency and high specificity for skin, subcutaneous tissue and its constituent cells, a method for improving the efficiency of gene transfer into a living body, a method for regenerating skin appendages using gene transfer, and a method for regenerating the front end of a defective limb. Each of them can be used as a treatment method for the purpose of treating diseases, improving symptoms / promoting health, etc. based on gene transfer, and can also be used as a tool for researching / developing treatment methods based on gene transfer, and can further be used as a drug innovation research and biological research tool other than the purpose of developing a treatment method based on gene transfer.

[0023] Problems to be solved by the invention

[0024] The first problem to be solved is that there is no gene transfer method with high gene transfer efficiency and high specificity for the skin, subcutaneous tissue, skin ulcer surface and their constituent cells.

[0025] The second problem is that, more generally, it is difficult to obtain high gene transfer efficiency and high expression of target genes in gene transfer into living organisms.

[0026] The third problem is that it is difficult to regenerate the skin appendages morphologically and functionally, which are lost due to diseases caused by defects, disorders, and dysfunctions of the skin appendages, or due to physiological changes such as aging.

[0027] The fourth problem is that when the distal tissues of the upper and lower limbs (upper arms, forearms, hands, fingers, thighs, calves, feet, toes, etc.) are lost, it is difficult to regenerate the defective tissues morphologically and functionally.

[0028] Methods for solving problems

[0029] The main feature of the present invention is that, in order to be able to carry out gene introduction with high efficiency and high specificity into the skin, subcutaneous tissue, skin ulcer surface and their constituent cells, the present invention includes: a new AAV (AAV is the abbreviation of adeno-associated virus, the same below) capsid with high gene introduction efficiency and high specificity into the cells of the skin / subcutaneous tissue and skin ulcer surface; a new AAV capsid with high gene introduction efficiency and high specificity into the epidermal tissue and its constituent cells, namely keratinocytes; a new tissue-specific promoter with high gene expression and high specificity into the epidermal tissue and its constituent cells, namely keratinocytes.

[0030] In addition, the main features of the present invention are: a method for improving the efficiency of gene introduction into organs / tissues / cells throughout the body and achieving high gene expression by using steroids systemically or locally before, simultaneously with, or early after gene introduction into various organisms such as AAV, retroviral vectors, lentiviral vectors, adenoviral vectors, Sendai virus vectors, naked DNA such as plasmid DNA, minicircle DNA, and the most recent mRNA (also including substances carried by lipid nanoparticles (LNP)), as well as research reagents and pharmaceuticals that can achieve this method.

[0031] In addition, the main features of the present invention are: a method for inducing regeneration, regeneration, and functional improvement of skin appendages by in vivo gene introduction to achieve sufficient skin appendage function for diseases such as skin ulcers and skin appendage defects, and various states of reduced skin appendage function represented by age-related changes, as well as research reagents and pharmaceuticals that can achieve this method.

[0032] In addition, the main features of the present invention are that it includes: a method for morphologically and functionally regenerating defective tissues in the case of loss of peripheral tissues of upper limbs and lower limbs (upper arms, forearms, hands, fingers, thighs, calves, feet, toes, etc.); research reagents and medicines that can implement this method; and a research and development platform for developing regeneration methods.

[0033] Effects of the Invention

[0034] The AAV of the present invention, namely AAVDJ1, which has excellent gene introduction efficiency into cells on the skin ulcer surface, has the following advantages: for cells on the skin ulcer surface, compared with other capsids including AAVDJ, it shows high gene introduction efficiency and excellent directivity into cells on the skin ulcer surface in vivo, cells of subcutaneous tissue, and their constituent cells under culture conditions.

[0035] The AAV of the present invention, namely AAVDJ2, which has excellent gene transfer efficiency into keratinocytes and epidermal tissues, has the following advantages: compared with AAV2, AAV6, and AAVDJ, which are known to show high gene transfer efficiency into keratinocytes, it shows high gene transfer efficiency and excellent directivity, and shows high gene transfer efficiency, excellent directivity, and high specificity into epidermal tissues in vivo and keratinocytes under culture conditions.

[0036] The promoters with high specificity for keratinocytes / epidermal tissues of the present invention, namely, the K14SCP3 promoter, the K16SCP3 promoter, and the K16P5 short promoter, bring higher or equivalent gene expression compared to the CAG promoter known to bring high gene expression in any tissue / cell type represented by keratinocytes / epidermal tissues, but on the other hand, only bring relatively low gene expression in keratinocytes represented by mesenchymal cells near the epidermis and tissues / cells other than epidermal tissues, and therefore have advantages as tissue-specific promoters for epidermal tissues and cultured keratinocytes.

[0037] The method of inducing regeneration, regeneration and functional improvement of skin appendages in vivo by gene introduction of the present invention has the following advantages: it can induce regeneration, regeneration and functional improvement of skin appendages lost due to physiological changes such as diseases caused by defects, disorders and dysfunctions of skin appendages, and aging.

[0038] The method of regenerating peripheral tissues of upper and lower limbs of the present invention has the following advantages: it is capable of morphologically and functionally regenerating a portion of peripheral tissues of upper and lower limbs that cannot be formed in mammals, represented by humans, thereby becoming a research and development platform for developing a complete regeneration method for the entire defective tissue that has not existed to date. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The results of a comparative study of the gene transfer efficiency of AAVDJ and AAVDJ1 on the ulcer surface of the back skin of mice (Example 1) show that AAVDJ1 has a higher gene transfer efficiency than AAVDJ, especially in the fat layer and muscle layer, which are deep tissues. (a) The upper section shows a diagram of the combination of the introduced gene and capsid administered to each group of mice, and shows a representative stereomicroscope photograph under visible light observation and a stereomicroscope photograph under fluorescence observation of each group. The left graph of (b) is a graph of counting the gene transfer positive cells in the superficial layer, fat layer, and muscle layer as seen in the tissue after AAVDJ encoding GFPNLS (GFP) and AAVDJ1 encoding mCherryNLS (RFP) are mixed and administered, and the right graph of (b) is a graph of counting the gene transfer positive cells in the superficial layer, fat layer, and muscle layer as seen in the tissue after AAVDJ encoding mCherryNLS and AAVDJ1 encoding GFPNLS are mixed and administered, and the image analysis is used to count the gene transfer positive cells in the superficial layer, fat layer, and muscle layer as seen in the tissue.

[0040] Figure 2 The results of a comparative study of gene transfer efficiency of new AAVDJ variants and known AAVs for mouse / human cultured keratinocytes (Example 2) show that new AAVDJ variants AAVDJK1, AAVDJK2, AAVDJK3, AAVDJK8, especially AAVDJK2, have high gene transfer efficiency compared to known AAVs. (a) is a diagram of the amino acid sequences of AAV2, AAV2K1, AAV2K2, AAV2K3, AAV2K8, AAV6, AAVDJ, and new AAVDJK1, AAVDJK2, AAVDJK3, and AAVDJK8, which are known AAVs with excellent gene transfer efficiency for keratinocytes. (b) is a phase contrast microscope photograph of a colony (the portion surrounded by the dotted line) of mouse / human keratinocytes under feeding and culture conditions. (c) is the result of gene transfer experiments on mouse keratinocytes using GFPNLS expression vectors prepared from each capsid, showing that the new AAVDJ variants AAVDJK1, AAVDJK2, AAVDJK3, AAVDJK8, and especially AAVDJK2 have higher gene transfer efficiency than known AAVs. (d) is the result of gene transfer experiments on human keratinocytes using GFPNLS expression vectors prepared from each capsid, showing that the new AAVDJ variants AAVDJK1, AAVDJK2, AAVDJK3, AAVDJK8, and especially AAVDJK2 have higher gene transfer efficiency than known AAVs.

[0041] Figure 3The results of comparative study experiments (Example 3) on the gene transfer efficiency of known AAV2, AAV2K2, AAVDJ and new AAVDJK2 in mouse / human cultured mesenchymal cells show that the high gene transfer efficiency for mesenchymal cells confirmed in AAVDJ and AAV2 is not confirmed in the new AAVDJK2. (a) The results of gene transfer experiments on mouse adipose-derived mesenchymal cells using GFPNLS expression vectors made from various capsids show that the high gene transfer efficiency for mesenchymal cells confirmed in AAVDJ is not confirmed in the new AAVDJK2. (b) The results of gene transfer experiments on human fibroblasts using GFPNLS expression vectors made from various capsids show that the high gene transfer efficiency for mesenchymal cells confirmed in AAV2 and AAANDJ is not confirmed in the new AAVDJK2.

[0042] Figure 4 The results of the injection test of fluorescent silica particles into the back skin of mice (reference example 1) show that most of the particles injected during intradermal injection will not be distributed in the epidermis. (a) is the case of intradermal injection using a surgical microscope. (b) is a diagram of the injection test of fluorescent silica particles into the back skin of mice. (c) is the situation of recovering the skin tissue 1 hour and 3 hours after the injection of fluorescent silica particles, and investigating the distribution of fluorescent silica particles on tissue sections. It can be seen that most of the injected particles are distributed from the intradermis to the subcutaneous part. (d) is an enlarged image of the epidermis of the same test object. It can be seen that most of the injected fluorescent silica particles will not be distributed in the epidermis.

[0043] Figure 5 The results of a comparative experiment on the gene transfer efficiency of AAVDJK2 and AAVDJ in the dorsal skin of mice show that AAVDJK2 has higher gene transfer efficiency and specificity in epidermal tissue than AAVDJ. (a) is a diagram of the experiment. AAVDJK2 expressing GFPNLS under the CAG promoter and AAVDJ expressing mCherryNLS were co-transfected with the same titer (10 11 (b) is a fluorescent stereomicroscope image of the tissue at the injection site collected 2 days later. (c) shows the histological findings that GFPNLS introduced by AAVDJK2 is strongly expressed in the epidermis and weakly expressed in the subcutaneous tissue, while mCherry introduced by AAVDJ is weakly expressed in the epidermis and strongly expressed in the subcutaneous tissue.

[0044] Figure 6The results of comparative study experiments (Example 5) on gene expression when AAV with AAVDJK2 capsid expressing GFPNLS via novel K14SCP3 promoter, K16SCP3 promoter, K16P5 short promoter and known CAG promoter, K14 promoter, K14 short promoter and K16P5 promoter was used as a vector for gene transfer into mouse / human cultured keratinocytes and mesenchymal cells show that the novel promoter has high gene transfer efficiency and / or high specificity for keratinocytes. (a) is a diagram of the GAPNLS expression AAV plasmid sequence carrying novel K14SCP3 promoter, K16SCP3 promoter, K16P5 short promoter and known CAG promoter, K14 promoter, K14 short promoter and K16P5 promoter. (b) is the result of gene transfer experiment on mouse keratinocytes using AAVDJK2 capsid vectors expressing GFPNLS from various promoters, showing that K14SCP3 promoter has higher gene transfer efficiency than CAG promoter, K14 promoter, and K14 short promoter, and K16SCP3 promoter and K16P5 short promoter have higher gene transfer efficiency than K14 promoter, K14 short promoter, and K16P5 promoter. (c) is the result of gene transfer efficiency investigated by flow cytometry after gene transfer on mouse keratinocytes using AAVDJK2 capsid vectors expressing GFPNLS from various promoters, showing that K14SCP3 promoter has higher gene transfer efficiency than CAG promoter, K14 promoter, and K14 short promoter, and K16SCP3 promoter and K16P5 short promoter have higher gene transfer efficiency than K14 promoter, K14 short promoter, and K16P5 promoter. (d) is the result of gene transfer experiment on mouse adipose-derived mesenchymal cells using AAVDJK2 capsid vectors expressing GFPNLS from various promoters, showing that the gene transfer efficiency of keratin-specific promoters including the new K14SCP3 promoter, K16SCP3 promoter, and K16P5 short promoter is much lower than that of CAG promoter. (e) is the result of gene transfer efficiency investigated by flow cytometry after gene transfer into mouse adipose-derived mesenchymal cells using AAVDJK2 capsid vectors expressing GFPNLS from various promoters, showing that the gene transfer efficiency of keratin-specific promoters including the new K14SCP3 promoter, K16SCP3 promoter, and K16P5 short promoter is much lower than that of CAG promoter.

[0045] Figure 7This is the result of a gene transfer experiment in which AAVDJK2 expressing GFPNLS under the K14SCP3 promoter was intradermally injected into the back skin of mice, showing that the vector introduced genes into the epidermal tissue with high efficiency and high specificity. In the tissue image of the back skin of mice on the second day after 10 μl of steroid (Kenacort injection) was subcutaneously administered to the abdomen, high GFPNLS expression was consistently confirmed in the epidermal tissue (white arrow) in the back skin. If the epidermal tissue of the hair follicles is removed, gene transfer from the dermis to the subcutaneous tissue is suppressed to a low frequency.

[0046] Figure 8 The figures show an experiment in which a skin ulcer surface isolated from the surrounding skin by installing a chamber on the dorsal fascia of mice was used to introduce genes using AAV to induce skin with skin appendages. The figure shows that skin with skin appendages was induced on the skin ulcer surface isolated from the surrounding skin. (a) is a diagram of the experiment. The date when the skin ulcer was created and the chamber was installed is set as D0, and the timing of gene introduction using AAV is shown. DJ1 and DJ represent the capsid of the AAV used (AAVDJ1 and AAVDJ, respectively). The part written in italics represents the introduced gene. The numerical value on the right end of each virus (5, 10, 20, 25, 50, 100) represents the titer of the administered AAV (×10 10 GC, gene copy number). (b) is an image showing the appearance of the ulcer surface over time. It can be seen that epidermal tissue has been induced on the ulcer surface since D21. (c) is a stereomicroscope image of the skin ulcer surface at D28. Hair growth (arrow) can be seen. (d) is tissue observation. HE represents hematoxylin staining, KRT14 represents keratin 14 as an epidermal marker, and DAPI represents cell nuclei. It can be seen that in the epidermal part induced on the ulcer surface independent of the surrounding skin, a skin appendage structure composed of hair follicles and sebaceous glands is formed.

[0047] Figure 9 The figures show an experiment in which a skin ulcer surface isolated from the surrounding skin by installing a chamber on the dorsal fascia of mice was used to introduce genes using AAV to induce skin with skin appendages. The growth of black hair was confirmed on the skin ulcer surface isolated from the surrounding skin. (a) is a diagram of the experiment. The date when the skin ulcer was made and the chamber was installed is set as D-1, which shows the timing of gene introduction using AAV. DJ1, DJ, and DJK2 represent the capsids of the AAV used (AAVDJ1, AAVDJ, and AAVDJK2, respectively). The parts written in italics represent the introduced genes. The right-hand end values ​​of each virus (10, 25, 50, 100) represent the titer of the AAV administered (×10 10GC, gene copy number). (b) shows the appearance of the ulcer surface over time. It can be seen that epidermal tissue (arrow) is induced on the ulcer surface from D21. (c) is a stereomicroscope image of the skin tissue induced on the ulcer surface at D30. Black hair growth (arrow) is seen.

[0048] Figure 10 This is a graphic illustration of an experiment on the effect of steroids on the efficiency of AAV-based gene delivery into skin ulcers. 11 The day before GC / 100μl, a skin ulcer was made on the back of the mouse and a silicon chamber was installed. A steroid-free group, a distal administration group in which 10μl of Corninger A (40mg / ml) as a steroid was injected subcutaneously into the abdomen, and a local administration group in which 10μl of Corninger was administered into the chamber were prepared. On the 7th day after AAV administration, the skin ulcer was observed with a fluorescent stereomicroscope, and tissues including the ulcer were collected.

[0049] Figure 11 The images are stereomicroscopic observations of the ulcer surface under visible light and fluorescence on day 7 after AAV administration, showing that strong GFP expression was observed on the ulcer surface in the steroid distal administration group.

[0050] Figure 12 It is the histological findings on the 7th day after AAV administration, showing that a large number of GFP-positive cells were confirmed in the distal administration group, especially in the deep layer. (a) is a representative finding of the ulcer tissue of each group, showing that the number of GFP-positive cells in the subcutaneous and deep parts of the steroid-free group is small, while a large number of GFP-positive cells were confirmed in the distal administration group, especially in the deep layer, and the number of positive cells in the shallow and deep layers in the local administration group was small. (b) is the number of GFP-positive cells counted on the tissue findings in the central part of the ulcer. It shows that if the shallow and deep layers are combined, significantly more GFP-positive cells are confirmed in the distal group compared with the steroid-free group. (c) It shows that if it is limited to the deep layer (subcutaneous + muscle layer), significantly more GFP-positive cells are confirmed in the distal group compared with the steroid-free group and the local administration group.

[0051] Figure 13 The results of a study on the effect of different concentrations of steroids on the efficiency of AAV-based gene delivery to skin ulcers were obtained after administration of AAVDJ-CAG-GFP virus for 10 11The skin ulcer surface was made on the back of the mouse the day before GC / 100μl, and a silicon chamber was installed. A steroid non-administered group, a distal administration group in which 1μl and 10μl of Corninger A (40mg / ml) as a steroid was injected subcutaneously into the abdomen, and a local administration group in which 1μl and 10μl of Corninger were administered into the chamber. On the 6th day after AAV administration, the skin ulcer surface was observed with a fluorescent stereomicroscope, and tissues including the ulcer surface were collected. As a result, the number of positive cells was higher in the distal administration group at 10μl, while the number of positive cells was higher in the local administration group at 1μl.

[0052] Figure 14 The results of the experiment on the effect of steroid administration on the efficiency of AAV-based gene transfer into the back skin of mice show that the gene transfer efficiency is improved by steroid administration. (a) is a diagram of the experiment. A distal administration group and a steroid-free group were prepared in which 10 μl of Conacton A (40 mg / ml) as a steroid was injected subcutaneously into the abdomen. One day later, the back was depilated, and then AAVDJK2-K14SCP3-GFPNLS10 was injected intradermally into the back skin. 11 GC / 35μl. Observation and tissue collection were performed on the second day after AAV injection. (b) Based on observations based on a stereomicroscope, it was confirmed that GFP expression was stronger in animals administered with steroids. (c) Histologically, GFPNLS-positive cells were confirmed with high efficiency and continuously in the epidermis in animals administered with steroids. In animals without steroids, GFPNLS-positive cells were scattered at a low frequency.

[0053] Figure 15 The results of an experiment on the effect of different concentrations of steroids on the efficiency of AAV-based gene transfer into the skin show that if the amount of steroid administered to the distal subcutaneous part of the abdomen is extremely reduced (0.01 μl), the effect of improving the gene transfer efficiency brought about by the steroid is reduced.

[0054] Figure 16 The figure shows the surgical operation of shifting the left upper limb to the dorsal side after the left upper limb is amputated in a mouse, with the purpose of avoiding the mouse's own invasion of the stump and keeping the local area as quiet as possible. The surgical method of each stage is shown in the order of left segment from top to bottom, middle segment from top to bottom, and right segment from top to bottom. The brachial plexus and the main arteries and veins to the upper limb are preserved during the operation. Through this surgical method, the left upper limb is shifted to the dorsal side.

[0055] Figure 17The results of the experiment on the effect of steroid administration on the efficiency of gene introduction based on AAV and retrovirus in the forearm amputation stump of mice show that the gene introduction efficiency is improved by the administration of steroids. (a) Shows the appearance of the recovered forearm amputation tissue and representative tissue observations. There is bone in the central part, muscle in the surrounding area, and skin and subcutaneous fat cover the outside. The black scale bar represents 2mm. (b) It is the tissue observation when GFPNLS is introduced into the gene using AAV and retroviral vectors after no steroid administration, local administration, and distal administration. In AAV and retrovirus, the gene introduction efficiency is greatly improved by local administration and distal administration of steroids. In particular, in the study of AAV, a significant improvement in the efficiency of gene introduction was confirmed in a wide range of tissues that constitute the limbs, such as subcutaneous, fascia, muscle, and periosteum. The scale bar of the low-magnification image represents 2mm, and the scale bar of the high-magnification image represents 500μm.

[0056] Figure 18 The results of the experiment on the effect of steroid administration on the efficiency of gene introduction based on mRNA in the front thigh muscle of mice show that the gene introduction efficiency is improved by steroid administration. (a) is a stereoscopic observation 12 hours after the steroid was administered to the distal end of the abdomen, and the mRNA expressing EGFP treated with Lipofectamine 2000 was injected into the front thigh muscle of nude mice. It shows that the fluorescent signal that can be confirmed by stereomicroscope was confirmed on the muscle surface of the steroid distal administration group. (b) is the observation of the tissue of the muscle body, showing that stronger GFP expression was confirmed in the steroid administration group.

[0057] Figure 19 The results of the experiment on the effect of steroid administration on the efficiency of AAV-based gene transfer into in vitro cultured cells showed that higher gene transfer can be achieved with appropriate concentrations of steroids. In the presence of 1 and 10 μg / ml steroids, more GFP-positive cells were confirmed compared to the absence of steroids, while in the presence of 100 and 1000 μg / ml steroids, the number of cells and the number of positive cells decreased due to the steroid effect on cells.

[0058] Figure 20The results of the experiment on the effect of steroid administration on the dynamic generation of subcutaneously injected fluorescent silica particles show that the local clearance rate of locally injected fluorescent silica particles is reduced by steroid administration. (a) is a diagram of the experiment, in which a steroid-free group and a distal administration group in which 10 μl of the steroid Corning A (40 mg / ml) was subcutaneously injected into the abdomen one day before were prepared. After 30 μl of fluorescent silica particles (sicastar (registered trademark)-redF, plain (unmodified), 30 nm, 25 mg / ml) with the same size as AAV were subcutaneously injected into the back of the mouse, local observation was performed using a fluorescent stereo microscope immediately, 1, 3, 6, 9, 12, 18, and up to 24 hours later to analyze the effect of steroids on the local retention of fluorescent silica particles. The left side of (b) is the result of taking fluorescence microscope images over time according to the exposure time (30 msec), and the right side is the result of taking images with a longer exposure time when the tissue is recovered after 24 hours. It shows that after the injection of steroid-injected animals, silica particles are obviously locally retained. In the observation at the time point of 24 hours, in the animals without steroids, it is also shown that the particles further diffuse to the periphery. (c) is the tissue section of the tissue collected at the time point of 24 hours. Hematoxylin and eosin staining (HE), nuclear staining using DAPI and silica, and fluorescence observation of silica alone are shown side by side. The tissue observation also shows that in the animals injected with steroids, the diffusion of silica particles to the surrounding tissues is inhibited compared with the animals without steroids, and the local retention is more obvious.

[0059] Figure 21 Illustration showing the method of treating the stump of the cut part of the nude mouse forearm. On the skin surface of the nude mouse forearm, there is a structure with a gentle protrusion and slight redness in the part proximal to the wrist joint (arrow in the right photo). In newborn mice, there is also a protrusion in this part, which is characterized by the growth of hair from birth (arrow in the left photo), and is a structure that is consistently present in anatomy. By setting the cutting height to a position proximal to the structure, it is possible to reliably cut at a position proximal to the wrist joint. Regarding the cut part, a simple transverse cut is made, or the surrounding skin of the stump is excised after cutting, or the nerve is retained for a longer time when cutting. The wound surface after cutting is set as an open wound, or the surrounding skin is crimped or sutured with the fingers. In the case of a lot of bleeding from the stump, hemostasis can be promoted by installing a silicon cap.

[0060] Figure 22The results of the experiment were that the left upper limb of the nude mouse was displaced dorsally, the forearm was cut horizontally, and then the cut surface was set as an open wound, showing the appearance of the stump and the tissue findings. No large morphological changes of the stump were confirmed in appearance until 70 days after the cut. On the tissue section, the stump of the bone was partially closed by cortical bone. The soft tissue was healed by the contraction of the surrounding skin. No significant tissue regeneration was confirmed. The upper scale bar represents 5 mm, and the lower scale bar represents 1 mm. The dotted line represents the cut surface judged based on the morphology of the bone.

[0061] Figure 23 The results of the experiment of gene introduction were performed after the left upper limb of the nude mouse was displaced dorsally, and the forearm was cut horizontally. The cut surface was set as an open wound, and the appearance and tissue findings of the stump were shown. The genes of POLE4, NFIB, PPARD, FGF10, FGF20, and FGF2 were introduced using retrovirus, PRRX1 and HDAC2 were introduced using AAVDJ1, and LEF1 and SHH were introduced using AVDJ. For retrovirus, 10 local injections were made from just before the cut to the fifth day after the cut. For AAVDJ-SHH, it was injected locally once immediately after the cut. For other AAV vectors, the three were mixed, applied to the wound twice on the day of the cut, injected twice on the next day, and injected three times on the second day. After 4 weeks after the cut, the tissue of the front part began to elongate axially. In the tissue findings of the tissues recovered at the 36th day, the axial extension of the bone in the tissue of the front part was confirmed. Furthermore, the front part exceeds the front part of the bone and is formed by a soft tissue mass (enlarged image). The scale bar in the tissue view indicates 10 mm. From the top, the first section: the front view of the dorsal appearance, the second section: the side view, the third, fourth, and fifth sections: the continuous slice images and enlarged images of the tissue view.

[0062] Figure 24The results of the experiment of gene introduction were performed after the left upper limb of the nude mouse was displaced dorsally, and the forearm was cut transversely. The cut surface was set as an open wound, and the appearance and tissue findings of the stump were shown. The genes of PRDX2, POLE4, NFIB, PPARD, FGF10, FGF20, FGF2, BMP5, and SHH were introduced using retrovirus, PRRX1 and HDAC2 were introduced using AAVDJ1, and LEF1 was introduced using AVDJ. Regarding retrovirus, 5 local injections were performed immediately before and from the second to fifth day after cutting. Regarding AAV vectors, 3 types were mixed and injected twice immediately before and the next day. After 3 weeks after cutting, the tissue at the front end began to elongate axially. In the tissue findings of the tissue recovered at the 40th day, a soft tissue mass was formed at the distal end of the bone cut surface, and the regeneration of bone with trabecular structure was confirmed from the inside (enlarged image). Starting from the top, section 1: dorsal and frontal appearance images, section 2: lateral appearance images, sections 3, 4, and 5: serial section images and magnified images of tissue findings.

[0063] Figure 25 The results of the experiment of gene introduction were performed after the left upper limb of the nude mouse was displaced to the dorsal side, and the forearm was cut horizontally. The cut surface was set as an open wound, and the appearance and tissue observation of the stump were shown. The genes of FGF10, FGF20, FGF2, NFIB, PPARD, and POLE4 were introduced into the stump using retrovirus. The gene of SHH was introduced into the outside of the stump using retrovirus. The gene of FGF8 was introduced into the stump using AAVDJK2. For the retrovirus expressing 6 genes, 18 local injections were performed from just before the cut to the 5th day. For the retrovirus expressing SHH, 11 local injections were performed from just after the cut to the 3rd day. For AAVDJK2 expressing FGF8, 7 local injections were performed from the 5th day to the 23rd day after the cut. Starting from 3 weeks after the cut, the shape of the front part became flat, and branched white structures were seen when observed from the palm side. In the histological view of the tissue collected at the 28th day, bone marrow cells (arrows) are clustered in the soft tissue mass in the same position as the white structure. From the top, the first section: the front view of the dorsal appearance, the second section: the front view of the ventral appearance, the left side of the third section: the palm side of the appearance on the 28th day, the right side of the third section: histological view.

[0064] Figure 26The results of the experiment in which the left upper limb of the nude mouse was displaced dorsally, the forearm was cut horizontally, and the skin around the cut surface was pressed with fingers to perform gene introduction. The appearance of the stump, representative tissue findings, and magnified images are shown. FGF10, FGF20, FGF2, and OCT4 genes were introduced into the stump using retrovirus. SHH gene was introduced into the outside of the stump using AAVDJ, and FGF8 gene was introduced into the stump from the inside to the front end using AAVDJ. AAVDJ expressing SHH and FGF8 was injected locally four times at intervals of 7 days from the day before cutting to the 20th day after cutting. Retrovirus expressing FGF10, FGF20, FGF2, and OCT4 was injected locally 20 times from the time of cutting to the 4th day. Then, retrovirus expressing OCT4 was injected locally 16 times from the 5th day to the 10th day after cutting. When the first injection of retrovirus solution was injected just before cutting, Conicton A was mixed and injected. About 10 weeks after cutting, a protrusion-like structure appeared at the front end and continued to elongate. In the tissue observation of the tissue collected at the 105th day, a cell structure running longitudinally along the axial direction in the elongated tissue was confirmed. From the top, the first paragraph: the front image of the dorsal appearance, the second paragraph: the side image of the appearance, the left side of the third paragraph: the tissue observation including the protrusion-like structure, the right side of the third paragraph: the enlarged image of the protrusion-like structure.

[0065] Figure 27The results of the experiment of gene introduction were obtained by displacing the left upper limb of the nude mouse to the dorsal side, cutting it transversely at the forearm, and pressing the surrounding skin with fingers on the cut surface. The appearance of the stump, representative tissue findings, and magnified images are shown. The genes of FGF10, FGF20, FGF2, OCT4, and SOX2 were introduced into the stump using retrovirus. The gene of SHH was introduced into the outside of the stump using AAVDJ, and the gene of FGF8 was introduced into the inside of the stump to the front part using AAVDJ. AAVDJ expressing SHH and FGF8 was injected locally four times at intervals of 7 days from the day before cutting to the 20th day after cutting. The retrovirus expressing FGF10, FGF20, FGF2, OCT4, and SOX2 was injected locally 20 times from the time of cutting to the 4th day. Then, the retrovirus expressing OCT4 and SOX2 was injected locally 16 times from the 5th day after cutting to the 10th day after cutting. When the first dose of retrovirus solution is injected just before the cut, Corning A is mixed and injected. About 8 weeks after the cut, a protrusion-like structure appears at the front end, and then the front end continues to extend in the shape of a branch. In the tissue observation of the tissue recovered at the 70th day, a cell structure running longitudinally along the axial direction in the extended branch was confirmed. From the top, the first paragraph: the frontal image of the dorsal appearance, the second paragraph: the side image of the appearance, the left side of the third paragraph: the tissue observation of the structure containing the branch extension, the right side of the third paragraph: the enlarged image of the structure of the branch extension.

[0066] Figure 28The results of the experiment in which the left upper limb of the nude mouse was displaced dorsally, the forearm was cut horizontally, and the skin around the cut surface was pressed with fingers to perform gene introduction. The appearance of the stump, representative tissue findings, and magnified images are shown. The genes of FGF10, FGF20, FGF2, OCT4, SOX2, and TBX6 were introduced into the stump using retrovirus. The gene of SHH was introduced into the outside of the stump using AAVDJ, and the gene of FGF8 was introduced from the inside of the stump to the front end using AAVDJ. For AAVDJ expressing SHH and FGF8, it was locally injected 4 times at intervals of 7 days from the day before cutting to the 20th day after cutting. For retrovirus expressing FGF10, FGF20, FGF2, OCT4, SOX2, and TBX6, it was locally injected 20 times from the time of cutting to the 4th day. Next, retrovirus expressing OCT4, SOX2, and TBX6 was locally injected 16 times from the 5th day to the 10th day after amputation. When the first injection of retrovirus solution was injected just before amputation, Conicton A was mixed and injected. About 8 weeks after amputation, a protruding structure appeared at the front end, and then the front end continued to extend in the shape of branches. In the tissue observation of the tissue recovered at the 98th day, a tissue structure composed of cartilage cells in a shape similar to the distal phalanx of the finger and a trabecular structure accompanied by bone regeneration were confirmed in the extended branch part along the axial direction. From the top, the first paragraph: the frontal image of the dorsal appearance, the second paragraph: the side image of the appearance, the third paragraph: the frontal image of the ventral appearance, the left side of the fourth paragraph: the enlarged image of the front part at the 98th day, the middle of the fourth paragraph: the tissue observation of the structure containing the branch extension, the right side of the fourth row: the enlarged image of the tissue observation of the structure of the branch extension.

[0067] Figure 29The results of the experiment in which the left upper limb of the nude mouse was displaced dorsally, the forearm was cut horizontally, and the skin around the cut surface was pressed with fingers to perform gene introduction. The appearance of the stump, representative tissue findings, and magnified images are shown. The genes of FGF10, FGF20, FGF2, OCT4, SOX2, TBX6, and SHH were introduced into the stump using retrovirus. The gene of FGF8 was introduced into the front end of the stump using AAVDJ. For AAVDJ expressing FGF8, it was locally injected four times at intervals of 7 days from the day before cutting to the 20th day after cutting. For retrovirus expressing FGF10, FGF20, FGF2, OCT4, SOX2, and TBX6, it was locally injected 16 times from the time of cutting to the 5th day after cutting. The retrovirus expressing SHH was locally injected four times from the time of cutting to the 3rd day. Next, retrovirus expressing OCT4, SOX2, and TBX6 was locally injected seven times from the 6th day to the 8th day after amputation. When the retrovirus solution was injected after amputation, Corninger A was mixed and injected. In the tissue observation of the tissue recovered at the 56th day after amputation, a bag-like structure was formed at the distal end of the bone stump, and new muscle bundles independent of the cartilage tissue and surrounding muscles were confirmed inside. From the top, the first paragraph: the frontal image of the dorsal appearance, the second paragraph: the frontal image of the ventral appearance, the middle of the third paragraph: the overall image of the tissue, the left side of the third paragraph: the enlarged image of the bag-like structure at the distal end of the bone stump, the arrow is the cartilage tissue, the right side of the third paragraph: the enlarged image of the bag-like structure at the distal end of the bone stump, the arrow is the new muscle tissue.

[0068] Figure 30 The results of the experiment show the appearance and tissue findings of the stump after the left upper limb of the nude mouse was displaced dorsally, and gene introduction based on FGF2 injection and virus liquid injection was performed at the predetermined cutting site. The forearm was cut horizontally, and the cut surface was set as an open wound. The gene introduction was also performed after cutting. The results show the appearance of the stump and the tissue findings. Before cutting, the gene introduction of HMGB3, DMNT, 1HDAC2, PRDX2, and FGF10 was performed once a day for 3 days using a retrovirus mixed with FGF2. Just before cutting, a retrovirus liquid expressing HMGB3, DMNT, 1HDAC2, PRDX2, and FGF10 mixed with FGF2 was also locally injected. The gene introduction of SHH was performed on the outside of the stump after cutting using AAVDJ. On the first and second days after cutting, the retrovirus liquid was applied to the cut surface once a day. In appearance, the front end began to protrude and elongate over time. Histologically, cartilage tissue with endochondral ossification-like tissue similar to intra-articular cartilage was induced in the bone stump. From the top, Section 1: dorsal frontal image, Section 2: lateral image, Sections 3 and 4: serial section images and magnified images of tissue findings.

[0069] Figure 31 The results of the experiment show the appearance and tissue findings of the stump after the left upper limb of the nude mouse was displaced dorsally, gene introduction was performed by injecting FGF2 and virus solution at the planned cutting site, and the forearm was cut horizontally. The cut surface was set as an open wound, and gene introduction was performed after cutting. The results show the appearance and tissue findings of the stump. Gene introduction of FGF10 and injection of FGF2 using retrovirus were performed from 4 days before cutting to 2 days before cutting. When cutting, the nerve stump was kept long and cut, and the skin around the stump was removed and the virus solution was injected again. One week after cutting, SHH gene introduction was performed on the outside using AAVDJ1. Histologically, cartilage tissue was induced continuously in the muscular layer near the stump. From the top, the first paragraph: the frontal image of the dorsal appearance, the second paragraph: the side image of the appearance, the third paragraph: the enlarged image of the front end of the tissue section, and the fourth paragraph: the tissue findings including the front part.

[0070] Figure 32 The results of the experiment show the appearance and tissue findings of the stump after the left upper limb of the nude mouse was displaced dorsally, and gene introduction was performed by injecting FGF2 and virus solution at the planned cutting site. The forearm was cut horizontally, and the cut surface was set as an open wound. The gene introduction was also performed after cutting. The results show the appearance and tissue findings of the stump. Gene introduction of HMGB3, DMNT1, HDAC2, and FGF10 using retrovirus and FGF2 injection were performed from 4 days before cutting to 2 days before cutting. When cutting, the nerve stump was kept long and cut, and the skin around the stump was removed and the virus solution was injected again. One week after cutting, the SHH gene was introduced on the outside using AAVDJ1. Histologically, cartilage tissue was continuously induced from the skin muscle layer near the stump, and the cartilage-like tissue covered the bone stump. From the top, the first paragraph: the frontal image of the dorsal appearance, the second paragraph: the side image of the appearance, the left side of the third paragraph: the tissue findings of the section including the front part, and the right side of the third paragraph: the enlarged image of the tissue findings of the front part.

[0071] Figure 33The results of the experiment show the appearance and tissue findings of the stump after the left upper limb of the nude mouse was displaced dorsally, and gene introduction was performed by injecting FGF2 and virus solution at the planned cutting site. The forearm was cut horizontally, and the cut surface was set as an open wound. The gene introduction was also performed after cutting. The results show the appearance and tissue findings of the stump. Gene introduction of MSX1, MSX2, LIN28A, MEIS1, and FGF10 and FGF2 injection using retrovirus were performed from 8 days before cutting to 1 day before cutting. When cutting, the nerve stump was kept long and cut, and the skin around the stump was removed, and the virus solution was injected again. 11 days after cutting, SHH gene introduction was performed on the outside using AAVDJ1. 14 days after cutting, LEF1 and FGF8 gene introduction were performed on the front part using AAVDJK2. Histologically, axial extension of the bone stump was induced, and cartilage tissue with endochondral ossification-like tissue similar to intra-articular cartilage was induced at the bone stump. From the top, the first section: dorsal and frontal appearance image, the second section: lateral appearance image, the third section: adjacent tissue sections containing the front end part (the first and third from the left) and enlarged images of each front end part (the second and fourth from the left).

[0072] Figure 34 The results of an experiment in which the left upper limb of a nude mouse was displaced dorsally, and gene transduction was performed by injecting FGF2 and virus solution at the planned cutting site. The forearm was cut transversely, and the cut surface was set as an open wound. Gene transduction was also performed after cutting. The results show the appearance and tissue findings of the stump. From 5 days before cutting to 2 days before cutting, retrovirus was used to introduce genes for MSX1, MSX2, LIN28A, WNT7A, and FGF10, and inject FGF2. One day before cutting, SHH gene transduction was performed using AAVDJ1, and LEF1 gene transduction was performed using AAVDJK2. After injecting retroviral solution, the stump was cut transversely, and the skin around the stump was removed. Retroviral solution was also injected the day after cutting. Histologically, muscle tissue was induced under the skin around the cut stump. From the top, the first paragraph: the frontal image of the dorsal appearance, the second paragraph: the side image of the appearance, the left side of the third paragraph: the tissue findings of the section including the front part, and the right side of the third paragraph: the enlarged image of the tissue findings of the front part.

[0073] Figure 35The results of an experiment in which the left upper limb of a nude mouse was displaced dorsally, and gene transduction was performed by injecting FGF2 and virus solution at the planned cutting site. The forearm was transversely cut, and the cut surface was set as an open wound. Gene transduction was also performed after cutting. The results show the appearance and tissue findings of the stump. From 5 days before cutting to 2 days before cutting, LEF1, HDAC2, PRDX2, WNT7A, and FGF10 gene transduction and FGF2 injection were performed using retrovirus. One day before cutting, SHH gene transduction was performed using AAVDJ1, and LEF1 gene transduction was performed using AAVDJK2. After injecting the retrovirus solution, the stump was transversely cut, and the skin around the stump was removed. The retrovirus solution was also injected the day after cutting. Histologically, muscle tissue was induced under the skin around the cut stump. From the top, the first paragraph: the frontal image of the dorsal appearance, the second paragraph: the side image of the appearance, the left side of the third paragraph: the tissue findings of the section including the front part, and the right side of the third paragraph: the enlarged image of the tissue findings of the front part.

[0074] Figure 36 The left upper limb of a nude mouse was displaced dorsally and then cut transversely at the forearm. The cut surface was set as an open wound, and the gene was introduced using AAV after cutting. The results show the appearance of the stump and the tissue findings. From the second day after cutting, the gene of LEF1, PRRX1, HDAC2, PRDX2, PARD, FGF10, FGF20, FGF2, BMP5, and SHH was introduced into the stump using AAVDJ. The front part was enlarged with a rounded shape, and the proliferation of fat tissue was confirmed in the muscle tissue from the subcutaneous to the deep fascia. From the top, the first section: the front view of the dorsal appearance, the second section: the side view, and the third and fourth sections: the continuous section images of the tissue findings. DETAILED DESCRIPTION

[0075] 1. Use of AAV with excellent gene transfer efficiency in cells on skin ulcer surface

[0076] In the present invention, "cells on the skin ulcer surface" are cells present on the skin ulcer surface of skin defects. In addition to cells physiologically present in the skin and soft tissues such as dermal-derived fibroblasts, mesenchymal-derived adipose tissue, adipose-derived mesenchymal cells, adipocytes, cells present in fascia, mesenchymal cells present in muscle tissue, muscle cells, and tissue macrophages, they also include circulating blood-derived cells such as monocytes and macrophages that gather on the skin ulcer surface through trauma signals.

[0077] It is known that AAV has high gene transfer efficiency, i.e., targeting, for specific cell types and tissues depending on the structure of its capsid. In particular, AAVDJ, an artificially developed capsid, shows significantly better targeting than other capsids for cells of mouse skin and soft tissues, as well as skin ulcer surfaces (see non-patent document 2, Grimm D, Lee JS, Wang L, Desai T, Akache B, Storm TA, Kay MA. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol. 2008 Jun; 82(12): 5887-911.).

[0078] In the present invention, "gene therapy" not only refers to a therapeutic method that supplements the functional deficiency of a target gene by expressing the target gene, but also refers to a therapeutic method that achieves tissue regeneration by expressing the target gene, a therapeutic method that introduces artificial genes with specific functions such as microRNA and siRNA, a therapeutic method that introduces genes for the purpose of altering genomic genes and editing the genome, and all other therapeutic methods that introduce genes for the purpose of treating diseases, improving physiological functions, preventing / improving aging, etc.

[0079] In the present invention, "gene introduction vector" refers to a medium or vector that delivers DNA or RNA into the cells of animals and plants in order to manipulate gene expression in the cells of animals and plants, or to manipulate the gene itself. Representative gene introduction vectors include viral vectors such as AAV, adenovirus vectors, lentivirus vectors, retrovirus vectors, Sendai virus vectors, plasmid vectors, mRNA, lipid nanoparticles composed of mRNA, etc.

[0080] In the present invention, "gene transfer medicines" refer to substances used for the purpose of treating or preventing diseases, morbid disorders, or recovery from physiological states, or further improvement from normal states in subjects who need to treat or prevent diseases, morbid disorders, or functional decline from physiological states, or subjects who expect further improvement from normal states (for example, recovery from aging-related changes). Quasi-drugs, cosmetics, etc. intended to affect the structure or function of humans or animals are also included in this category.

[0081] In the present invention, "a research and development tool that can be used as a gene therapy" does not only refer to a tool used for demonstration experiments in experimental animals for gene therapy. It also includes the use of gene introduction for the development of therapeutic methods and effect verification of other methods that ultimately envision inducing gene expression (various viral vectors, plasmid DNA administration, microRNA, siRNA, mRNA administration, etc.). In particular, it also includes the use of gene introduction as part of a screening method such as CRISPR screening.

[0082] In the present invention, "can be used as a drug innovation research and biological research tool for purposes other than the development of therapeutic methods based on gene introduction" includes the following situations: using gene introduction as a research and development tool for the purpose of preparing pathological models, controlling molecular biological signal pathways, etc., and ultimately using it as a therapeutic method development and biological research and development tool other than gene therapy such as low molecular weight compounds, medium / high molecular weight compounds, protein preparations, antibody pharmaceuticals, cell pharmaceuticals.

[0083] The AAV of the present invention, i.e., AAVDJ1, which has excellent gene introduction efficiency for cells on the skin ulcer surface, has the following advantages: for cells on the skin ulcer surface, compared with other capsids including AAVDJ, it shows higher gene introduction efficiency and excellent directivity for cells on the skin ulcer surface, so in addition to being used as a gene introduction vector in gene therapy for skin ulcer surfaces in vivo, it can also be used as a research and development tool for gene therapy for skin ulcer surfaces. In addition, it can be used as a drug innovation research and biological research tool for purposes other than the development of therapeutic methods based on gene introduction. Since it also shows high directivity when culturing various cells present on the skin ulcer surface, it can be used as a gene introduction vector in gene therapy using cultured cells as a gene introduction vector for cultured cells, and in addition, it can also be used as a research and development tool for gene therapy. In addition, it can be used as a drug innovation research and biological research tool for purposes other than the development of therapeutic methods based on gene introduction.

[0084] The AAV of the present invention having excellent gene transfer efficiency into skin ulcer surface is developed by using directed evolution method for capsid-modified AAVDJ library, wherein the capsid-modified AAVDJ library is prepared by using a plasmid, wherein a part of the base sequence of the Rep-Cap capsid of AAVDJ is modified in such a way that QRG is encoded at the amino acid position 589 and 590 of the AAVDJ capsid, i.e., the position of NR, followed by encoding 7 random amino acids, followed by 11 bases of A (QRGXXXXXXXA (SEQ ID NO. 1), where X is a random amino acid) (see Muller OJ, Kaul F, Weitzman MD, Pasqualini R, Arap W, Kleinschmidt JA, Trepel M. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat Biotechnol. 2003; 21(9): 1040-6.). After the capsid-modified AAVDJ library was infected with the skin ulcer surface on the back of the mouse, the infected ulcer surface tissue was recovered and primary mesenchymal cells were isolated and cultured therefrom. DNA containing the viral genome was extracted from the cells, and the extracted DNA was subjected to PCR to amplify the fragment containing the randomized base sequence, which was cloned into the backbone vector, and AAV was made again, and the cycle was repeated. The fragments remaining after repeating 4 series of independent cycles were sequenced, and AAV expressing GFPNLS was prepared for the modified AAVDJ with multiple candidate sequences detected from multiple systems at the randomized sequence site. The result of studying the gene introduction efficiency using mouse fat-derived mesenchymal cells and the skin ulcer surface made on the back of the mouse was that EPKARAP (sequence number 2) showed the best gene introduction efficiency, so the AAV with this peptide was used as AAVDJ1 with excellent directivity to the cells of the skin ulcer surface. That is, among the replacement peptides of AAVDJ1, the sequence of EPKARAP (sequence number 2) in particular plays an important role in infectivity.

[0085] 2. Method for producing AAV with excellent gene transfer efficiency into cells on skin ulcer surface

[0086] The AAVDJ1 of the present invention, which has excellent directivity to cells on the skin ulcer surface, is an AAV having a capsid (sequence number 5: amino acid sequence 2) in which the 589th and 590th amino acids, i.e., the NR site of the known AAVDJ capsid (sequence number 3: amino acid sequence 1) are replaced by QRGEPKARAPA (sequence number 4). AAV expressing the target gene is prepared by simultaneous transfection with a Rep-Cap plasmid encoding AAVDJ1 as a capsid sequence, a vector plasmid carrying a target gene, an auxiliary plasmid, or a general method for manufacturing AAV such as a baculovirus-based AAV manufacturing method. The base sequence encoding AAVDJ1 (sequence number 6: capsid base sequence 1) and the base sequence of the Rep-cap plasmid (sequence number 7: plasmid base sequence 1) are shown.

[0087] The amino acid sequence of the known AAVDJ capsid (SEQ ID NO: 3) is as follows: The 589th and 590th NR positions are underlined.

[0088] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLLVEEAKTAPGKKRPVE HSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS PRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID QYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRG NRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL

[0089] The amino acid sequence of the AAVDJ1 capsid of the present invention (SEQ ID NO: 5) is as follows: The substituted portion of QRGEPKARAPA (SEQ ID NO: 4) is underlined.

[0090] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLLVEEAKTAPGKKRPVE HSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS PRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID QYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRG QRGEPKARAPAQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL*

[0091] An example of the base sequence encoding the AAVDJ1 capsid of the present invention (SEQ ID NO: 6) is as follows: the modified sites are underlined.

[0092] CAGAGAGGCGAACCAAAAGCACGTGCACCAGCCCAGGCGGCC ACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCGCCTCAGATCCTGATCAAGAACACGCCTTGTACCTGCGGATCCTCCGACCACCTTCAACCAGT CAAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA

[0093] An example of the base sequence of the Rep-cap plasmid encoding the AAVDJ1 capsid of the present invention (SEQ ID NO: 7) is as follows: The underlined portion corresponds to the AAVDJ1 capsid.

[0094] ATGGCTGCCGATGGTTATCTTCCAGATTGG CTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGA GCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGG GAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGAC AACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAA CCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGG CTCCTGGAAAGAAGAGGCCTGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAG CAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTCCCAGACCCTCAACCAATCGGAGA ACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACG AGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACC ACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGG ATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACT TTTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAGCTTCAAGCTCTTC AACATCCAGGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCATCCAGGT GTTTACGGACTCGGAGTACCAGCTGCCGTACGTTCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGG ACGTGTTCATGATTCCCCAGTACGGCTACCTAACACTCAACAACGGTAGTCAGGCCGTGGGACGCTCCTCCTTCTAC TGCCTGGAATACTTTCCTTCGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCC TTTCCACAGCAGCTACGCCCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAGTACCTGTACTACT TGTCTCGGACTCAAACAACAGGAGGCACGACAAATACGCAGACTCTGGGCTTCAGCCAAGGTGGGCCTAATACAATG GCCAATCAGGCAAAGAACTGGCTGCCAGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAA CAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGG CCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCA GAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTAC GGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCCAGAGAGGCGAACCAAAAGCACGTGCACCAGCCCAGG CGGCCACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGG CCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACA CCCTCCGCCTCAGATCCTGATCAAGAACACGCCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGA ACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAG CGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGG CGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA

[0095] 3. Experimental Results 1

[0096] Hereinafter, the present invention will be described in detail based on Examples and the like, but the present invention is not limited to these Examples.

[0097] Example 1

[0098] Figure 1 This is the result of a comparative study of the gene transfer efficiency of AAVDJ and AAVDJ1 in the ulcer surface of the mouse back skin. Prepare capsid plasmids, vector plasmids expressing GFPNLS (NLS is the abbreviation of nuclear localization signal, and the fluorescent dye with this sequence emits fluorescence in unison with the cell nucleus) and mCherryNLS under the CAG promoter (sequence number 8: plasmid base sequence 2, sequence number 9: plasmid base sequence 3), and pAD5 plasmid as an auxiliary plasmid, and prepare AAV by transfecting 293AAV cells using the calcium phosphate method according to conventional methods, and concentrate by ultracentrifugation separation (refer to non-patent document 2), prepare AAVDJ-GFPNLS vector, AAVDJ-mCherryNLS vector, AAVDJ1-GFPNLS vector, and AAVDJ1-mCherry vector, and then prepare 10 1150 μl of virus solution of the combined vectors of GC 1) AAVDJ-GFPNLS + AAVDJ-mCherryNLS, 2) AAVDJ1-GFPNLS + AAVDJ1-mCherryNLS, 3) AAVDJ-GFPNLS + AAVDJ1-mCherryNLS, 4) AAVDJ1-GFPNLS + AAVDJ-mCherryNLS was inoculated on the ulcer surface in the silicon chamber mounted on the back of the mouse, with 5 mice in each group. After 4 days, the ulcer surface was photographed using a fluorescent stereomicroscope (Axio Zoom.V16, Zeiss), and the tissue containing the ulcer surface was collected, fixed with 4% paraformaldehyde, and embedded in OCT complex to make frozen sections. The sections stained with DAPI for nuclei and the adjacent sections stained with HE were read as image files using a fluorescent slide scanner (VS200, Olympus). At this time, samples from the group that had undergone gene introduction using the same capsid virus were used to adjust the exposure time of green fluorescence photography and red fluorescence photography. Using the continuity of adjacent HE staining as an indicator, the areas of the DAPI-stained slices were classified into the surface layer (on the fascia above the subcutaneous fat tissue), the fat layer (subcutaneous fat tissue), and the muscle layer (muscles such as the trapezius muscle). In order to segment the nuclei of each layer, a UNet++ model was generated that was learned using the RMS prop optimizer and the binary cross entropy / Dice loss (BCE-dice-loss) function. In order to generate learning data, the image was cut out of 512×512 pixels and repeated 400 times (epoch), a batch size of 8, and 10 -4 The learning rate is applied to model learning. After learning, the model is applied to all images to predict the nuclear region, and the probability of each pixel is calculated in the range of [0,1]. Pixels with a probability in the range of [0.5,1] are extracted as the nuclear region, and then the nuclear region is segmented using the Watershed algorithm. Nuclei with more than 5 fluorescent positive pixels are defined as nuclei of gene-transferred positive cells. For each region and each layer, the number of gene-transferred positive cells is counted. For each sample, 10 to 16 slices (average 14.4) were analyzed.

[0099] As a result, according to the fluorescence stereomicroscope observation of the ulcer surface, in the group using the same capsid for gene introduction, similar signals were observed in both green fluorescence observation and red fluorescence observation, but in the group using different capsids for gene introduction, different signals were observed in green fluorescence observation and red fluorescence observation. In the analysis of the number of gene introduction-positive nuclei observed based on tissue, in the test objects of any group of AAVDJ-GFPNLS+AAVDJ1-mCherryNLS and AAVDJ1-GFPNLS+AAVDJ-mCherryNLS, especially in the fat layer and muscle layer, it was confirmed that the number of gene introduction-positive cells of the virus with AAVDJ1 capsid was higher than that of the virus with AAVDJ capsid. It shows that AAVDJ1 has a higher gene introduction efficiency than AAVDJ, especially in the fat layer and muscle layer which are deep tissues.

[0100] The sequence of the vector plasmid pAAV-CAG-GFPNLS expressing GFPNLS (SEQ ID NO: 8) is as follows.

[0101]

[0102] The sequence of the vector plasmid pAAV-CAG-mCherryNLS expressing mCherryNLS (SEQ ID NO: 9) is as follows.

[0103]

[0104] 4. Use of AAV with excellent gene transfer efficiency in keratinocytes / epidermal tissue

[0105] The AAV of the present invention, i.e., AAVDJK2, which has excellent gene introduction efficiency for keratinocytes / epidermal tissues, shows higher gene introduction efficiency and excellent directivity compared to AAV2, AAV6, and AAVDJ, which are known to show high gene introduction efficiency for keratinocytes, and therefore can be used as a gene introduction vector in gene therapy for epidermal tissues in vivo, and can also be used as a research and development tool for gene therapy for epidermal tissues. In addition, it can be used as a drug innovation research and biological research tool for purposes other than the development of therapeutic methods based on gene introduction. Since the cultured keratinocytes constituting the epidermal tissue also show high directivity, it can be used as a gene introduction vector in regenerative medicine / gene therapy using cultured keratinocytes, and can also be used as a research and development tool for gene therapy using cultured keratinocytes. In addition, it can be used as a drug innovation research and biological research tool for purposes other than the development of therapeutic methods based on gene introduction using cultured keratinocytes.

[0106] 5. Method for producing AAV with excellent gene transfer efficiency into keratinocytes / epidermal tissue

[0107] The AAV of the present invention having excellent targeting to keratinocytes / epidermal tissue is an AAV having the following capsids: a capsid (AAVDJK2) (sequence number 14: amino acid sequence 3) in which the 589th and 590th amino acids, i.e., the NR sites of the known AAVDJ capsid (sequence number 3: amino acid sequence 1) are replaced by NAAAPRGDLAPAAR (sequence number 10), a capsid (AAVDJK1) (sequence number 15: amino acid sequence 4) replaced by NAAARGDTATLAAR (sequence number 11), a capsid (AAVDJK3) (sequence number 16: amino acid sequence 5) replaced by NAAARGDQQSLAAR (sequence number 12), and a capsid (AAVDJK8) (sequence number 17: amino acid sequence 6) replaced by NAAAPRGDLRPAAR (sequence number 13). AAV expressing the target gene is prepared by co-transfection with a Rep-Cap plasmid encoding each AAV as a capsid sequence, a vector plasmid carrying the target gene, and a helper plasmid, or by a general method for manufacturing AAV such as a baculovirus-based AAV manufacturing method. Hereinafter, as a reference, an example of a base sequence encoding AAVDJK2 (SEQ ID NO. 18: capsid base sequence 2), an example of a base sequence encoding AAVDJK1 (SEQ ID NO. 19: capsid base sequence 3), an example of a base sequence encoding AAVDJK3 (SEQ ID NO. 20: capsid base sequence 4), an example of a base sequence encoding AAVDJK8 (SEQ ID NO. 21: capsid base sequence 5), and an example of a base sequence of a Rep-cap plasmid encoding AAVDJK2 (SEQ ID NO. 22: plasmid base sequence 4) are shown.

[0108] The amino acid sequence of AAVDJK2 capsid (SEQ ID NO: 14) is as follows: The substituted portion of NAAAPRGDLAPAAR (SEQ ID NO: 10) is underlined.

[0109] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLLVEEAKTAPGKKRPVE HSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS PRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID QYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRG NAAAPRGDLAPAAR QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL

[0110] The amino acid sequence of AAVDJK1 capsid (SEQ ID NO: 15) is as follows: The substituted portion of NAAARGDTATLAAR (SEQ ID NO: 11) is underlined.

[0111] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLLVEEAKTAPGKKRPVE HSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS PRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID QYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRG NAAARGDTATLAAR QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL

[0112] The amino acid sequence of AAVDJK3 capsid (SEQ ID NO: 16) is as follows: The substituted portion of NAAARGDQQSLAAR (SEQ ID NO: 12) is underlined.

[0113] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLLVEEAKTAPGKKRPVE HSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS PRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID QYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRG NAAARGDQQSLAAR QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL

[0114] The amino acid sequence of AAVDJK8 capsid (SEQ ID NO: 17) is as follows: The substituted portion of NAAAPRGDLRPAAR (SEQ ID NO: 13) is underlined.

[0115] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLLVEEAKTAPGKKRPVE HSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS PRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID QYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRG NAAAPRGDLRPAAR QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL

[0116] An example of a base sequence encoding the AAVDJK2 capsid (SEQ ID NO: 18) is shown below, and the modified sites are underlined.

[0117] AACGCAGCAGCACCACGTGGTGATCTGGCACCAGCAGCAAGACAAGCAGCT ACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCGCCTCAGATCCTGATCAAGAACACGCCTTGTACCTGCGGATCCTCCGACCACCTTCAACCAGT CAAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA

[0118] An example of a base sequence encoding AAVDJK1 capsid (SEQ ID NO: 19) is shown below, and the modified sites are underlined.

[0119] AACGCAGCAGCACGTGGTGATACTGCAACTCTGGCAGCAAGACAAGCAGCT ACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCGCCTCAGATCCTGATCAAGAACACGCCTTGTACCTGCGGATCCTCCGACCACCTTCAACCAGT CAAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA

[0120] An example of a base sequence encoding the AAVDJK3 capsid (SEQ ID NO: 20) is shown below, and the modified sites are underlined.

[0121] AACGCAGCAGCACGTGGTGATCAGCAGTCTCTGGCAGCAAGACAAGCAGCT ACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCGCCTCAGATCCTGATCAAGAACACGCCTTGTACCTGCGGATCCTCCGACCACCTTCAACCAGT CAAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA

[0122] An example of a base sequence encoding the AAVDJK8 capsid (SEQ ID NO: 21) is shown below, and the modified sites are underlined.

[0123] AACGCAGCAGCACCACGTGGTGATCTGCGTCCAGCAGCAAGACAAGCAGCT ACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCGCCTCAGATCCTGATCAAGAACACGCCTTGTACCTGCGGATCCTCCGACCACCTTCAACCAGT CAAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA

[0124] An example of a Rep-Cap plasmid base sequence encoding AAVDJK2 capsid (SEQ ID NO: 22) is as follows: The underlined portion corresponds to the AAVDJK2 capsid.

[0125] ATGGCTGCCGATGGTTATCTTCCAGATTGG CTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGA GCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGG GAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGAC AACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAA CCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGG CTCCTGGAAAGAAGAGGCCTGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAG CAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTCCCAGACCCTCAACCAATCGGAGA ACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACG AGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACC ACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGG ATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACT TTTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAGCTTCAAGCTCTTC AACATCCAGGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCATCCAGGT GTTTACGGACTCGGAGTACCAGCTGCCGTACGTTCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGG ACGTGTTCATGATTCCCCAGTACGGCTACCTAACACTCAACAACGGTAGTCAGGCCGTGGGACGCTCCTCCTTCTAC TGCCTGGAATACTTTCCTTCGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCC TTTCCACAGCAGCTACGCCCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAGTACCTGTACTACT TGTCTCGGACTCAAACAACAGGAGGCACGACAAATACGCAGACTCTGGGCTTCAGCCAAGGTGGGCCTAATACAATG GCCAATCAGGCAAAGAACTGGCTGCCAGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAA CAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGG CCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCA GAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTAC GGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACGCAGCAGCACCACGTGGTGATCTGGCACCAGCAG CAAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTAC CTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGG ACTTAAACACCCTCCGCCTCAGATCCTGATCAAGAACACGCCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGT CAAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAA AACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAA TACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA

[0126] 6. Experimental Results 2

[0127] Hereinafter, the present invention will be described in detail based on Examples and the like, but the present invention is not limited to these Examples.

[0128] Example 2

[0129] Figure 2The results of a comparative study of the gene delivery efficiency of the new AAVDJ variant and known AAVs in mouse / human cultured keratinocytes. As capsid plasmids, capsid plasmids were prepared in which new AAVDJ variants AAVDJK1 (sequence number 19: capsid base sequence 3), AAVDJK2 (sequence number 18: capsid base sequence 2), AAVDJK3 (sequence number 20: capsid base sequence 4), AAVDJK8 (sequence number 21: capsid base sequence 5), and known AAVs having excellent gene transfer efficiency into keratinocytes, namely AAV2, AAV2K1, AAV2K2, AAV2K3, AAV2K8, AAV6, and AAVDJ, were integrated into a backbone plasmid of plasmid base sequence 4 (sequence number 22) as capsid sequences (see Sallach J, Di Pasquale G, Larcher F, Niehoff N, Rubsam M, Huber A, Chiorini J, Almarza D, Eming SA, Ulus H, Nishimura S, Hacker UT, Hallek M, Niessen CM, Buning H. Tropism-modified AAV vectorsovercome barriers to successful cutaneous therapy. Mol Ther. 2014 May; 22(5):929-39., Melo SP, Lisowski L, Bashkirova E, Zhen HH, Chu K, Keene DR, Marinkovich MP, KayMA, Oro AE. Somatic correction of junctional epidermolysis bullosa by a highly recomogenic bin AAV variant. Mol Ther. 2014Apr; 22(4):725-33. and Bonafont J, Mencía A, Chacón-Solano E, Srifa W, Vaidyanathan S, Romano R, Garcia M, Hervás-Salcedo R, Ugalde L, Duarte B, Porteus MH, Del Rio M, Larcher F, Murillas R.Correction of recessive dystrophic epidermolysis bullosa by homology-directed repair-mediated genome editing. Mol Ther.2021Jun 2; 29(6): 2008-2018.), using a vector plasmid expressing GFPNLS under the CAG promoter and a pAD5 plasmid as an auxiliary plasmid, AAV production was performed by transfecting 293AAV cells using the calcium phosphate method according to conventional methods, and concentrated by ultracentrifugation to obtain a vector solution containing each AAV. For mouse keratinocytes, cells cultured from the back skin of 3-5 week-old C57BL / 6J Jcl mice under culture conditions on feeder cells according to conventional methods were used (refer to non-patent document 2). For human keratinocytes, commercially available cells, i.e., normal human epidermal keratinocytes (adult donors, pooled, product code: C12006) (PromoCell, Heidelberg, Germany) were maintained under the same culture conditions as mouse keratinocytes and used. Mouse and human keratinocytes were cultured in 24-well plates, and when the confluence reached 50%, the medium was replaced with a medium containing 1.0×10. 9 GC / well of AAV culture medium. Four wells were used for each AAV and the operation was performed under the same conditions. Phase contrast microscope photos and fluorescent photos were taken in four fields of view after 2 days, 4 days, and 8 days. Using ImageJ software, the area of ​​the region where keratinocytes formed colonies was calculated on the image. The number of GFPNLS-positive cells in the same area was counted on the image. The number of GFPNLS-positive cells per unit area in the keratinocyte area was calculated. The results showed that the new AAVDJ variants AAVDJK1, AAVDJK2, AAVDJK3, AAVDJK8, and especially AAVDJK2 had high gene introduction efficiency compared to known AAVs, whether for mouse keratinocytes or human keratinocytes.

[0130] Example 3

[0131] Figure 3This is the result of a comparative study of the gene introduction efficiency of known AAV2, AAV2K2, AAVDJ and new AAVDJK2 for mouse / human cultured mesenchymal cells. AAVs with various capsids expressing GFPNLS under the CAG promoter were prepared by the same method as in Example 2. Mouse adipose-derived mesenchymal cells used cells that were primary cultured from the inguinal skin of 3-5 week-old C57BL / 6J Jcl mice according to conventional methods (refer to non-patent document 2). Regarding human dermal fibroblasts (dermal fibroblasts are dermal-derived mesenchymal cells), commercially available cells, namely normal human dermal fibroblasts (young foreskin, product code: C12300) (PromoCell, Heidelberg, Germany) were maintained under the same culture conditions as mouse adipose-derived mesenchymal cells and used. Mouse / human mesenchymal cells were cultured in 24-well plates, and at the stage of 50% confluence, the culture medium was replaced with a medium containing 1.0×10 9 GC / well of AAV culture medium. Four wells were used for each AAV and the operation was performed under the same conditions. Phase contrast microscope photos and fluorescence photos were taken in four fields of view for 8 consecutive days. Each fluorescence photo (area 1.99mm 2 ) in the number of GFPNLS-positive cells. In mouse adipose-derived mesenchymal cells, the high gene transfer efficiency confirmed in AAVDJ for mesenchymal cells was not confirmed in the new AAVDJK2. In human fibroblasts, the high gene transfer efficiency confirmed in AAV2 and AAVDJ for mesenchymal cells was not confirmed in the new AAVDJK2.

[0132] [Reference Example 1]

[0133] Figure 4This is the result of an injection test of fluorescent silica particles into the back skin of mice. A liquid was prepared in which fluorescent silica particles (sicastar (registered trademark)-greenF, plain, 30nm, 25mg / ml, Filgen) of the same size as AAV were diluted to 20 times with phosphate-buffered saline. Under a surgical microscope, 20μl of the dilution was injected intradermally into the back of the mouse at a depth as close to the surface as possible using a 29G injection needle. The tissue was recovered at 0 hours, 1 hour, and 3 hours after the injection, and buried in an OCT complex for freezing. Sections were collected from the frozen tissue at a thickness of 10μm every 200μm, washed with phosphate-buffered saline, and then stained with DAPI for nuclei. On the skin tissue sections immediately after the injection of the fluorescent silica particles, 1 hour, and 3 hours later, most of the injected particles were distributed from the skin to the subcutaneous layer, and not distributed in the epidermis. That is, it was found that although intradermal injection is a standard method for administering AAV to the epidermis, it is not an excellent method in terms of efficiency and specificity.

[0134] Example 4

[0135] Figure 5 The results of a comparative test of gene transfer efficiency between AAVDJK2 and AAVDJ in mouse back skin. The AAVDJK2 vector expressing GFPNLS under the CAG promoter and the AAVDJ vector expressing mCherryNLS under the CAG promoter were prepared by the concentrated AAV production method described in Example 2. 11 GC (gene copy number) / 35μl) containing two AAV solutions was intradermally injected into the back of mice. Two days after the injection, the tissue including the injection site was collected while observing the injection site with a fluorescent stereomicroscope. After fixation with 4% paraformaldehyde, it was embedded in OCT complex and frozen sections were made. The adjacent sections were stained with HE, nuclear staining was performed with DAPI, and read as image files using a fluorescent slide scanner (VS200, Olympus). From the histological observation, GFPNLS introduced by AAVDJK2 was strongly expressed in the epidermis and weakly expressed in the subcutaneous tissue. In contrast, mCherry introduced by AAVDJ was weakly expressed in the epidermis and strongly expressed in the subcutaneous tissue. It can be seen that compared with AAVDJ, AAVDJK2 has higher gene introduction efficiency and specificity for epidermal tissue.

[0136] 7. Use of promoters showing high specificity for keratinocytes / epidermal tissue

[0137] The promoters of the present invention, i.e., K14SCP3 promoter, K16SCP3 promoter, and K16P5 short promoter, which show high specificity to keratinocytes / epidermal tissues, bring higher or equivalent gene expression compared to the CAG promoter known to bring high gene expression to keratinocytes / epidermal tissues regardless of tissue or cell type. On the other hand, only relatively low gene expression is brought in tissues and cells other than keratinocytes / epidermal tissues represented by mesenchymal cells near the epidermis, so it can be used as a gene introduction vector when gene therapy is performed on cultured keratinocytes, a gene introduction vector when gene therapy is performed on epidermal tissues in vivo, and can also be used as a research and development tool for these gene therapy methods. In addition, it can be used as a drug innovation research and biological research tool other than the development of a therapy based on gene introduction. In gene therapy for keratinocytes / epidermal tissues and research and development thereof, it is preferred in most cases that gene expression (non-specific gene expression) other than keratinocytes / epidermal tissues is suppressed to a lower level, so it is expected that there are more cases of effectiveness of the promoters of the present invention showing high specificity to keratinocytes / epidermal tissues.

[0138] The promoter of the present invention may be not only the K14SCP3 promoter, the K16SCP3 promoter, or the K16P5 short promoter, but may also be a sequence having a high identity to these base sequences. Such identity is usually 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more.

[0139] 8. Method for producing a gene transfer vector having a promoter showing high specificity for keratinocytes / epidermal tissue

[0140] The promoters of the present invention, i.e., K14SCP3 promoter, K16SCP3 promoter, and K16P5 short promoter, which show high specificity for keratinocytes / epidermal tissues, are used as promoter sequences of AAV, lentiviral vectors, naked plasmids, etc. to prepare gene introduction vectors, thereby expressing genes with high specificity for keratinocytes / epidermal tissues. For example, in the case of AAV expressing GFPNLS as a target gene using K14SCP3 as a promoter, the K14SCP3 promoter sequence (sequence number 23: promoter base sequence 1) is used in the vector plasmid, and a plasmid (sequence number 24: plasmid base sequence 5) is designed, and an AAV expressing the target gene is prepared by simultaneous transfection with a capsid-encoding Rep-Cap plasmid and an auxiliary plasmid, or by a general method for manufacturing AAV such as a baculovirus-based AAV manufacturing method. The same is true for the K16SCP3 promoter (sequence number 25: promoter base sequence 2) and the K16P5 short promoter (sequence number 26: promoter base sequence 3).

[0141] The K14SCP3 promoter sequence (SEQ ID NO: 23) is as follows.

[0142] CTCCGGAGCTTCTATTCCTGATCCCTGCATAAGAAGGAGACATGGTGGTGGTGGTGGTGGGTGGGGGTGGTGGGGCACAGAGGAAGCCGGTACTGGGCTCTGCACCCCATTCCCGCTCCCAGATCCCTCTGGACACAGCATTTTTCTCCAGTGAGCACAGCCTCCCCTTGCCCCACAGCCAACAGCAACATGCCTCCCAACAAAAGCATCTGTCCCTCAGCCAAAACCCCTGTTGCCTCTCTCTGGGGAAATTGTAGGGCTGGGCCAGGGTGGGGGGACCATTCTCTGCAGGGAGATTAGGAGTGTCTGTCAGGGGCGGGTGGAGCGGGGTGGGGCCCTGGCTTACTCACATCCTTGAGAGTCCTTTGCTGGCAGATTTGGGGAGCCCACAGCTCAGATGTCTGTCTCAGCATTGTCTTCCAAGCTCCTAGGCCACAGTAGTGGGGGGCTCCCTTCTCTGGCTTCTTCTTTGGTGACAGTCAAGGTGGGGTTGGGGGTGACAGAGGGTCCTGCTTCTCTTCTAGGAGCAGTTGATCCCAGGAAGAGCATTGGAGCCTCCAGCAGGGGCTGTTGGGGCCTGTCTGAGGAGATAGGATGCGTCAGGCAGCCCCAGACACGATCACATTCCTCTCAACATGCCTGCCGGGGTCTGTGGAGCCTAGGGGCTGATGGGAGGGTGGGGTGGGGGCCGGAAGGGTTTAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGTCCGCCTGGAGACCTCGAGCCGAGTGGTCGTGCCTCCATAGAA

[0143] An example of the base sequence of the pAAV-K14SCP3-GFPNLS plasmid (SEQ ID NO: 24) is as follows. The underlined part corresponds to the K14SCP3 promoter.

[0144] CTCCGGAGCTTCTATTCCTGATCCCTGCATAAGAAGGAGACATGGTGGTGGTGGTGGTGGGTGGGGGTGG TGGGGCACAGAGGAAGCCGGTACTGGGCTCTGCACCCCATTCCCGCTCCCAGATCCCTCTGGACACAGCATTTTTCT CCAGTGAGCACAGCCTCCCCTTGCCCCACAGCCAACAGCAACATGCCTCCCAACAAAAGCATCTGTCCCTCAGCCAA AACCCCTGTTGCCTCTCTCTGGGGAAATTGTAGGGCTGGGCCAGGGTGGGGGGACCATTCTCTGCAGGGAGATTAGG AGTGTCTGTCAGGGGCGGGTGGAGCGGGGTGGGGCCCTGGCTTACTCACATCCTTGAGAGTCCTTTGCTGGCAGATT TGGGGAGCCCACAGCTCAGATGTCTGTCTCAGCATTGTCTTCCAAGCTCCTAGGCCACAGTAGTGGGGGGCTCCCTT CTCTGGCTTCTTCTTTGGTGACAGTCAAGGTGGGGTTGGGGGTGACAGAGGGTCCTGCTTCTCTTCTAGGAGCAGTT GATCCCAGGAAGAGCATTGGAGCCTCCAGCAGGGGCTGTTGGGGCCTGTCTGAGGAGATAGGATGCGTCAGGCAGCC CCAGACACGATCACATTCCTCTCAACATGCCTGCCGGGGTCTGTGGAGCCTAGGGGCTGATGGGAGGGTGGGGTGGG GGCCGGAAGGGTTTAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGTCCGCCTGGAGACCTCGAGCCGAGT GGTCGTGCCTCCATAGAA

[0145] The K16SCP3 promoter sequence (SEQ ID NO: 25) is as follows.

[0146] CTCCGGAGCTTCTATTCCTGATCCCTGCAGAAGAAGGAGACGGTGGTGGTGGTGGGTGGGGGTGGCAGGGCACAGAGGAAGCCAGTACCGGGCCCTGCACCCCATTCCCACTCCCAGATCCCTCTGGACACAGCATTTTTCTCCAGTGAGCACAGCCTTCCCTTGCCCCACAGCCAACAGCAACATGGCTCCCAACAAAAGCATCTGCCCCTCAGCCAAAACCCCTGTTGCCTCTCTCTGGGGAAATTGTAGGACTGGGTCAGGGTGGGGGAACCATTCTCTGCAGGGAGATTAGGAGTGTCTGTCAGGGGTGGGTGGAGCGGGGTGGGGCCCTGGCTTACTCACATCCTCGAGAGTCCTTTGCTGGCAGATTTGGGGAGCCCACAGCACAGGTGTCTGTCTCAGTATTGTCTTCCAAGCTCCTAGGCCACAGTAGTGGGGGGCTCCCCTCTCTGGCTTTTTCTTTGGTGACAGTCAAGGTTGGGGGTGGGGTGAGAGAGGGTCCTGCTTTTCTTCTAGGAACAGTTGATCCCAGGAAGAGCAGTGGAGCCTCCAGCAGGGGCTGTTGGGGCCTGTCTGAGGAGATAGGACGCGTCAGGCAGCCCCAGACACGACCACATTCCTCCCAACATGCCTGCCGGGGTCTGTGGAGCCCAGGGGCTGAGGGGAGGGTGGGGTGGGGGCCGGAAGGGTTTAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGTCCGCCTGGAGACCTCGAGCCGAGTGGTCGTGCCTCCATAGAA

[0147] The K16P5 short promoter sequence (SEQ ID NO: 26) is as follows.

[0148]

[0149] The K14 promoter sequence (SEQ ID NO: 27) is as follows.

[0150]

[0151] The K14 short promoter sequence (SEQ ID NO: 28) is as follows.

[0152]

[0153] The K16P5 promoter sequence (SEQ ID NO: 29) is as follows.

[0154]

[0155] 9. Experimental results 3

[0156] Hereinafter, the present invention will be described in detail based on Examples and the like, but the present invention is not limited to these Examples.

[0157] Example 5

[0158] Figure 6 The present invention is the result of a comparative study of gene expression during gene introduction into mouse / human cultured keratinocytes and mesenchymal cells using AAV having an AAVDJK2 capsid as a vector. The AAVDJK2 capsid expresses GFPNLS through the newly developed K14SCP3 promoter (sequence number 23), K16SCP3 promoter (sequence number 24), K16P5 short promoter (sequence number 25) and the known CAG promoter, K14 promoter (sequence number 27), K14 short promoter (sequence number 28), and K16P5 promoter (sequence number 29). AAVDJK2 vectors expressing GFPNLS under each promoter were prepared by the concentrated AAV production method described in Example 2. Mouse cultured keratinocytes were prepared by the method of Example 2, and mouse adipose-derived mesenchymal cells were prepared by the method of Example 3. Mouse keratinocytes were cultured in a 24-well plate, and at the stage of 50% confluence, the culture medium was replaced with a medium containing 1.0×10 9 GC / well AAV culture medium. 4 wells were used for each AAV and the operation was performed under the same conditions. The culture medium was replaced after 1 day and 3 days. Phase contrast microscope photos and fluorescent photos were taken with 4 fields of view after 2 days and 4 days. Using ImageJ software, the area of ​​the region where keratinocytes form colonies was calculated on the image. The number of GFPNLS-positive cells in the same area was counted on the image. The number of GFPNLS-positive cells per unit area in the keratinocyte region was calculated. The results show that for mouse cultured keratinocytes, the K14SCP3 promoter has a gene introduction efficiency greater than that of the CAG promoter, the K14 promoter, and the K14 short promoter. In addition, the K16SCP3 promoter and the K16P5 short promoter have a gene introduction efficiency greater than that of the K14 promoter, the K14 short promoter, and the K16P5 promoter.

[0159] Mouse keratinocytes were cultured in 24-well plates. When the cells reached 50% confluence, the medium was replaced with 1.0 × 10 9GC / well AAV culture medium. 4 wells were used for each AAV and the operation was performed under the same conditions. The culture medium was replaced after 1 day and 3 days. After 4 days, the keratinocytes were trypsinized in the same way as the passage to prepare a suspension. After passing through a 35μm filter, it was spun down and a single cell suspension was prepared using phosphate buffered saline with 5% FBS and 2mM EDTA as the buffer. Flow cytometry analysis was performed using a FACS Melody cell sorter (BD, USA) and FlowJo (BD, USA), and a plot was collected with the ratio of GFP-positive cells and the fluorescence intensity of GFP as variables. The results show that the K14SCP3 promoter has a gene introduction efficiency greater than that of the CAG promoter, the K14 promoter, and the K14 short promoter. In addition, the K16SCP3 promoter and the K16P5 short promoter have a gene introduction efficiency greater than that of the K14 promoter, the K14 short promoter, and the K16P5 promoter.

[0160] Mouse adipose-derived mesenchymal cells were cultured in 24-well plates. When the cells reached 50% confluence, the culture medium was replaced with 1.0×10 9 GC / well of AAV culture medium. Use 4 wells for each AAV and operate under the same conditions. Replace the culture medium after 1 day and 3 days. Take phase contrast microscopy and fluorescence photos in 4 fields of view after 2 days and 4 days. Take phase contrast microscopy and fluorescence photos in 4 fields of view. Count each fluorescent photo (area 1.99mm 2 ) The number of GFPNLS-positive cells was found to be significantly lower than that of the CAG promoter.

[0161] Mouse adipose-derived mesenchymal cells were cultured in 24-well plates. When the cells reached 50% confluence, the medium was replaced with 1.0 × 10 9GC / well AAV culture medium. Four wells were used for each AAV and the operation was performed under the same conditions. The culture medium was replaced after 1 day and 3 days. After 4 days, the mouse cell-derived mesenchymal cells were trypsinized in the same way as the passage to prepare a suspension. After passing through a 35μm filter, the suspension was spun down and a single cell suspension was prepared using phosphate-buffered saline with 5% FBS and 2mM EDTA as the buffer. Flow cytometry analysis was performed using a FACS Melody cell sorter (BD, USA) and FlowJo (BD, USA), and graphs with the ratio of GFP-positive cells and the fluorescence intensity of GFP as variables were collected. It can be seen that the gene introduction efficiency of the keratin-specific promoter containing the new K14SCP3 promoter, K16SCP3 promoter, and K16P5 short promoter is much lower than that of the CAG promoter. In short, it can be seen that the new promoter has high gene introduction efficiency and high specificity for keratinocytes.

[0162] Example 6

[0163] Figure 7 The results of a gene transfer experiment in which AAVDJK2 expressing GFPNLS under the K14SCP3 promoter was intradermally injected into the back skin of mice. The AAVDJ2 vector expressing GFPNLS under the K14SCP3 promoter was prepared by the concentrated AAV production method described in Example 2. The back skin of mice that had been subcutaneously injected with 10 μl of the steroid Conacton A (40 mg / ml) into the abdomen was shaved with a razor and then AAVDJK2-K14SCP3-GFPNLS10 was intradermally injected. 11 GC / 35μl of virus solution. After 2 days, the surrounding skin tissue including the injection site was collected, fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for one night, replaced with 30% sucrose / phosphate buffered saline, embedded in OCT complex and frozen. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C (9), 2.5cm C-FP094), sections were collected from the frozen tissue at a thickness of 10μl every 200μl, washed with phosphate buffered saline, and nuclear staining was performed with DAPI. Use a slide scanner (VS200, Olympus) to read as an image file. High GFPNLS expression was consistently confirmed in the epidermal tissue. On the other hand, if the epidermal tissue of the hair follicles was removed, the GAPNLS expressing cells from the dermis to the subcutaneous tissue were suppressed to a low frequency. It can be seen that AAV with AAVDJK2 as the capsid and K14SCP3 as the promoter acts as a gene introduction vector with high efficiency and specificity for the epidermal tissue.

[0164] 10. Use of the method for inducing skin appendages in vivo

[0165] Skin appendages are composed of hair follicles, sebaceous glands, sweat glands, etc., and play the role of protection from mechanical obstacles, heat preservation, moisturizing, and temperature regulation. Skin appendages are formed by the interaction of epithelial tissue and mesenchymal tissue during the fetal period and organ production (refer to non-patent document 4). As representative conditions caused by defects, disorders, and dysfunctions of skin appendages, there are alopecia, sebum deficiency, etc. In addition to temperature regulation disorders and itching caused by dry skin, it also becomes a cause of cosmetic surgery obstacles. In particular, these conditions are also caused in the process of physiological aging changes. It is difficult to regenerate lost skin appendages clinically, and it is usually necessary to transplant existing appendages from other parts of the body through skin grafting, flap formation, etc. In this regard, if the regeneration and regeneration of skin appendages can be induced in a living body, it can become a therapeutic method to induce the regeneration, regeneration and functional improvement of skin appendages and achieve sufficient skin appendage function for diseases such as skin ulcers with skin and skin appendage defects, including alopecia, sebum deficiency, and various conditions of reduced skin appendage function including age-related changes. In addition, it can also be used as a drug innovation research and biological research tool for the purpose of regeneration, regeneration and functional improvement of skin appendages.

[0166] 11. Methods for inducing skin appendages in vivo

[0167] The method for inducing skin appendages in vivo of the present invention is characterized in that it includes: a step of imparting skin appendage inducing ability by introducing genes, administering proteins, or administering compounds into somatic cells that do not have skin appendage inducing ability. Hereinafter, the gene introduced in gene introduction is referred to as an "introduced gene". Here, the "introduced gene" includes not only genes encoding proteins, but also non-coding RNAs such as microRNAs.

[0168] The genes that impart skin appendage induction ability include at least the following protein-encoding genes: DNP63A gene, GRHL2 gene, TFAP2A gene, cMYC gene, LEF1 gene, SOX2 gene, HOXC4 gene, HOXC9 gene, HOXC13 gene, JARID2 gene, HEY1 gene, HEY2 gene, FOXO1 gene, FOXD1 gene, EGR3 gene, MEF2C gene, LHX2 gene, PRRX1 gene, PRRX2 gene, CREB3 gene, ETV1 gene, TBX6 gene, MSX2 gene, PRDM1 gene, SHH gene.

[0169] The proteins that impart skin appendage induction ability include at least proteins encoded by the following genes: DNP63A gene, GRHL2 gene, TFAP2A gene, cMYC gene, LEF1 gene, SOX2 gene, HOXC4 gene, HOXC9 gene, HOXC13 gene, JARID2 gene, HEY1 gene, HEY2 gene, FOXO1 gene, FOXD1 gene, EGR3 gene, MEF2C gene, LHX2 gene, PRRX1 gene, PRRX2 gene, CREB3 gene, ETV1 gene, TBX6 gene, MSX2 gene, PRDM1 gene, SHH gene.

[0170] The base sequences of any gene used in the present invention are known (Table 1). In addition, in this specification, NCBI is the abbreviation of the National Center for Biotechnology Information of the United States, and the accession numbers in Table 1 are also registered in the database provided by NCBI.

[0171]

[0172] Most of the sources of these genes exist in mammals including humans, and genes of any mammalian origin can be used, preferably appropriately selected according to the source of the somatic cells to be introduced. For example, when humans are the subjects, the above-mentioned introduced genes and proteins are preferably of human origin. In addition, in addition to wild-type genes and proteins, the above-mentioned introduced genes and proteins may also be variant genes in which several (for example, 1 to 10, preferably 1 to 6, more preferably 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2) amino acids in the amino acid sequence are replaced, deleted and / or inserted, and encode variant gene products having the same function as the wild-type gene product. In addition, sequences obtained by changing / optimizing codons in a manner that encodes the same amino acids as those encoded by each gene may also be used.

[0173] In the present invention, the above-mentioned introduced gene can be prepared according to conventional methods based on known sequence information. For example, RNA is extracted from cells of mammalian origin and cloned according to conventional methods, thereby preparing cDNA of the target gene. In addition, it can also be synthesized as an artificial gene. When synthesizing an artificial gene, codon optimization can also be performed according to the source animal of the somatic cell introduced.

[0174] In the present invention, the type of "somatic cells" to be induced into cells having skin appendage induction ability is not particularly limited, and may include somatic cells derived from any tissue or site. In particular, as somatic cells that can be expected to be the induction origin in the present invention, for example, somatic cells derived from tissues such as skin, subcutaneous fat, muscle, fascia, and blood cells can be exemplified, and more specifically, skin fibroblasts, mesenchymal cells derived from subcutaneous fat tissue (subcutaneous fat cells), adipocytes, muscle cells, fascia cells, tissue monocytes, and tissue macrophages can be exemplified.

[0175] The introduction of the above-mentioned introduced gene into somatic cells can be carried out by the method commonly used in the transfection of animal cells. Specifically, as a method for introducing the above-mentioned introduced gene into somatic cells, a method using a vector, a method based on mRNA using a suitable delivery method such as lipid nanoparticles, etc. can be exemplified. Among them, from the viewpoint of introduction efficiency, a method using a vector is preferably used. In the case of using a vector to introduce the above-mentioned introduced gene into somatic cells, as a vector, a viral vector, a non-viral vector (including a plasmid (DNA) vector), an artificial virus, etc. can be used. From the viewpoint of gene introduction efficiency, a viral vector such as adeno-associated virus, a retrovirus, and a lentivirus is preferably used. From the viewpoint of safety, a method based on mRNA is preferably used. It should be noted that when the above-mentioned introduced gene is multiple, it can be integrated into different vectors, mRNAs, respectively, or more than two introduced genes can be integrated into one vector, mRNA.

[0176] Administration of the above-mentioned protein to somatic cells can be carried out by administration of the protein itself depending on the properties of the protein, or by protein transfection using a cell-penetrating peptide.

[0177] In the present invention, by introducing genes, administering proteins, or administering compounds to somatic cells present in a living body, cells having skin appendage induction ability can be induced from somatic cells in the living body, thereby conferring regeneration and regeneration of skin appendages. The introduced genes and proteins may be genes and proteins that are relatively strongly expressed in epithelial cells having skin appendage induction ability, or genes and proteins that are relatively strongly expressed in mesenchymal cells having skin appendage induction ability, but both genes and proteins may be introduced and administered simultaneously.

[0178] The method of inducing skin appendages in vivo of the present invention is characterized in that it includes: a step of imparting skin appendage inducing ability by introducing a gene, administering a protein, or administering a compound into a somatic cell that does not have skin appendage inducing ability. The present invention is not only used for diseases such as skin ulcers where the skin and skin appendages themselves are defective, but also for various states of reduced skin appendage function including alopecia, sebum deficiency, and age-related changes associated with these diseases, and can quantitatively and functionally improve the state of reduced skin appendage function. However, in experimental animals such as wild-type mice that do not have genetic abnormalities, it is difficult to produce a state of quantitatively reduced skin appendage function such as alopecia and sebum deficiency, and it is difficult to accurately evaluate the degree of improvement based on therapeutic intervention.

[0179] Therefore, in the test of the effect of imparting skin appendage induction ability by introducing genes, administering proteins, or administering compounds into somatic cells that do not have skin appendage induction ability of the present invention, a test of newly inducing skin tissue with skin appendages on a skin ulcer surface isolated from the surrounding skin by a chamber was adopted. As confirmed in Non-Patent Document 2, physiologically, even skin / epidermal tissue cannot be regenerated on a skin ulcer surface isolated from the surrounding skin, so it is clear that the skin appendages accompanying the induced skin are caused by the effect of the present invention.

[0180] 12. Experimental results 4

[0181] Hereinafter, the present invention will be described in detail based on Examples and the like, but the present invention is not limited to these Examples.

[0182] Example 7

[0183] Figure 8 The results of an experiment in which a cavity was installed on the dorsal fascia of mice to create a skin ulcer surface isolated from the surrounding skin, and AAV was used for gene introduction to induce skin with skin appendages. A skin ulcer was created on the back of 4-5 week old C57BL / 6J Jcl mice, and the cavity was sutured and fixed to the deep fascia, and then the surrounding skin and the cavity were sutured to create a skin ulcer surface isolated from the surrounding skin. On the skin ulcer surface isolated from the surrounding skin by the cavity, the skin / epidermal structure will not regenerate physiologically, and the ulcer surface will not heal (refer to non-patent document 2). The AAV vectors used in gene introduction were prepared using the concentrated AAV preparation method described in Example 2. After the cavity was installed, the AAV vector was inoculated into the cavity to introduce the gene into the ulcer surface. The capsid, introduced gene, and titer of each vector were set to AAVDJ1-DNP63A1×10 12 GC (gene copy number), AAVDJ1-GRHL2 1×10 11GC, AAVDJ1-TFAP2A5×10 11 GC, AAVDJ1-cMYC 5×10 11 GC, AAVDJ1-LEF12×10 11 GC. On the 9th day after the chamber was installed and AAV was first administered, 2.5×10 11 GC, AAVJD-PRDM1 2.5×10 11 GC, AAVDJ1-LEF1 1×10 11 GC, AAVDJ1-SHH 5×10 10 The AAV vector of GC was inoculated into the chamber to introduce genes into the ulcer surface. On the ulcer surface, the induction of epidermal tissue was observed from the 21st day after the installation of the chamber and the initial administration of AAV. On the 28th day, it was clearly observed that epidermal tissue was formed in a part of the ulcer surface, and hair was confirmed to sprout from a part. After photographing the ulcer surface using a stereomicroscope (Axio Zoom.V16, Zeiss), the tissue including the ulcer surface was collected, fixed with 4% paraformaldehyde and embedded in OCT complex to make frozen sections. For the sections, immunostaining was performed using cytokeratin 14 antibody (ab181595, Abcam) and secondary antibody according to conventional methods, and then nuclear staining and sealing were performed using DAPI Fluoromount-G (Southern Biotech). Fluorescent images were read using a fluorescent slide scanner (VS200, Olympus) and a confocal microscope (Zeiss LSM900), and then the seal was removed by slowly washing the sealant. The slices were stained with hematoxylin and eosin, then resealed, and the tissue was read using a slide scanner. In terms of tissue observation, it was confirmed that skin appendages such as hair follicle structures and sebaceous gland structures were newly induced on the ulcer surface and independent of the surrounding epidermis. Since skin with skin appendages was confirmed on the ulcer surface where even the epidermal structure cannot be regenerated physiologically, it is known that skin with skin appendages can be induced by gene introduction using AAV.

[0184] Example 8

[0185] Fig. 9 Results of an experiment in which a chamber was installed on the dorsal fascia of mice to isolate the skin from the surrounding skin and to induce skin with skin appendages by gene transfer using AAV. The experiment was conducted in the same manner as in Example 7, except that the timing of chamber installation and gene transfer, and the vector used for gene transfer were different. On the day after the chamber was installed, the AAV vector was inoculated into the chamber to introduce genes into the ulcer surface. The capsid, gene, and timing of each vector were set to AVDJ-DNP63A1×10 12GC (gene copy number), AAVDJ-GRHL2 1×10 11 GC, AAVDJ-TFAP2A5×10 11 GC, AAVDJ-cMYC 5×10 11 GC, AAVDJ-LEF1 2×10 11 GC. On day 9 after the initial administration of AAV, 2.5×10 11 GC, AAVDJ-PRDM1 2.5×10 11 GC, AAVDJK2-LEF1 2.5×10 11 GC, AAVDJ1-SHH 1×10 11 The GC AAV vector was inoculated into the chamber to introduce genes into the ulcer surface. On the ulcer surface, epidermal tissue induction was observed on the 21st day after the chamber was installed and AAV was first administered. On the 28th day, epidermal tissue was clearly observed to be formed in a part of the ulcer surface, and on the 30th day, black hair was confirmed to sprout from a part. It can be seen that gene introduction using AAV can induce skin tissue accompanied by the growth of black hair in an epidermal part independent of the surrounding area.

[0186] It can be understood from Examples 7 and 8 that skin with skin appendages can be induced in vivo by introducing five genes, namely, DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene, and then introducing FOXD1 gene, PRDM1 gene, LEF1 gene, and SHH gene into a skin ulcer surface isolated from the surrounding skin. As confirmed in Non-Patent Document 2, physiologically, even the skin and epidermal structure cannot be regenerated on a skin ulcer surface isolated from the surrounding skin, so it can be understood that the skin appendages accompanying the induced skin are caused by the effect of the present invention.

[0187] According to Patent Document 1 (International Publication No. 2022 / 244502), it is shown that cells having the ability to induce skin appendages, particularly epithelial cells, can be induced by introducing DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene and LEF1 gene, or DNP63A gene, GRHL2 gene, TFAP2A gene and LEF1 gene into primary cultured mesenchymal cells derived from adult fat of mice, and it is shown that cells having the ability to induce skin appendages, particularly mesenchymal cells, can be induced by introducing genes in Group 1 or 2 shown in Table 2 into primary cultured mesenchymal cells derived from adult fat of mice. By transplanting a mixture of these induced epithelial cells having the ability to induce skin appendages and induced mesenchymal cells having the ability to induce skin appendages into a silicon chamber installed on the back of an immunodeficient animal, reconstruction and regeneration of skin tissue having skin appendages can be achieved; and the results of Examples 7 and 8 suggest that by introducing five genes, namely, DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene, or four genes, namely, DNP63A gene, GRHL2 gene, TFAP2A gene, and LEF1 gene, into a skin ulcer surface isolated from the surrounding skin, and then introducing the genes of mesenchymal cells having the ability to inducing hair follicles listed in the table, skin with skin appendages can be induced in vivo.

[0188] [Table 2]

[0189] First induced mesenchymal cells Second, induced mesenchymal cells SHH LEF1 ETV1 FOXD1 PRDM1 S H ETV1 FOXD1 PRDM1 SHH LEF1 ETV1 FOXD1 SHH LEF1 FOXD1 PRDM1 SHH LEF1 ETV1 PRDM1 SHH ETV1 FOXD1 PRDM1 SHH LEF1 ETV1 FOXD1 PRDM1 SHH LEF1 SHH LEF1 PRDM1 S H FOXD1 PRDM1 SHH PRDM1 SHH FOXD1 SHH ETV1 FOXD1 PRDM1

[0190] In Examples 7 and 8, in order to clearly show that the induced skin appendages are caused by the effects of the present invention, the skin ulcer surface isolated from the surrounding skin, where even the surrounding skin / epidermal tissue cannot be confirmed to regenerate physiologically, induced the regeneration of the skin and skin appendages. It is shown that the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene and LEF1 gene were introduced to induce cells, especially epithelial cells, having the ability to induce skin appendages. However, when the present invention is used for various states of reduced skin appendage function including alopecia, sebum deficiency, and age-related changes associated with these, since skin / epidermal tissue exists at the treatment target site, the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene and LEF1 gene, or all of the DNP63A gene, GRHL2 gene, TFAP2A gene and LEF1 gene are not required. In particular, it is suggested that, as confirmed in non-patent document 2, since the DNP63A gene, GRHL2 gene, TFAP2A gene and c-MYC gene play a function of inducing epidermal cells from mesenchymal cells, when the present invention is used for various conditions of reduced skin appendage function including alopecia, sebum deficiency, and age-related changes associated with these diseases in which skin / epidermal tissue exists in the treatment target site, by introducing the LEF1 gene alone, or introducing a part of the DNP63A gene, GRHL2 gene, TFAP2A gene and c-MYC gene in addition to the LEF1 gene, epithelial cells with skin appendage inducing ability can be induced from the skin / epidermal tissue originally existing in the treatment target site.

[0191] That is, it is suggested that when the present invention is used for various states of reduced skin appendage function including alopecia, sebum deficiency, and age-related changes associated with these in the skin / epidermal tissue of the treatment target area, the state of reduced skin appendage function can be improved quantitatively and functionally by introducing the LEF1 gene alone, or by introducing the DNP63A gene, GRHL2 gene, TFAP2A gene, and part of the c-MYC gene in addition to the LEF1 gene, and the genes in Group 1 or 2 shown in Table 2.

[0192] 13. Use of the method for improving the efficiency of gene introduction into organisms

[0193] The method for improving the efficiency of gene introduction in vivo of the present invention can be used as a therapeutic method in the treatment of diseases using gene introduction, and can also be used as a research and development tool for gene therapy. In addition, it can be used as a drug innovation research and biological research tool other than the purpose of developing a therapeutic method based on gene introduction. As a gene introduction method in vivo, various methods such as viral vectors such as AAV, retroviral vectors, lentiviral vectors, adenoviral vectors, Sendai virus vectors, naked DNA such as plasmid DNA, minicircle DNA, and recent mRNA (also including lipid nanoparticles (LNP) as carriers) are used, but most of them are difficult to achieve gene introduction with high efficiency. The method for improving the efficiency of gene introduction in vivo of the present invention is a method for achieving a gene introduction efficiency exceeding the previous method when the gene is introduced in vivo, so it not only includes the application of skin, skin ulcers, limbs constituent tissues, muscle tissues, etc. listed in the embodiments, but also includes the application of cranial nerves such as central nervous system, ocular nerves, sensory organs represented by eyeballs, retina, etc., abdominal viscera, thoracic viscera, etc. It can be used as a gene therapy for all organs / tissues / cells in the body, a research and development tool for gene therapy, innovative drug research other than the development of therapeutic methods based on gene delivery, and a biological research tool.

[0194] 14. Methods for improving the efficiency of gene transfer in organisms

[0195] The method for improving the efficiency of gene introduction in the organism of the present invention provides the following method: by using steroids systemically or locally before, at the same time or in the early stage after the introduction of genes in the organism, high gene introduction efficiency and high gene expression are achieved. Systemic administration is carried out by methods generally used in the systemic administration of steroid preparations such as intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, oral administration, etc. Local administration can be carried out by methods generally used in the local administration of steroid preparations such as intradermal injection, subcutaneous injection, intramuscular injection, intraarticular injection, coating on the skin, intraluminal dissemination, eye drops, ear drops, nose drops, etc., and can also include: local administration of gene introduction, such as intracranial administration, administration to the brain parenchyma during brain parenchyma (in addition to injection, local dissemination during surgery, endoscope-assisted administration), etc., which are not commonly used as the treatment method using steroid preparations in the past, and are direct administration methods to the target site. The steroid preparations to be administered may include all preparations having adrenocortical hormone and steroid hormone effects, represented by the following substances: methylprednisolone, methylprednisolone sodium succinate, methylprednisolone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone butyrate, hydrocortisone butyrate propionate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone sodium succinate, prednisolone acetate valerate, clobetasone butyrate, flumethasone, triamcinolone, triamcinolone acetonide, alclomethasone propionate, dexamethasone, dexamethasone sodium phosphate, dexamethasone sodium meta-sulfobenzoate, dexamethasone palmitate, dexamethasone propionate, dexamethasone valerate, dexamethasone cipecilate, betamethasone, betamethasone sodium phosphate, betamethasone valerate, betamethasone dipropionate, betamethasone butyrate propionate, fluocinolone acetate acetonide), diflucortolone valerate, fludroxycortide, fluocinonide, budesonide, diflorasone acetate, amcinonide, mometasone furoate, mometasone furoate monohydrate, beclomethasone propionate, fluticasone propionate, fluticasone furoate, difluprednate, clobetasol propionate, cortisone acetate, ciclesonide, deproton propionate, fludrocortisone acetate, halobetasol propionate. The method of administering steroid preparations (including usage, dosage, number of times, time, period, etc.) is a matter to be optimized according to the preparation administered, the organ / tissue / cell for the purpose of gene introduction, the effect for the purpose of gene introduction, etc. The present invention includes all administration methods for improving gene introduction efficiency and gene expression by using steroids in combination with genes in vivo.

[0196] 15. Experimental results 5

[0197] Hereinafter, the present invention will be described in detail based on Examples and the like, but the present invention is not limited to these Examples.

[0198] Example 9

[0199] Fig.10 This is a graphic illustration of a study on the effect of steroids on the efficiency of AAV-based gene delivery into skin ulcers. Fig.11 This is the stereomicroscope observation of the ulcer surface under visible light and fluorescence observation on the 7th day after AAV administration in this experiment. Fig.12 This is the histological finding on the 7th day after AAV administration in this experiment. In order to investigate the effect of steroid administration on the efficiency of gene introduction when AAV is used to introduce genes into the skin ulcer surface, a skin ulcer surface was made on the back of the mouse, and a model animal with a silicone chamber was made. The silicone chamber was made and installed by the method described in Du Z, Shen Q, Mito D, Kato M, Okazaki M, Kurita M. Optimized 3D-printed template design for production of silicone skin chambers. J Dermatol Sci. 2022 Jan; 105 (1): 55-57. When the silicon chamber was installed, a steroid-free group, a distal administration group in which 10 μl of Corninger A (40 mg / ml) as a steroid was injected subcutaneously into the abdomen, and a local administration group in which 10 μl of Corninger A was administered into the chamber was made. The AAV vector used was an AAVDJ virus vector expressing GFP under the CAG promoter, which was purchased from a vector builder. The day after the chamber was installed and the steroid was administered, 10 11GC / 100μl was inoculated into the chamber of each animal. On the 7th day after AAV administration, the skin ulcer surface was observed with a fluorescent stereomicroscope (Axio Zoom.V16, Zeiss). As a result, in the distal administration group, stronger GFP expression was confirmed compared with the steroid non-administered group and the local administration group. Next, the tissue of the ulcer surface was collected, fixed with 4% paraformaldehyde and phosphate buffer (ph7.4) for one night, replaced with 30% sucrose / phosphate buffered saline, and embedded in OCT complex for freezing. Using Kawamoto films (SECTION-LAB, Cryofilm type 2C(9), 2.5cm C-FP094), sections were collected from frozen tissues at a thickness of 10μm every 200μm, washed with phosphate buffered saline, and then stained with DAPI for nuclei. Adjacent sections were prepared for each section and stained with hematoxylin and eosin. Use a slide scanner (VS200, Olympus) to read as an image file. In terms of tissue findings, a large number of GFP-positive cells were confirmed in the distal administration group. In order to quantitatively compare the tissue findings of each group, the number of GFP-positive cells on the enlarged image of the central part of the ulcer was counted. At this time, the adjacent hematoxylin and eosin staining findings were used as a reference, and were classified into the surface layer (fascia on the subcutaneous fat tissue), the fat layer (subcutaneous fat tissue), and the muscle layer (muscles such as the trapezius muscle), and the number of GFP-positive cells in each area was counted. For each experimental animal / tissue, 5 sections were counted and the average value was obtained. The difference in the average value of 3 animals in each group was explored using t-test. Regarding the number of GFP-positive cells in all areas, in the distal administration group, significantly more GFP-positive cells were confirmed than when there was no steroid. Especially when limited to deep tissues (fat layer + muscle layer), in the distal administration group, significantly more GFP-positive cells were confirmed than when there was no steroid and the local administration group.

[0200] Example 10

[0201] Fig.13The present invention is an experimental result on the effect of different concentrations of steroids on the efficiency of AAV-based gene introduction into skin ulcers. A steroid-free group, a distal administration group in which 1 μl and 10 μl of Corninger A (40 mg / ml) as a steroid was injected subcutaneously into the abdomen, and a local administration group in which 1 μl and 10 μl of Corninger were administered into the cavity were prepared. In addition, the experiment was conducted in the same manner as in Example 9 until tissue observation. In terms of tissue observation, a large number of GFP-positive cells were confirmed in the distal administration group at 10 μl, and a large number of GFP-positive cells were confirmed in the local administration group at 1 μl. Especially in the local area where inflammation exists, such as the skin ulcer surface, since steroids have an anti-inflammatory effect, it is assumed that the number of cells itself will change according to the concentration of action. In order to improve the efficiency of gene introduction, it is necessary to study the optimal dosage according to the distal and local administration methods.

[0202] Embodiment 11

[0203] Fig.14 The results of an experiment on the effect of steroid administration on the efficiency of AAV-based gene introduction into the back skin of mice were presented. A steroid-free group and a distal administration group were prepared in which 10 μl of Conacore A (40 mg / ml) as a steroid was injected subcutaneously into the abdomen. As an AAV vector, an AAV with AAVDJK2 as the capsid and GFPNLS expressed under the K14SCP3 promoter was prepared. One day after the steroid was administered to the distal administration group, the back skin of each group of mice was shaved, and then AAVDJK2-K14SCP3-GFPNLS10 was injected intradermally. 11 GC / 35μl. On the second day after AAV injection, while observing the injection site with a fluorescent stereo microscope (Axio Zoom.V16, Zeiss), tissues including the surrounding skin were collected, fixed with 4% paraformaldehyde and phosphate buffer (ph7.4) for one night, replaced with 30% sucrose / phosphate buffered saline, and embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C(9), 2.5cm C-FP094), sections were collected from frozen tissues at a thickness of 10μm every 200μm, washed with phosphate buffered saline, and then stained with DAPI for nuclei. Sections adjacent to each section were prepared and stained with hematoxylin and eosin. Use a slide scanner (VS200, Olympus) to read as an image file. In terms of tissue observation, in the steroid distal administration group, GFPNLS-positive cells were observed efficiently and continuously in the epidermal tissue, while in the steroid non-administration group, GFPNLS cells were scattered at a low frequency. It is known that the efficiency of gene introduction into the skin by AAV is improved by the action of steroids.

[0204] Example 12

[0205] Fig.15 The present invention is an experimental result on the effect of different concentrations of steroids on the efficiency of AAV-based gene introduction into the skin. A distal administration group was prepared in which 10 μl, 0.1 μl, and 0.01 μl of Corninger A (40 mg / ml) as a steroid were injected subcutaneously into the abdomen, and the experiment was conducted in the same manner as in Example 11. In terms of tissue observation, in animals to which 10 μl and 0.1 μl of Corninger A were distally administered, GFPNLS-positive cells were confirmed centered on the epidermal tissue, while in the observation of animals to which 0.01 μl of Corninger A was distally administered, no significant difference was confirmed with that of animals without steroids. It can be seen that if the amount of steroid is reduced (0.01 μl), the effect of improving the gene introduction efficiency brought about by the steroid is reduced.

[0206] Embodiment 13

[0207] Fig.16 The surgical procedure of shifting the left upper limb to the dorsal side before cutting off the upper limb in mice is shown in order to avoid the mouse's own invasion of the stump and keep the local area as quiet as possible. The surgical method of each stage is shown in the order of left segment from top to bottom, middle segment from top to bottom, and right segment from top to bottom. After inhalation anesthesia with isoflurane, the traction line is hung on the left hand, and the upper limb is flexed and abducted to the side to ensure the surgical posture. The skin is cut longitudinally in front of the axilla, and the muscle body on the front side of the shoulder joint is cut with a bipolar electric knife to expose the joint capsule of the shoulder joint. Enter the joint from the front side of the joint capsule, push the cutting of the joint capsule in the up-down direction, and grasp the stump of the scapula. While pulling the scapula, the surrounding muscles are peeled off and the scapula is removed. After cutting off the muscles such as the teres major connecting the humerus and the chest wall, the skin incision is extended to the dorsal skin of the root of the upper limb. The body position is changed to the right prone position, and the muscle group connecting the humerus and the chest wall is cut off to free the upper limb. During a series of tissue excisions, the brachial plexus and the main arteries and veins to the upper limb are preserved. The free upper limb is displaced to the dorsal side, and the ventral muscles such as a part of the deltoid muscle around the shoulder joint are fixed to the chest wall while the skin is sutured. When suturing the skin, the remaining skin is appropriately trimmed. Usually 4-6 stitches are enough to close the wound. Through a series of operations, the left upper limb is displaced to the dorsal side while preserving the main nerves and blood vessels.

[0208] In the present embodiment, an upper limb amputation model animal is prepared for mice. The upper limb amputation model animal can be prepared by displacing the upper limb of a non-human mammal and then cutting off the upper limb in the upper arm / forearm / hand / finger plane, or by displacing the upper limb of a non-human mammal and then cutting off the upper limb in the forearm plane. The upper limb amputation model animal is preferably carried out while maintaining the main trunk nerves, arteries and veins of the upper limb. The upper limb displacement can be performed at the shoulder joint, or it can be accompanied by the removal of the scapula. By applying the substance to be tested to such an upper limb amputation model animal and studying the effect on the amputation site, a screening method for a pharmaceutical substance can be achieved. In particular, the pharmaceutical substance can be a gene introduction vector for the purpose of gene introduction, or it can be a substance for the regeneration of the severed tissue. As non-human mammals, for example, rabbits, dogs, cats, guinea pigs, hamsters, rats or mice are included.

[0209] Embodiment 14

[0210] Fig.17 The results of an experiment on the effect of steroid administration on the efficiency of AAV and retrovirus-based gene introduction into the forearm stump of mice that had been amputated after the left upper limb was moved dorsally by the surgical method of Example 13. After collecting the forearm tissue, it was fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for one night, and then embedded in an OCT complex and frozen. Using Kawamoto films (SECTION-LAB, Cryofilm type 2C (9), 2.5 cm C-FP094), sections were collected from the frozen tissue at a thickness of 10 μm every 200 μm, and stained with hematoxylin and eosin, or adjacent sections were washed with phosphate-buffered saline and nuclear stained with DAPI. The slide scanner (VS200, Olympus) was used to read as an image file. In terms of tissue appearance, the forearm amputation tissue has bones in the central part, muscles around it, and skin and subcutaneous fat covering its outer side. In the AAV-based gene introduction, an AAV having an AAVDJ capsid and expressing GFPNLS under the CAG promoter was prepared by the concentrated AAV production method described in Example 2. A steroid-free group, a distal administration group in which 10 μl of Corninger A (40 mg / ml) as a steroid was injected subcutaneously into the abdomen, and a local administration group in which 5 μl of Corninger A (40 mg / ml) was injected into the cut end were prepared. The day after the left upper limb was displaced dorsally, the upper limb was cut off on the forearm, and 1 hour later, steroids were administered according to the settings. The next day, 3 μl of AAVDJ-CAG-GFPNLS10 was injected into each animal at intervals of 2 hours. 11GC / 9μl, injected into the cut part 3 times. Tissue was recovered one week after AAV injection. In terms of fluorescent tissue observation, the number of GFPNLS-positive cells in the cut tissue increased by local and distal administration of steroids. It can be seen that through the use of steroids, when AAV is used for gene introduction, a significant improvement in gene introduction efficiency is confirmed in a wide range of tissues / cells such as subcutaneous, fascia, muscle, and periosteum that constitute the cut part of the limbs. For retrovirus, Lipofectamine 2000 (Thermo Fisher Scientific) was used to transfect 293FT cells (Thermo Fisher Scientific) with the PMXs retroviral backbone plasmid encoding GFPNLS and the packaging plasmid (pCMV-gagpol-PA, pCMV-VSVg) according to conventional methods, and the cell supernatant after replacing the culture medium was used as the retroviral solution (refer to non-patent document 2). For each animal, 2 ml of the retroviral solution was concentrated to 100 μl of the virus concentrate by polyethylene glycol concentration. The day after the left upper limb was displaced dorsally, the upper limb was cut off on the forearm, and the virus solution was injected for the first time 1 hour later, and 10 μl of Corninger A (40 mg / ml) was injected into the subcutaneous abdomen in the distal administration group, and 5 μl of Corninger A (40 mg / ml) was mixed and injected into the virus solution in the local administration group. For each animal, the retrovirus was injected 10 times at intervals of 3 hours over 2 days. The tissue was recovered 1 week after the virus solution was injected. In fluorescent tissue observation, the number of GFPNLS-positive cells in the cut-off tissue increased by local and distal administration of steroids. It can be seen that by the use of steroids, when using retrovirus to introduce genes, the improvement of gene introduction efficiency is confirmed in the tissues / cells constituting the cut-off part of the limbs.

[0211] Embodiment 15

[0212] Fig.18The results of the experiment were about the effect of steroid administration on the efficiency of mRNA-based gene introduction into the front thigh muscle of mice. The mRNA used in the experiment was eGFP mRNA manufactured by Arcalis. Regarding the Lipofectamine 2000 transfection reagent, a complex was prepared using 1 μg of mRNA, 1.2 μl of lipofectamine, and a total of 100 μl of Opti-MEM medium according to the method described in the attached instructions, and 50 μl of it was used as the complex reagent for mRNA transfection per muscle body. Nude mice (BALB / cAJcl-nu / nu) were used in the experiment. A steroid-free group before mRNA transfection and a distal administration group in which 10 μl of Corninger A (40 mg / ml) as a steroid was injected subcutaneously into the abdomen the day before were prepared. The complex reagent for mRNA transfection was prepared just before the injection and injected into the muscle body of the front thigh 5-15 minutes later. After the animals were euthanized at 12 hours after the injection, the muscle body was removed and the surface of the muscle body was observed using a fluorescent stereomicroscope (AxioZoom.V16, Zeiss). As a result, a strong green fluorescence was confirmed in the distal administration group. After fixing with 4% paraformaldehyde and phosphate buffer (ph7.4) for one night, the tissue was embedded in OCT complex and frozen. Using Kawamoto films (SECTION-LAB, Cryofilm type 2C (9), 2.5cm C-FP094), slices were collected from the frozen tissue with a thickness of 10μm at a cross section perpendicular to the direction of the muscle fibers. After washing with phosphate-buffered saline, nuclear staining was performed with DAPI. Hematoxylin and eosin staining was performed on adjacent slices. The prepared tissue slices were read as image files using a slide scanner (VS200, Olympus). As a result, a strong GFP signal was confirmed in a part of the muscle body in the distal administration group, and the signal was observed to diffuse centrifugally, while only a weak signal was observed in the steroid non-administered group. It is known that the use of steroids also improves the efficiency of gene transfer into in vivo tissues using mRNA.

[0213] Example 16

[0214] Fig.19 The results of the experiment were about the effect of steroid administration on the efficiency of AAV-based gene introduction into in vitro cultured cells. 293AAV cells (Cell Biolabs) were used as cultured cells. AAV vectors used were AAVDJ viral vectors expressing GFP under the CAG promoter, which were customized and purchased from Vector Builder. 293AAV cells were cultured in DMEM medium supplemented with 10% newborn bovine serum according to conventional methods. 1.5×10 5On day 1, the culture medium was replaced with a medium containing 1 μg / ml, 10 μg / ml, 100 μg / ml, or 1 mg / ml of Corninger A. On day 2, half of the culture medium was replaced with a medium containing 10 10 GC / well AAV culture medium. On the 3rd day, all culture media were replaced with culture media that did not contain steroids and AAV. On the 6th day, photos under phase contrast and fluorescence observation were taken with a fluorescence microscope (IX73, Olympus). GFP-positive cells were observed in the green fluorescence observation image, and non-specific fluorescence expression was observed in the red fluorescence observation image. In the presence of 1, 10μg / ml steroids, higher GFP-positive cells were confirmed compared with those without steroids, and in the presence of 100μg / ml, 1mg / ml steroids, the number of cells and the number of positive cells decreased due to the steroid effect on cells. It can be seen that in the presence of appropriate concentrations of steroids, the efficiency of AAV-based gene introduction in in vitro cultured cells is improved.

[0215] Embodiment 17

[0216] Fig. 20These are the test results on the effect of steroid administration on the dynamic production of subcutaneously injected fluorescent silica particles. A steroid-free group and a distal administration group were prepared in which 10 μl of Corninger A (40 mg / ml), a steroid, was injected subcutaneously into the abdomen. On the day after steroid administration, a large area of ​​the mouse back was shaved, and 30 μl of red fluorescent silica particles (sicastar (registered trademark) -redF, plain, 30 nm, 25 mg / ml) of the same size as AAV were subcutaneously injected. Immediately after injection, 1, 3, 6, 9, 12, 18, and up to 24 hours later, local observations were performed using a fluorescent stereomicroscope (Axio Zoom.V16, Zeiss) to analyze the effect of steroids on the local retention of fluorescent silica particles. According to the exposure time (30msec), the fluorescence microscope images were taken over time, and the red fluorescence signals on the images were compared. As a result, it was found that in the steroid-free group, the red fluorescence signal was weakened from the early stage, while in the distal group, the red fluorescence signal remained relatively strongly. In addition, the images taken at a time point of 24 hours with a longer exposure time were compared and studied, and it was found that in animals without steroids, the weak fluorescence signal diffused in a wider range. In order to evaluate the diffusion state of fluorescent silica particles in more detail in the tissue, skin tissue was collected at a time point of 24 hours, and the tissue was quickly embedded in the OCT complex and frozen. Kawamoto slices (SECTION-LAB, Cryofilm type 2C (9), 2.5cm C-FP094) were used to collect slices from frozen tissue with a thickness of 10m, and after washing with phosphate-buffered saline, nuclear staining was performed with DAPI. Hematoxylin and eosin staining of adjacent slices was performed. A slide scanner (VS200, Olympus) was used to read as an image file. The same is true for tissue observation. In the distal administration group, a stronger fluorescence signal was observed in a more limited area, while in the steroid-free animals, a weak fluorescence signal was spread over a wider range. It can be seen that in the steroid-injected animals, the diffusion of silica particles into the surrounding tissues was suppressed and the local retention was more obvious compared to the steroid-free animals.

[0217] In the present invention, by using steroids systemically or locally before, at the same time, or in the early stage after the introduction of genes in the organism, high gene introduction efficiency and high gene expression can be achieved. In gene therapy using viral vectors, in order to suppress the existing immune response to viral vectors, the immune response caused by repeated treatment, and to enable efficient gene introduction, steroids are sometimes used (refer to Yanda MK, Tomar V, Cebotaru CV, Guggino WB, Cebotaru L. Short-Term Steroid Treatment of Rhesus Macaque Increases Transduction. Hum Gene Ther. 2022 Feb; 33 (3-4): 131-147.). As shown in Example 9, Example 10, Example 11, Example 12, Example 14, and Example 15, the effect of the present invention has been confirmed at the time of the initial gene introduction, so the suppression of immune response based on repeated treatment is not used as the main mechanism of action. In addition, in the experimental animal breeding environment, AAV non-specifically infects experimental animals, and the possibility of having an existing immune response to AAV is low. That is, the possibility that the present invention is merely the effect of suppressing the existing immune response is low. Furthermore, in the gene transfer experiments based on retroviral vectors in Example 14 and mRNA in Example 15, there is no possibility that the existing immune response is induced by the administration of exogenous retroviruses or exogenous mRNA, and it can be seen that the steroid administration in the present invention is different from the known steroid use method and is highly novel.

[0218] According to Example 16, it was confirmed that the efficiency of gene introduction was improved and high gene expression was achieved by the action of steroids in cultured cells. That is, it was suggested that this effect was caused by the action of steroids at the cellular level in addition to the effect on the systemic immune system. It is possible that the endogenous cGAS-STING pathway induced by exogenous gene introduction was inhibited.

[0219] According to Example 17, the local retention tendency of subcutaneously administered fluorescent silica particles increases and the clearance rate of local injection decreases by the administration of steroids. It can also be seen from the improvement of inflammatory edema that steroids affect the body fluid circulation in peripheral tissues, so it is suggested that the efficiency of gene introduction can be improved by increasing the local retention time of the gene introduction vector administered to the peripheral tissues, reducing the local clearance rate, increasing the chance of action, and increasing the duration of action.

[0220] Steroids are physiologically active substances that have many effects at the cellular level and the individual level. Therefore, through the anti-inflammatory and anti-immune effects at the individual level, which are the characteristic effects of steroids, the biological reactions that weaken the effects of gene introduction vectors are suppressed, and the gene introduction efficiency in the body is improved. In the present invention, it is suggested that the mechanism of achieving high gene introduction efficiency and high gene expression by using steroids systemically or locally before, simultaneously with, or early after gene introduction in the body involves many aspects.

[0221] 16. Use of the method for regenerating tissue from severed limbs

[0222] In mammals, wound healing occurs in the severed limb stumps. That is, scar healing occurs in the bone stumps, muscle stumps, and skin / subcutaneous tissues through primary wound healing or secondary wound healing, and no functional tissue regeneration occurs. In contrast, in the present invention, functional tissue regeneration is produced in the severed limbs of mammals by adding interventions such as gene introduction, protein administration, compound administration, and cell therapy, thereby providing a model related to medical treatment (therapy), biology, tissue regeneration technology, or future technology development. Mammals include, for example, humans, monkeys, rats, or mice. In particular, mice are preferably used as a model for research and development.

[0223] In the present invention, "tissue regeneration of amputated limbs in mammals" refers to the regeneration of tissues that cannot be regenerated by physiological primary wound healing and secondary wound healing on the bone stump cut surface, muscle stump cut surface, and skin / subcutaneous tissue cut surface when the limbs are cut. It refers to the regeneration / hyperplasia / regeneration of various tissues contained in the peripheral tissues of the limbs, represented by the regeneration / hyperplasia of fat tissue, the regeneration / hyperplasia of bone tissue, the regeneration / hyperplasia of cartilage tissue, the regeneration / hyperplasia of muscle tissue, and the regeneration of limb-like structures, which are beyond the physiological range.

[0224] The "model for developing a method for regenerating tissue of amputated limbs in mammals" in the present invention refers to a research and development model that can be used as a method for medical (therapeutic) research and development based on gene therapy, administration of proteins / compounds, etc., and drug innovation / biological research.

[0225] 17. Method for regenerating tissue from severed limbs

[0226] The present invention includes: a method for regenerating tissue of a severed limb in a mammal, an animal model that can serve as a model for developing the method (including an experimental surgical method for preparing the model and a severed limb method), an introduced gene / an administered protein / an administered compound, an auxiliary physiologically active substance, and a gene introduction method / a substance administration method.

[0227] In the present invention, a mouse forearm amputation model was developed as an animal model for studying the effects of gene introduction, protein, and compound administration on tissue regeneration in mammals. By the surgical operation described in Example 13, after upper limb amputation in mice, the invasion of the mouse itself on the stump can be avoided, and the local quietness can be maintained as much as possible.

[0228] Fig.21 Illustration showing the method of treating the stump of the cut part of the forearm of a nude mouse. The forearm is cut in a plane proximal to the skin structure with gentle protrusion and redness existing on the skin surface of the forearm of a nude mouse. Regarding the cut part, a simple transverse cut is made, or the skin around the stump is excised after the cut, or the nerve is retained for a long time during the cut. The wound surface after the cut is set as an open wound, or the surrounding skin is crimped or sutured with the fingers. In the case of heavy bleeding from the stump, bleeding can be stopped by installing a silicon cap.

[0229] Through the surgical operation described in Example 13 of the present invention, and Fig.21 The method for treating severed stumps illustrated in a diagrammatic manner provides a research and development platform for developing methods for regenerating severed limb parts of mammals.

[0230] The method of the present invention for regenerating tissue of a severed limb in a mammal is characterized by comprising the steps of introducing or causing to act a substance into somatic cells of a severed limb that do not have the ability to regenerate tissue physiologically (including cells of the blood system that migrate locally upon severance), wherein the substance is a gene / protein that is relatively strongly expressed in cells that have the ability to regenerate tissue at the distal end of a severed limb (e.g., cells present in limb buds and lateral plate mesoderm in the fetal period of mammals, and cells of amphibians that have a higher ability to regenerate tissue physiologically, such as Ambystoma mexicanum, Cynops pyrhogaster, and Xenopus laevis); or a step of causing the substance to act in an environment where tissue regeneration occurs (e.g., cells present in limb buds and lateral plate mesoderm in the fetal period of mammals, and cells of amphibians that have a higher ability to regenerate tissue physiologically, such as Ambystoma mexicanum, Cynops pyrhogaster, and Xenopus laevis); mexicanum, Japanese salamander (Cynops pyrhogaster), African clawed frog (Xenopus laevis) and other amphibians with higher tissue regeneration ability physiologically (tissue regeneration after limb amputation), cytokines, hormones, and compounds with equivalent / similar functions to them. Hereinafter, the introduced gene / protein is referred to as "introduced gene / introduced protein". The protein that plays a role is referred to as "action protein", and the compound that plays a role is referred to as "action compound".

[0231] The introduced genes, introduced proteins, active proteins, and active compounds can be used in parallel in multiple ways, or the following substances can be combined: genes and proteins that are relatively weakly expressed in cells with tissue regeneration ability, or that do not act as master regulatory genes in the differentiation / production process of each cell; cytokines and hormones that do not act strongly in the environment where tissue regeneration occurs, and compounds that do not have equivalent / similar functions to them.

[0232] The genes and proteins in the present invention that are relatively strongly expressed in cells with tissue regeneration ability or that act as master regulatory genes in the differentiation / production process of each cell, and the cytokines and hormones that act strongly in an environment where tissue regeneration occurs include at least the genes listed in Table 3 and the proteins encoded by the genes.

[0233] [Table 3]

[0234] BMP2 HMGB3 Sp8 NR2C2 WNT3A BMP4 HMGN1 SP9 TBX15 WNT5A BMP5 HMGN2 STMN1 BCL11A ZIC3 BMP7 HOXA10 TRIM69 SMARCA1 POU5F1 BMP9 HOXA9 XB5727893 SMARCA4 SOX2 FGF2 HSPA8 ZNF326 CIRBP KLF4 FGF4 LEF1 TBX5 LIN28A cMYC FGF8 MARCKSL1 TCF7L2 HOXB9 PBX1 FGF10 MEIS1 NFIB SALL4 FGF20 MSX1 SHOX2 FOXD1 S H MSX2 ZNF449 PRDM1 RSPO2 MYCN EGR1 EGR3 BRCA1 nG HDAC1 WNT7A DNMT1 PRDX1 POLE4 IL6 E2F8 PRDX2 ATF1 FOXA1 ETV2 Prod1 PPARD FOXA2 GTF2F1 PRRX1 TIMELESS MBD4 HAND2 SAP30 TFDP1 NR2F2 HDAC2 SMC3 TFDP2 PA2G4 HMGB2 SNRPF KAZALD1 SPDEF

[0235] Most of the sources of these genes exist in mammals including humans, and genes of any mammalian origin can be used, preferably appropriately selected according to the source of the somatic cells to be introduced. For example, in the case of humans, the above-mentioned introduced genes and proteins are preferably of human origin. In addition, in addition to wild-type genes and proteins, the above-mentioned introduced genes and proteins may also be variant genes encoding variant gene products in which several (for example, 1 to 10, preferably 1 to 6, more preferably 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2) amino acids in the amino acid sequence are replaced, deleted and / or inserted and have the same function as the wild-type gene product. In addition, sequences in which codons are changed / optimized in a manner that encodes the same amino acids as those encoded by each gene may also be used.

[0236] In the present invention, the above-mentioned introduced gene can be prepared according to conventional methods based on known sequence information. For example, RNA is extracted from cells of mammalian origin and cloned according to conventional methods, thereby preparing cDNA of the target gene. In addition, it can also be synthesized as an artificial gene. When the artificial gene is synthesized, codon optimization can also be performed according to the source animal of the somatic cell introduced.

[0237] 18. Experimental results 6

[0238] Hereinafter, the present invention will be described in detail based on Examples and the like, but the present invention is not limited to these Examples.

[0239] Embodiment 18

[0240] Fig. 22The results of the experiment were as follows: the left upper limb of a nude mouse was displaced dorsally, the forearm was transversely cut, and then the cut surface was set as an open wound. On the 5th day after the left upper limb of a 4-5 week old BALB / cAJcl-nu / nu (from the Central Research Institute for Experimental Animals) mouse was displaced dorsally by the surgical method of Example 13, the proximal end of the gently protruding and reddened skin structure present on the skin surface of the forearm was transversely cut, and the cut surface was set as an open wound. Every 7 days, photos were taken under observation with a surgical microscope (MM100-YOH manufactured by Sanko Hawki Co., Ltd.), and the process was observed until the 70th day after the cut. After the forearm tissue was collected, it was fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for one night, and then embedded in OCT complex for freezing. Using Kawamoto films (SECTION-LAB, Cryofilm type 2C (9), 2.5 cm C-FP094), sections were collected every 200 μm from frozen tissue at a thickness of 10 μm, stained with hematoxylin and eosin, and the obtained slides were read as image files using a slide scanner (VS200, Olympus). In terms of tissue observation, the stump of the bone was closed by cortical bone, and the soft tissue was healed by contraction and closure of the surrounding skin. No significant tissue regeneration was confirmed in the amputated part of the limbs.

[0241] Embodiment 19

[0242] Fig.23The results of the experiment were as follows: the left upper limb of the nude mouse was displaced dorsally, the forearm was cut transversely, and then the cut surface was set as an open wound to introduce genes. Retrovirus was used to introduce genes for POLE4, NFIB, PPARD, FGF10, FGF20, and FGF2, AAVDJ1 was used to introduce genes for PRRX1 and HDAC2, and AAVDJ was used to introduce genes for LEF1 and SHH. For retrovirus, PMXs retrovirus backbone plasmids encoding each gene and packaging plasmids (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to conventional methods, and the cell supernatant after replacing the culture medium was used as the retrovirus solution. For each animal, 2 ml of retroviral solution was concentrated to a total of 500 μl of virus concentrate by polyethylene glycol concentration for POLE2, NFIB, and PPARD, and 1 ml of retroviral solution was concentrated to a total of 500 μl of virus concentrate by polyethylene glycol concentration for FGF10, FGF20, and FGF2. Regarding AAV, AAV with a specified capsid expressing each gene under the CAG promoter was produced and used by the concentrated AAV production method described in Example 2. On the 5th day after the left upper limb of a 4-5-week-old BALB / cAJcl-nu / nu (from the Central Research Institute for Experimental Animals) mouse was displaced dorsally by the surgical method of Example 13, there was a skin structure with a gentle protrusion and redness on the skin surface of the forearm, and the proximal end of the skin structure was set as the cutting high position. After the initial injection of retrovirus in the predetermined cutting line, a transverse cut was performed, and the cut surface was set as an open wound. For retrovirus, local injection was performed 10 times from the first injection immediately before amputation to the fifth day after amputation. For AAVDJ-SHH, 2.5×10 11 GC / 8μl of virus solution. 1218 μl of virus solution of AAVDJ-LEF1, AAVDJ1-PRRX1, and AAVDJ1-HDAC2 of GC was applied to the wound twice on the day of amputation, 2 μl each time, and then injected twice on the next day and three times on the second day for local injection. After amputation, photos were taken under observation under a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.) every 7 days. After taking photos on the 36th day after amputation, the tissue of the forearm was collected, fixed with 4% paraformaldehyde and phosphate buffer (ph7.4) for one night, and then embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C (9), 2.5 cm C-FP094), sections were collected every 200 μm at a thickness of 10 μm from the frozen tissue, stained with hematoxylin and eosin, and the obtained slides were read as image files using a slide scanner (VS200, Olympus). From the tissue observation, a blastema-like soft tissue mass was formed at the front end, which is similar to the blastema found in the severed limbs of amphibians such as Ambystoma mexicanum, Cynops pyrhogaster, and Xenopus laevis, which have higher tissue regeneration ability physiologically. It was confirmed that the bone extended axially into the tissue.

[0243] Embodiment 20

[0244] Fig.24The results of the experiment were as follows: the left upper limb of the nude mouse was displaced dorsally, the forearm was cut transversely, and then the cut surface was set as an open wound to carry out gene introduction. The genes of PRDX2, POLE4, NFIB, PPARD, FGF10, FGF20, FGF2, BMP5, and SHH were introduced using retrovirus, the genes of PRRX1 and HDAC2 were introduced using AAVDJ1, and the gene of LEF1 was introduced using AVDJ. For retrovirus, the PMXs retrovirus backbone plasmid encoding each gene and the packaging plasmid (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the conventional method, and the cell supernatant after replacing the culture medium was used as the retrovirus solution. For each animal, PRDX2, POLE2, NFIB, PPARD, FGF10, FGF20, FGF2, and BMP5 all used a virus concentrate obtained by concentrating 1 ml of retroviral solution to a total of 400 μl using polyethylene glycol concentration. Regarding AAV, an AAV with a specified capsid expressing each gene under the CAG promoter was produced by the concentrated AAV production method described in Example 2 and used. On the second day after the left upper limb of a 4-5 week-old BALB / cAJcl-nu / nu (from the Central Research Institute for Laboratory Animals) mouse was displaced dorsally by the surgical method of Example 13, a skin structure with a gentle protrusion and redness was present on the skin surface of the forearm, and the proximal end of the skin structure was set as the high cutting position. At the predetermined cutting line, 100 μl of the retroviral solution was injected as the first dose of retrovirus, and then 1.0×10 11 GC was cut transversely with 5 μl solution of AAVDJ-LEF1, AAVDJ1-PRRX1, and AAVDJ1-HDAC2. The cut surface was set as an open wound and a silicon cap was installed to stop bleeding. The next day, 1.0×10 115 μl solution of AAVDJ-LEF1, AAVDJ1-PRRX1, and AAVDJ1-HDAC2 of GC was injected twice. From the 2nd day to the 5th day, the retrovirus solution was injected into the front part once a day. After amputation, photos were taken under observation with a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.) every 7 days. After taking photos on the 40th day after amputation, the tissue of the forearm was collected, and then fixed with 4% paraformaldehyde and phosphate buffer (ph7.4) for 1 night, and then embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C (9), 2.5 cm C-FP094), sections were collected every 200 μm at a thickness of 10 μm from the frozen tissue, stained with hematoxylin and eosin, and the obtained slides were read as image files using a slide scanner (VS200, Olympus). Histologically, new bone tissue formation was observed in the distal part of the bone stump, accompanied by a blastema-like soft tissue mass and abundant trabecular structures extending from the inside.

[0245] Embodiment 21

[0246] Fig.25The results of the experiment were as follows: the left upper limb of the nude mouse was displaced dorsally, the forearm was cut transversely, and then the cut surface was set as an open wound to carry out gene introduction. The genes of FGF10, FGF20, FGF2, NFIB, PPARD, and POLE4 were introduced into the center of the stump using retrovirus. The gene of SHH was introduced into the outside of the stump using retrovirus. The gene of FGF8 was introduced into the stump using AAVDJK2. For retrovirus, the PMXs retrovirus backbone plasmid encoding each gene and the packaging plasmid (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) according to the conventional method using Lipofectamine 2000 (Thermo Fisher Scientific), and the cell supernatant after replacing the culture medium was used as the retrovirus solution. For each animal, a virus concentrate concentrated by polyethylene glycol concentration based on 4 ml of retrovirus solution was used for FGF10, FGF20, FGF2, and SHH, and a virus concentrate concentrated by polyethylene glycol concentration based on 12 ml of retrovirus solution was used for NFIB, PPARD, and POLE4. Regarding AAV, a virus with an AAVDJK2 capsid expressing FGF8 under the K14 promoter was produced by the concentrated AAV production method described in Example 2 and used. On the second day after the left upper limb of a 4-5 week-old BALB / cAJcl-nu / nu (from the Central Research Institute for Experimental Animals) mouse was displaced dorsally by the surgical method of Example 13, a skin structure with gentle protrusion and redness was present on the skin surface of the forearm, and the proximal end of the skin structure was set as the cutting high position. After injecting the retrovirus solution expressing FGF10, FGF20, FGF2, NFIB, PPARD, and POLE4 into the part of the planned cutting line, the transverse cutting was performed, and the cut surface was set as an open wound. After injecting the retrovirus solution expressing FGF10, FGF20, FGF2, NFIB, PPARD, and POLE4 again, the retrovirus solution expressing SHH was injected into the anterolateral part of the forearm. The retrovirus solution expressing FGF10, FGF20, FGF2, NFIB, PPARD, and POLE4 injected before and after the cutting was 20μl, and the retrovirus solution expressing SHH was 2μl. Then, 4μl of each retrovirus solution expressing FGF10, FGF20, FGF2, NFIB, PPARD, and POLE4 was locally injected 18 times during the period including before and after the cutting until the 5th day of the cutting. 2μl of each retrovirus solution expressing SHH was locally injected 11 times to the anterior and lateral side during the period including after the cutting until the 3rd day of the cutting. For AAVDJK2 expressing FGF8 under the K14 promoter, 1.0 × 10 112 μl of GC solution was injected locally 7 times between the 5th and 23rd days after amputation. After amputation, photographs were taken under observation with a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.) every 7 days. Starting 3 weeks after amputation, the shape of the front end became flat, and branched white structures were seen when observed from the ventral side. After photographs were taken on the 28th day after amputation, the tissue of the forearm was collected, fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for one night, and then embedded in OCT complex and frozen. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C (9), 2.5 cm C-FP094), sections were collected every 200 μm at a thickness of 10 μm from the frozen tissue, stained with hematoxylin and eosin, and the resulting slides were read as image files using a slide scanner (VS200, Olympus). From the tissue observation, the presence of bone marrow cell aggregation was confirmed in the soft tissue block in accordance with the position of the white structure.

[0247] Embodiment 22

[0248] Fig.26The results of the experiment were as follows: the left upper limb of the nude mouse was displaced dorsally, the forearm was cut horizontally, and the skin around the cut surface was pressed with fingers to perform gene introduction. The genes of FGF10, FGF20, FGF2, and OCT4 were introduced into the stump using retrovirus. The gene of SHH was introduced into the outside of the stump using AAVDJ, and the gene of FGF8 was introduced into the inside of the stump to the front part using AAVDJ. For retrovirus, the PMXs retrovirus backbone plasmid encoding each gene and the packaging plasmid (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the conventional method, and the cell supernatant after the culture medium was replaced was used as the retrovirus solution. For each animal, the first 20 local injections were made using a virus concentrate concentrated by polyethylene glycol based on 4 ml of retrovirus solution expressing OCT4 and 24 ml of retrovirus solution for FGF10, FGF20, and FGF2. For the last 16 local injections, a virus concentrate concentrated by polyethylene glycol based on 24 ml of retrovirus solution expressing OCT4 was used. AAV with an AAVDJ capsid expressing SHH and FGF8 under the CAG promoter was purchased from a vector manufacturer. The day after the left upper limb of a 4-5 week old BALB / cAJcl-nu / nu (from the Central Research Institute of Laboratory Animals) mouse was displaced dorsally by the surgical method of Example 13, a gently protruding, red skin structure was found on the skin surface of the forearm. The proximal end of the skin structure was set as the high cutting position, and 1.0×10 AAVDJ-SHH was locally injected into the outer part of the forearm at the cutting line. 11 GC / 2μl, AAVDJ-FGF8 1.0×10 11 GC / 2μl. On the next day, 5μl of Conacton A (40mg / ml), a steroid, was mixed with 20μl of the retrovirus concentrate expressing FGF10, FGF20, FGF2, and OCT4 and injected. The mixture was injected into the subcutaneous tissue, intramuscular tissue, and peri-bone tissue. At the same time, the forearm was cut at the predetermined cutting height, and the skin on the front and back of the forearm was pressed with fingers to close the wound. Then, until the 4th day after the cut, 10μl of the retrovirus concentrate was locally injected 20 times. Next, from the 5th day to the 10th day after the cut, 10μl of the retrovirus concentrate expressing OCT4 was locally injected 16 times. On the 6th, 13th, and 20th days after the cut, 1.0×10 AAVDJ-SHH was locally injected into the outer part of the cut end. 11GC / 2μl, AAVDJ-FGF81.0×10 was injected locally into the inner part of the cut end 11 GC / 2μl. After amputation, photographs were taken every 7 days under a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.). About 10 weeks after amputation, a protrusion-like structure appeared at the front end, and then the front end continued to elongate. After taking photos on the 105th day after amputation, the forearm tissue was collected, fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for one night, and then embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C (9), 2.5 cm C-FP094), sections were collected every 200 μm at a thickness of 10 μm from the frozen tissue, stained with hematoxylin and eosin, and the resulting slides were read as image files using a slide scanner (VS200, Olympus). From the tissue observation, a cell structure running longitudinally along the axial direction in the elongated tissue was confirmed.

[0249] Embodiment 23

[0250] Fig. 27The results of the experiment were as follows: the left upper limb of the nude mouse was displaced dorsally, the forearm was cut horizontally, and the skin around the cut surface was pressed with fingers to perform gene introduction. The genes of FGF10, FGF20, FGF2, OCT4, and SOX2 were introduced into the stump using retrovirus. The gene of SHH was introduced into the outside of the stump using AAVDJ, and the gene of FGF8 was introduced from the inside of the stump to the front part using AAVDJ. For retrovirus, the PMXs retrovirus backbone plasmid encoding each gene and the packaging plasmid (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine2000 (Thermo Fisher Scientific) according to the conventional method, and the cell supernatant after the culture medium was replaced was used as the retrovirus solution. For each animal, the first 20 local injections were made using 4 ml of retrovirus solution expressing OCT4 and SOX2 and 12 ml of retrovirus solution concentrated by polyethylene glycol concentration for FGF10, FGF20, and FGF2. For the last 16 local injections, 12 ml of retrovirus solution expressing OCT4 and SOX2 was used as the basis for the concentrated virus concentrate. AAV with an AAVDJ capsid that expresses SHH and FGF8 under the CAG promoter was purchased and customized from a vector company. The day after the left upper limb of a 4-5 week old BALB / cAJcl-nu / nu (from the Central Research Institute of Laboratory Animals) mouse was displaced dorsally by the surgical method of Example 13, a gently protruding, red skin structure was found on the skin surface of the forearm. The proximal end of the skin structure was set as the high cutting position, and AAVDJ-SHH 1.0×10 11 GC / 2μl, AAVDJ-FGF8 1.0×10 11 GC / 2μl. On the next day, 5μl of Conacton A (40mg / ml), a steroid, was mixed with 20μl of the retrovirus concentrate expressing FGF10, FGF20, FGF2, OCT4, and SOX2 and injected. The mixture was injected into the subcutaneous tissue, intramuscular tissue, and peri-bone tissue. At the same time, the forearm was cut at the predetermined cutting height, and the skin on the front and back of the forearm was pressed with fingers to close the wound. Then, until the 4th day after the cut, 10μl of the retrovirus concentrate was locally injected 20 times. Next, from the 5th day to the 10th day after the cut, 10μl of the retrovirus concentrate expressing OCT4 and SOX2 was locally injected 16 times. On the 6th, 13th, and 20th days after the cut, AAVDJ-SHH 1.0×10 11GC / 2μl, AAVDJ-FGF8 1.0×10 11 GC / 2μl. After amputation, photographs were taken every 7 days under a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.). About 8 weeks after amputation, a protrusion-like structure appeared at the front end, and then the front end continued to elongate in the shape of a branch. After taking photos on the 70th day after amputation, the tissue of the forearm was collected, and then fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for one night, and then embedded in OCT complex and frozen. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C (9), 2.5cm C-FP094), sections were collected every 200μm at a thickness of 10μm from the frozen tissue, stained with hematoxylin and eosin, and the resulting slides were read as image files using a slide scanner (VS200, Olympus). From the tissue observation, a cell structure running longitudinally along the axial direction in the elongated branch was confirmed.

[0251] Embodiment 24

[0252] Fig.28The results of the experiment were as follows: the left upper limb of the nude mouse was displaced dorsally, the forearm was cut horizontally, and the skin around the cut surface was pressed with fingers to perform gene introduction. The genes of FGF10, FGF20, FGF2, OCT4, SOX2, and TBX6 were introduced into the stump using retrovirus. The gene of SHH was introduced into the outside of the stump using AAVDJ, and the gene of FGF8 was introduced into the inside of the stump to the front part using AAVDJ. For retrovirus, the PMXs retrovirus backbone plasmid encoding each gene and the packaging plasmid (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the conventional method, and the cell supernatant after the culture medium was replaced was used as the retrovirus solution. For each animal, the first 20 local injections were made using 4 ml of retrovirus solution expressing OCT4, SOX2, and TBX6 and 8 ml of retrovirus solution concentrated by polyethylene glycol concentration for FGF10, FGF20, and FGF2. For the last 16 local injections, 8 ml of retrovirus solution expressing OCT4, SOX2, and TBX6 was used as the basis for the concentrated virus concentrate. AAV with an AAVDJ capsid expressing SHH and FGF8 under the CAG promoter was purchased and customized from a vector company. The day after the left upper limb of a 4-5 week old BALB / cAJcl-nu / nu (from the Central Research Institute of Laboratory Animals) mouse was displaced dorsally by the surgical method of Example 13, a gently protruding, red skin structure was found on the skin surface of the forearm. The proximal end of the skin structure was set as the high cutting position, and 1.0×10 AAVDJ-SHH was locally injected into the outer part of the forearm at the cutting line. 11 GC / 2μl, AAVDJ-FGF8 1.0×10 11 GC / 2μl. On the next day, 5μl of Conacton A (40mg / ml), a steroid, was mixed with 20μl of the retrovirus concentrate expressing FGF10, FGF20, FGF2, OCT4, SOX2, and TBX6 and injected. The mixture was injected into the subcutaneous tissue, intramuscular tissue, and peri-bone tissue. At the same time, the forearm was cut at the predetermined cutting height, and the skin on the front and back of the forearm was pressed with fingers to close the wound. Then, until the 4th day after the cut, 10μl of the retrovirus concentrate was locally injected 20 times. Then, from the 5th day to the 10th day after the cut, 10μl of the retrovirus concentrate expressing OCT4, SOX2, and TBX6 was locally injected 16 times. On the 6th, 13th, and 20th days after the cut, AAVDJ-SHH 1.0×1011 GC / 2μl, AAVDJ-FGF81.0×10 was injected locally into the inner part of the cut end 11 GC / 2μl. After amputation, photographs were taken every 7 days under a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.). About 8 weeks after amputation, a protrusion-like structure appeared at the front end, and then the front end continued to extend in the shape of a branch. After taking photos on the 98th day after amputation, the forearm tissue was collected, fixed with 4% paraformaldehyde and phosphate buffer (ph7.4) for one night, and then embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C(9), 2.5cm C-FP094), sections were collected every 200μm at a thickness of 10μm from the frozen tissue, stained with hematoxylin and eosin, and the resulting slides were read as image files using a slide scanner (VS200, Olympus). From the histological observation, a tissue structure composed of cartilage cells and similar to the distal phalanx of a finger running axially in the elongated branch part and a trabecular structure accompanied by bone regeneration were confirmed.

[0253] Embodiment 25

[0254] Fig.29The results of the gene introduction test were performed by displacing the left upper limb of the nude mouse to the dorsal side, cutting it horizontally at the forearm, and pressing the surrounding skin with fingers on the cut surface. The appearance of the stump and representative tissue findings, as well as magnified images are shown. The genes of FGF10, FGF20, FGF2, OCT4, SOX2, TBX6, and SHH were introduced into the stump using retrovirus. The gene of FGF8 was introduced into the front end of the stump using AAVDJ. For retrovirus, the PMXs retrovirus backbone plasmid encoding each gene and the packaging plasmid (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the conventional method, and the cell supernatant after replacing the culture medium was used as the retrovirus solution. For each animal, as the first half injection amount, for FGF10, FGF20, and FGF2, a virus concentrate concentrated by polyethylene glycol concentration was used based on 4 ml of retrovirus solution expressing OCT4, SOX2, and TBX6 and 8 ml of retrovirus solution. For SHH, a virus concentrate was prepared separately from other gene-expressing retroviruses based on 6 ml of retrovirus solution. AAV with an AAVDJ capsid expressing FGF8 under the CAG promoter was purchased and customized from a vector company. After the left upper limb of a 4-5 week-old BALB / cAJcl-nu / nu (from the Central Research Institute for Experimental Animals) mouse was displaced dorsally by the surgical method of Example 13, a skin structure with a gentle protrusion and redness was present on the skin surface of the forearm. The proximal end of the skin structure was set as the high cutting position, and AAVDJ-SHH 2.0×10 11GC / 6μl. The next day, 20μl of the retrovirus concentrate expressing FGF10, FGF20, FGF2, OCT4, SOX2, and TBX6 was injected into the subcutaneous and intramuscular tissues around the bone at the predetermined cutting line, and then the forearm was cut at the predetermined cutting height. The skin of the front and back of the forearm was pressed with fingers to close the wound. Immediately afterwards, 3μl of the retrovirus concentrate expressing SHH was injected into the outer subcutaneous tissue near the stump. On the same day, at 2 hours after cutting, 2μl of Corning A (40mg / ml) was mixed with 8μl of the retrovirus solution expressing FGF10, FGF20, FGF2, OCT4, SOX2, and TBX6 for local injection. From the second day of cutting to the fifth day, 10μl of the retrovirus solution expressing FGF10, FGF20, FGF2, OCT4, SOX2, and TBX6 was locally injected 16 times. Regarding the retrovirus expressing SHH, 3 μl of the virus solution was injected subcutaneously three times outside the stump from the day after amputation to the day after amputation. From the day after amputation to the day after amputation, 8 μl of the retrovirus concentrate expressing OCT4, SOX2, and TBX6 was injected locally seven times. On the day after amputation, 1.0×10 AAVDJ-FGF8 was injected locally at the distal end of the amputation on the day after amputation, 13 days, and 20 days after amputation. 11 GC / 2.5μl. After amputation, photographs were taken under observation under a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.) every 7 days (no record was made on the 21st day after amputation). After taking photographs on the 56th day after amputation, the tissue of the forearm was collected, fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for one night, and then embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C (9), 2.5cm C-FP094), sections were collected every 200μm at a thickness of 10μm from the frozen tissue, stained with hematoxylin and eosin, and the resulting slides were read as image files using a slide scanner (VS200, Olympus). In terms of tissue observation, a bag-like structure was formed at the distal end of the bone stump, and new muscle bundles independent of the cartilage tissue and surrounding muscles were confirmed inside.

[0255] Embodiment 26

[0256] Fig.30The results of the experiment were that the left upper limb of the nude mouse was displaced dorsally, and gene introduction based on FGF2 injection and virus solution was performed at the predetermined cutting site. The forearm was cut horizontally, and the cut surface was set as an open wound. Gene introduction was also performed after cutting. HMGB3, DMNT, 1HDAC2, PRDX2, and FGF10 were introduced into the stump using retrovirus. SHH gene introduction was performed on the outside of the stump using AAVDJ. For retrovirus, PMXs retrovirus backbone plasmids encoding each gene and packaging plasmids (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to conventional methods, and the cell supernatant after the culture medium was replaced was used as the retrovirus solution. For each animal, a virus concentrate concentrated by polyethylene glycol concentration based on 6 ml of the retrovirus solution was used. Regarding AAV, an AAV with an AAVDJ capsid expressing SHH under the CAG promoter was prepared by the concentrated AAV production method described in Example 2 and used. As an FGF2 solution, a solution (500 μg / ml) prepared by dissolving Fiblast spray preparation (Kaiken Pharmaceutical Co., Ltd.) in PBS was used. On the 7th day of the dorsal displacement of the left upper limb of a 4-5-week-old BALB / cAJcl-nu / nu (from the Central Research Institute of Experimental Animals) mouse by the surgical method of Example 13, there was a skin structure with a gentle protrusion and redness on the skin surface of the forearm, and the proximal end of the skin structure was set as the high position of the cut. 30 μl of the retrovirus concentrate expressing HMGB3, DMNT, 1HDAC2, PRDX2, and FGF10 was locally injected into the predetermined cutting site. After 6 hours, 25 μl of FGF2 solution was locally injected. One day later and two days later, the retrovirus and FGF2 solution were also locally injected into the predetermined cutting site at intervals. Three days later, 30 μl of retroviral concentrate was injected locally, and then the wound was cut transversely to form an open wound. AAVDJ-SHH 5.0×10 11GC. On the first and second days after amputation, 2 μl of the retrovirus concentrate was applied to the cut section. After amputation, photographs were taken under observation under a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.) every 7 days. Starting from about 5 weeks after amputation, the front end began to protrude and elongate over time. After taking photographs on the 63rd day after amputation, the forearm tissue was collected and then fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for one night, and then embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C (9), 2.5 cm C-FP094), sections were collected every 200 μm at a thickness of 10 μm from the frozen tissue, stained with hematoxylin and eosin, and the resulting slides were read as image files using a slide scanner (VS200, Olympus). From the tissue observation, it was confirmed that cartilage tissue with endochondral ossification-like tissue images similar to intra-articular cartilage was induced at the bone stump.

[0257] Embodiment 27

[0258] Fig.31The results of the experiment were that the left upper limb of the nude mouse was displaced dorsally, and gene introduction based on FGF2 injection and virus solution was performed at the predetermined cutting site. The forearm was cut horizontally, and the cut surface was set as an open wound. The gene introduction was also performed after cutting. The FGF10 gene was introduced into the stump using retrovirus. The SHH gene was introduced outside the stump using AAVDJ. For retrovirus, PMXs retrovirus backbone plasmids encoding each gene and packaging plasmids (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to conventional methods, and the cell supernatant after the culture medium was replaced was used as the retrovirus solution. For each animal, 80 μl of the virus concentrate concentrated by polyethylene glycol concentration based on 6 ml of the retrovirus solution was used. Regarding AAV, a virus with an AAVDJ1 capsid expressing SHH under the CAG promoter was produced by the concentrated AAV production method described in Example 2 and used. As an FGF2 solution, a solution (500 μg / ml) prepared by dissolving Fiblast spray preparation (Kaiken Pharmaceutical Co., Ltd.) in PBS was used. On the 10th day after the left upper limb of a 4-5 week-old BALB / cAJcl-nu / nu (from the Central Research Institute of Experimental Animals) mouse was displaced dorsally by the surgical method of Example 13, there was a skin structure with a gentle protrusion and redness on the skin surface of the forearm, and the proximal end of the skin structure was set as the high position of the cut. After injecting 20 μl of retrovirus concentrate from 4 days before to 2 days before the cut, 20 μl of FGF2 solution was locally injected 3-5 hours later. When cutting, the nerve stump was kept longer and cut horizontally, and after removing the skin around the stump, 20 μl of retrovirus concentrate was injected. AAVDJ1-SHH was injected locally at the lateral aspect on the 7th day after resection. 11 GC. After amputation, photographs were taken under observation under a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.) every 7 days. Starting from about 2 weeks after amputation, the front end began to protrude over time. After taking photographs on the 63rd day after amputation, the forearm tissue was collected, fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for one night, and then embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilm type2C (9), 2.5 cm C-FP094), sections were collected from the frozen tissue at a thickness of 10 μm every 200 μm, stained with hematoxylin and eosin, and the resulting slides were read as image files using a slide scanner (VS200, Olympus). From the tissue observation, it was confirmed that cartilage tissue was continuously induced from the muscular layer near the stump.

[0259] Embodiment 28

[0260] Fig.32 The results of the experiment were that the left upper limb of the nude mouse was displaced dorsally, and gene introduction based on FGF2 injection and virus solution was performed at the predetermined cutting site. The forearm was cut horizontally, and the cut surface was set as an open wound. Gene introduction was also performed after cutting. HMGB3, DMNT1, HDAC2, and FGF10 genes were introduced into the stump using retrovirus. SHH gene introduction was performed on the outside of the stump using AAVDJ. For retrovirus, PMXs retrovirus backbone plasmids encoding each gene and packaging plasmids (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to conventional methods, and the cell supernatant after the culture medium was replaced was used as the retrovirus solution. For each animal, 80 μl of the virus concentrate concentrated by polyethylene glycol concentration based on 6 ml of the retrovirus solution was used. Regarding AAV, a virus with an AAVDJ1 capsid expressing SHH under the CAG promoter was produced by the concentrated AAV production method described in Example 2 and used. As an FGF2 solution, a solution (500 μg / ml) prepared by dissolving Fiblast spray preparation (Kenichi Pharmaceutical Co., Ltd.) in PBS was used. On the 10th day after the left upper limb of a 4-5 week-old BALB / cAJcl-nu / nu (from the Central Research Institute of Experimental Animals) mouse was displaced dorsally by the surgical method of Example 13, there was a skin structure with a gentle protrusion and redness on the skin surface of the forearm, and the proximal end of the skin structure was set as the high position of the cut. After injecting 20 μl of retrovirus concentrate from 4 days before to 2 days before the cut, 20 μl of FGF2 solution was locally injected 3-5 hours later. When cutting, the nerve stump was retained for a long time and cut horizontally. After removing the skin around the stump, 20 μl of retrovirus concentrate was injected. AAVDJ1-SHH was injected locally at the lateral aspect on the 7th day after resection. 11GC. After amputation, photographs were taken under observation under a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.) every 7 days. Starting from about 2 weeks after amputation, the front end began to protrude over time. After taking photographs on the 63rd day after amputation, the forearm tissue was collected, fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for one night, and then embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C (9), 2.5 cm C-FP094), sections were collected from the frozen tissue at a thickness of 10 μm every 200 μm, stained with hematoxylin and eosin, and the resulting slides were read as image files using a slide scanner (VS200, Olympus). From the tissue observation, it was confirmed that cartilage tissue was continuously induced from the muscular layer near the stump, and the cartilage-like tissue covered the bone stump.

[0261] Embodiment 29

[0262] Fig.33The results of the experiment were that the left upper limb of the nude mouse was displaced dorsally, and gene introduction based on FGF2 injection and virus solution injection was performed at the predetermined cutting site. The forearm was cut horizontally, and the cut surface was set as an open wound. Gene introduction was also performed after cutting. MSX1, MSX2, LIN28A, MEIS1, and FGF10 were introduced into the stump using retrovirus. SHH gene introduction was performed on the outside of the stump using AAVDJ1. LEF1 and FGF8 gene introduction was performed in the front part using AAVDJK2. For retrovirus, PMXs retrovirus backbone plasmids encoding each gene and packaging plasmids (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the conventional method, and the cell supernatant after the culture medium was replaced was used as the retrovirus solution. For each animal, 8 ml of retrovirus solution was used for MSX1, MSX2, and LIN28A, and 4 ml of retrovirus solution was used for MEIS1 and FGF10, respectively. Virus concentrates were prepared twice by polyethylene glycol concentration and used. Regarding AAV, viruses with AAVDJ1 capsid expressing SHH under the CAG promoter and AAVDJK2 capsid expressing LEF1 or FGF8 under the K14 promoter were prepared by the concentrated AAV preparation method described in Example 2 and used. As the FGF2 solution, a solution (500 μg / ml) prepared by dissolving Fiblast spray preparation (Kanagaku Seiyaku Co., Ltd.) in PBS was used. On the 5th day (8 days before amputation) when the left upper limb of a 4-5 week old BALB / cAJcl-nu / nu (from the Central Research Institute for Experimental Animals) mouse was displaced dorsally by the surgical method of Example 13, a gently protruding and red skin structure was found on the skin surface of the forearm, and the proximal end of the skin structure was set as the high position for amputation. After injecting 20 μl of retrovirus concentrate 8 days to 1 day before amputation, 20 μl of FGF2 solution was locally injected 3-5 hours later. When amputating, the nerve stump was retained longer and cut horizontally, and after removing the skin around the stump, 20 μl of retrovirus concentrate was injected. 11 days after amputation, SHH 5×10 11 GC / 4μl for gene introduction. 14 days after the cut, AAVDJK2 was used to introduce 5×10 11A total of 4 μl of LEF1 and FGF8 of GC were introduced for gene introduction. After amputation, photographs were taken every 7 days under observation with a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.). Starting from about 2 weeks after amputation, the front end began to protrude over time. After taking photos on the 56th day after amputation, the tissue of the forearm was collected, and then fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for 1 night, and then embedded in OCT complex and frozen. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C (9), 2.5 cm C-FP094), sections were collected from the frozen tissue at a thickness of 10 μm every 200 μm, stained with hematoxylin and eosin, and the resulting slides were read as image files using a slide scanner (VS200, Olympus). Histologically, it was confirmed that axial extension of the bone stump was induced, and cartilage tissue with endochondral ossification-like tissue images similar to intra-articular cartilage was induced at the bone stump.

[0263] Embodiment 30

[0264] Fig.34The results of the experiment were that the left upper limb of the nude mouse was displaced dorsally, and gene introduction based on FGF2 injection and virus solution injection was performed at the predetermined cutting site. The forearm was cut horizontally, and the cut surface was set as an open wound. Gene introduction was also performed after cutting. MSX1, MSX2, LIN28A, WNT7A, and FGF10 were introduced into the stump using retrovirus. One day before cutting, SHH gene introduction was performed on the outside of the stump using AAVDJ1, and LEF1 gene introduction was performed subcutaneously using AAVDJK2. For retrovirus, PMXs retrovirus backbone plasmids encoding each gene and packaging plasmids (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the conventional method, and the cell supernatant after the culture medium was replaced was used as the retrovirus solution. For each animal, 6 ml of retrovirus solution was used as the basis for MSX1, MSX2, and LIN28A, and 8 ml of retrovirus solution was used as the basis for WNT7A and FGF10, respectively. Virus concentrates concentrated by polyethylene glycol concentration were prepared and used. Regarding AAV, viruses with AAVDJ1 capsid expressing SHH under the CAG promoter and viruses with AAVDJK2 capsid expressing LEF1 under the K16P5 promoter were prepared and used by the concentrated AAV preparation method described in Example 2. As an FGF2 solution, a solution (500 μg / ml) obtained by dissolving Fiblast spray preparation (Kenichi Pharmaceutical Co., Ltd.) in PBS was used. On the 4th day after the left upper limb of a 4-5 week-old BALB / cAJcl-nu / nu (from the Central Research Institute of Experimental Animals) mouse was displaced dorsally by the surgical method of Example 13, there was a skin structure with redness accompanied by a gentle protrusion on the skin surface of the forearm, and the proximal end of the skin structure was set as the cutting high position. 20 μl of retrovirus concentrate was injected 5 to 2 days before the cut, and 20 μl of FGF2 solution was injected locally 3-5 hours later. 3.3×10 viral solution expressing SHH under CAG promoter was injected into the skeletal muscle of the rat using AAV DJ1 one day before the cut. 10 After GC / 4μl was injected into the outside, 1.6×10 11GC / 10μl was injected subcutaneously all over the body. 20μl of FGF2 solution was injected locally 12 hours later. Before cutting, 20μl of retroviral solution was injected into the planned cutting site, and then a transverse cut was made to remove the skin around the stump. 4μl of retroviral solution was injected into the front end the day after cutting. Pictures were taken every 7 days after cutting under a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.). Starting from about 2 weeks after cutting, the front end began to protrude over time. After taking pictures on the 56th day after cutting, the forearm tissue was collected, fixed with 4% paraformaldehyde and phosphate buffer (ph7.4) for one night, and then embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C(9), 2.5 cm C-FP094), sections were collected from frozen tissue at a thickness of 10 μm every 200 μm, stained with hematoxylin and eosin, and the obtained slides were read as image files using a slide scanner (VS200, Olympus). Histologically, it was confirmed that the thickness of the subcutaneous muscle tissue around the cut end increased and proliferated.

[0265] Embodiment 31

[0266] Fig.35The results of the experiment were that the left upper limb of the nude mouse was displaced dorsally, and gene introduction based on FGF2 injection and virus solution injection was performed at the predetermined cutting site. The forearm was cut horizontally, and the cut surface was set as an open wound. Gene introduction was also performed after cutting. LEF1, HDAC2, PRDX2, WNT7A, and FGF10 were introduced into the stump using retrovirus. One day before cutting, SHH gene introduction was performed on the outside of the stump using AAVDJ1, and LEF1 gene introduction was performed subcutaneously using AAVDJK2. For retrovirus, PMXs retrovirus backbone plasmids encoding each gene and packaging plasmids (pCMV-gagpol-PA, pCMV-VSVg) were transfected into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to conventional methods, and the cell supernatant after replacing the culture medium was used as the retrovirus solution. For each animal, 6 ml of retrovirus solution was used as the basis for LEF1, HDAC2, and PRDX2, and 8 ml of retrovirus solution was used as the basis for WNT7A and FGF10, respectively. Virus concentrates concentrated by polyethylene glycol concentration were prepared and used. Regarding AAV, viruses with AAVDJ1 capsids expressing SHH under the CAG promoter and viruses with AAVDJK2 capsids expressing LEF1 under the K16P5 promoter were prepared and used by the concentrated AAV preparation method described in Example 2. As an FGF2 solution, a solution (500 μg / ml) obtained by dissolving Fiblast spray preparation (Kenichi Pharmaceutical Co., Ltd.) in PBS was used. On the 4th day after the left upper limb of a 4-5 week-old BALB / cAJcl-nu / nu (from the Central Research Institute of Experimental Animals) mouse was displaced dorsally by the surgical method of Example 13, there was a skin structure with redness accompanied by a gentle protrusion on the skin surface of the forearm, and the proximal end of the skin structure was set as the cutting high position. 20 μl of retrovirus concentrate was injected 5 to 2 days before the cut, and 20 μl of FGF2 solution was injected locally 3 to 5 hours later. 3.3×10 viral solution expressing SHH under CAG promoter was injected into the scalp of the scalp using AAV DJ1 one day before the cut. 10 After GC / 4μl was injected into the outside, 1.6×10 11GC / 10μl was injected subcutaneously all over the body. 20μl of FGF2 solution was injected locally 12 hours later. Before cutting, 20μl of retroviral solution was injected into the planned cutting site, and then a transverse cut was made to remove the skin around the stump. 4μl of retroviral solution was injected into the front end the day after cutting. Pictures were taken every 7 days after cutting under a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.). Starting from about 2 weeks after cutting, the front end began to protrude over time. After taking pictures on the 56th day after cutting, the forearm tissue was collected, fixed with 4% paraformaldehyde and phosphate buffer (ph7.4) for one night, and then embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilm type 2C(9), 2.5 cm C-FP094), sections were collected from frozen tissue at a thickness of 10 μm every 200 μm, stained with hematoxylin and eosin, and the obtained slides were read as image files using a slide scanner (VS200, Olympus). Histologically, it was confirmed that the thickness of the subcutaneous muscle tissue around the cut end increased and proliferated.

[0267] Embodiment 32

[0268] Fig.36 The results of the experiment are as follows: after the left upper limb of a nude mouse was displaced dorsally, the forearm was transversely cut, the cut surface was set as an open wound, and gene introduction was performed even after cutting. AAVDJ was used to introduce LEF1, PRRX1, HDAC2, PRDX2, PPARD, FGF10, FGF20, FGF2, BMP5, and SHH genes into the stump. Regarding AAV, a virus with an AAVDJ capsid expressing LEF1, PRRX1, HDAC2, PRDX2, PARD, FGF10, FGF20, FGF2, BMP5, and SHH under the CAG promoter was prepared by the concentrated AAV production method described in Example 2 and used. On the second day after the left upper limb of a 4-5 week-old BALB / cAJcl-nu / nu (from the Central Research Institute for Experimental Animals) mouse was displaced dorsally by the surgical method of Example 13, there was a skin structure with gentle protrusion and redness on the skin surface of the forearm, and the proximal end of the skin structure was set as the cutting high position. After transverse cutting, the cut surface was set as an open wound. From cutting to the second day, 5×10 10 GC AAV expressing PRRX1, HDAC2, PRDX2, PPARD, FGF20, FGF2, SHH, containing 5×10 9 GC AAV expressing FGF10, containing 10 1125 μl of the virus solution of AAV expressing BMP5 in the GC was injected into the front part 10 times. After the amputation, photos were taken under observation under a surgical microscope (MM100-YOH manufactured by Mitaka Optical Instruments Co., Ltd.) every 7 days. From about 2 weeks after the amputation, the front end began to increase in size with roundness over time. After taking photos on the 70th day after the amputation, the tissue of the forearm was collected, and then fixed with 4% paraformaldehyde and phosphate buffer (pH 7.4) for 1 night, and then embedded in OCT complex for freezing. Using Kawamoto slides (SECTION-LAB, Cryofilmtype 2C (9), 2.5 cm C-FP094), sections were collected every 200 μm at a thickness of 10 μm from the frozen tissue, stained with hematoxylin and eosin, and the obtained slides were read as image files using a slide scanner (VS200, Olympus). Histologically, the proliferation of adipose tissue in the muscle tissue from subcutaneous to deep fascia was confirmed.

[0269] In the present invention, as shown in Example 18, Example 19, Example 20, Example 21, Example 22, Example 23, Example 24, Example 25, Example 26, Example 27, Example 28, Example 29, Example 30, Example 31, and Example 32, the surgical operation described in Example 13 and Fig.21 The method for treating severed stumps illustrated in the figure provides a research and development platform for developing methods for regenerating severed limbs in mammals.

[0270] In addition, in the present invention, by performing gene introduction into the severed stump of genes expressed in the fetal period of mammals and candidate genes obtained by analyzing the genes expressed in the regeneration period of amphibians (Table 3), protein administration, steroid administration and other interventions, the following tissue regeneration related to the regeneration of branched finger-like morphology and the regeneration of functional limbs including neuromuscular tissue can be obtained in the stump that is usually healed by contraction and closure of the cortical bone and surrounding skin and no significant tissue regeneration is confirmed (Example 18): the formation of a blastema-like soft tissue mass confirmed in the severed limb stump of amphibians that physiologically have a higher tissue regeneration ability (Example 19), the formation of a blastema-like soft tissue mass and the regeneration of the limb from the endogenous tissue (Example 20). The invention discloses the formation of bone tissue with rich trabecular structure with the front end being raised (Example 20), the formation of branched structure caused by bone marrow cells after the shape of the front end is changed to flat (Example 21), the elongation of the front end and the axially extending cell structure in the elongated tissue (Example 22), the elongation of the front end while branching and the axially overlapping cell structure in the elongated tissue (Example 23), the elongation of the front end in a branched shape and the axially extending trabecular structure with bone regeneration composed of cartilage cells in the shape similar to the distal phalanx of the finger in the branched elongated tissue (Example 24), and the bag-like structure with cartilage structure and independent muscle bundle regeneration inside (Example 25). The invention can be applied as a therapeutic method for the purpose of inducing regeneration of defective limbs and a research and development method for the purpose of therapeutic development.

[0271] In addition, in the present invention, the following significant regeneration of cartilage tissue can be obtained: protrusion and elongation of the front end portion accompanied by endochondral ossification-like tissue images similar to cartilage in the joint (Example 26), induction of cartilage tissue having continuity from the skin muscle layer near the stump (Example 27), a covering structure of cartilage-like tissue at the stump of the bone (Example 28), axial extension of the stump of the bone, induction of cartilage tissue accompanied by endochondral ossification-like tissue images similar to cartilage in the stump of the bone (Example 29), etc. Since a treatment method for degeneration of articular cartilage caused by aging, osteoarthritis, etc., induction of endochondral ossification at the stump of the bone, and elongation of the part can be obtained, it can be applied as a treatment method for lengthening limb bones, as well as a research and development method for the purpose of future treatment development and treatment development.

[0272] In addition, in the present invention, the following muscle tissue and fat tissue regeneration can be obtained: proliferation of subcutaneous muscle tissue around the cut end (Example 30, Example 31), proliferation of fat tissue in the muscle tissue from the cut end to the deep fascia (Example 32), etc. It can be applied as a treatment method for cosmetic changes / muscle strength reduction caused by thinning of subcutaneous fat tissue with aging, cosmetic changes caused by various reasons such as trauma and malignant tumor removal, muscle strength changes in paralytic diseases, etc., and a research and development method for the purpose of future treatment development and for the purpose of treatment development.

Claims

1. A non-naturally altered adeno-associated virus AAV capsid protein, which comprises a peptide insertion compared to the corresponding parent AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence EPKARAP, i.e., sequence number 2, and the insertion site is between amino acid residues 589 and 590 of VP1 of AAVDJ, or the corresponding position of the capsid protein of other AAV serotypes.

2. A non-naturally modified AAV capsid protein having as its amino acid sequence the amino acid sequence recorded in sequence number 5, sequence number 14, sequence number 15, sequence number 16 or sequence number 17, which contains a peptide insertion compared to the parent adeno-associated virus AAV capsid protein, i.e., AAVDJ.

3. A polynucleotide comprising a base sequence encoding the altered AAV capsid protein according to claim 1 or 2.

4. An expression vector comprising the polynucleotide of claim 3, wherein the base sequence encoding the altered AAV capsid protein in the polynucleotide is operably linked to a promoter sequence.

5. A recombinant virus or viral vector comprising the altered AAV capsid protein according to claim 1 or 2.

6. The recombinant virus or viral vector according to claim 5, which is AAV.

7. The recombinant virus or viral vector according to claim 5 or 6, which comprises a base sequence encoding a therapeutic gene product.

8. The recombinant virus or viral vector according to any one of claims 5 to 7, which has higher infectivity to cells on the skin ulcer surface or epidermal cells.

9. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the recombinant virus or viral vector according to any one of claims 5 to 8.

10. A method for treating or preventing a skin disease, a skin disorder or a decrease in skin function in a subject in need of such treatment or prevention, wherein the method include: The pharmaceutical composition of claim 9 is administered to the subject by intradermal injection, subcutaneous injection, or parenteral administration, optionally intravenously.

11. A promoter comprising a base sequence as recorded in sequence number 23 or sequence number 25, or a base sequence having at least 95% or at least 99% identity with the base sequence as recorded in sequence number 23 or sequence number 25, so that a target gene is expressed tissue / cell-specifically in mammalian cells.

12. A promoter comprising the base sequence recorded in SEQ ID NO: 26, or a base sequence having at least 95% or at least 99% identity with the base sequence recorded in SEQ ID NO: 26, which enables tissue / cell-specific expression of a target gene in mammalian cells. A recombinant vector comprising the promoter according to claim 11 or 12.

14. A recombinant vector comprising the promoter according to claim 11 or 12 and a base sequence encoding a therapeutic gene product.

15. The recombinant vector according to claim 13 or 14, which is AAV. 16 . The recombinant vector according to claim 13 , which has high gene expression in epidermal cells and epidermal tissues. 17 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the recombinant vector according to any one of claims 13 to 16.

18. A method for treating or preventing a skin disease, a skin disorder or a decrease in skin function in a subject in need of such treatment or prevention, wherein the method include: The pharmaceutical composition of claim 17 is administered to the subject by intradermal injection, subcutaneous injection, or parenteral administration, optionally intravenously.

19. A gene introduction vector, which induces skin tissue having skin appendages from a skin ulcer surface in a living body by introducing a gene encoding at least any one of DNP63A gene, GRHL2 gene, TFAP2A gene, cMYC gene, LEF1 gene, SOX2 gene, HOXC4 gene, HOXC9 gene, HOXC13 gene, JARID2 gene, HEY1 gene, HEY2 gene, FOXO1 gene, FOXD1 gene, EGR3 gene, MEF2C gene, LHX2 gene, PRRX1 gene, PRRX2 gene, CREB3 gene, ETV1 gene, TBX6 gene, MSX2 gene, PRDM1 gene, and SHH gene as a gene conferring skin appendage inducing ability.

20. A gene introduction vector, which induces skin tissue with skin appendages from a skin ulcer surface in a living body by introducing a gene encoding at least any one of the DNP63A gene, GRHL2 gene, TFAP2A gene, cMYC gene, LEF1 gene, FOXD1 gene, PRDM1 gene, and SHH gene as a gene that confers skin appendage inducing ability.

21. A gene introduction vector, which introduces genes encoding all or part of the DNP63A gene, GRHL2 gene, TFAP2A gene, cMYC gene, LEF1 gene, FOXD1 gene, PRDM1 gene, and SHH gene as genes that confer skin appendage inducing ability, thereby inducing the regeneration of skin appendages and improving the function of skin appendages in vivo.

22. A gene introduction vector, which introduces genes encoding all or part of the LEF1 gene, FOXD1 gene, PRDM1 gene, and SHH gene as genes conferring skin appendage induction ability, thereby inducing the regeneration of skin appendages and improving the function of skin appendages in vivo.

23. A gene transfer vector for inducing the regeneration of skin appendages and improving the function of skin appendages in vivo by transferring a gene encoding both or one of the FOXD1 gene and the PRDM1 gene as a gene conferring skin appendage induction ability. 24 . The gene transfer vector according to claim 19 , wherein the gene transfer vector is AAV or mRNA.

25. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the gene transfer vector according to any one of claims 19 to 24.

26. A method for treating or preventing a skin disease, a skin disorder or a decrease in skin function in a subject in need of such treatment or prevention, wherein the method include: The pharmaceutical composition of claim 25 is administered to the subject by spreading on the ulcer surface, intradermal injection or subcutaneous injection.

27. A pharmaceutical for gene transfer, comprising a gene transfer vector and a steroid in an amount effective for improving gene transfer efficiency or gene expression.

28. The pharmaceutical for gene transfer according to claim 27, wherein the steroid acts systemically or locally on the individual into which the gene is transferred.

29. The gene-transfer pharmaceutical according to claim 27 or 28, wherein the gene-transfer vector is a viral vector, AAV, a retroviral vector, mRNA treated with a liposome transfection agent, mRNA formulated in liposomes / lipid nanoparticles, or naked DNA. 30 . The gene transfer pharmaceutical according to claim 27 , wherein the gene transfer vector is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

31. A method for improving gene transfer efficiency. It is characterized in that In gene transfer into a living body, steroids are allowed to act systemically on the individual before gene transfer.

32. A method for treating or preventing a skin disease, skin disorder or reduced skin function in a subject in need of such treatment or prevention, wherein the method include: The steroid is allowed to act systemically or locally on the subject in advance, simultaneously or early after the introduction, and the gene transfer vector is administered to the subject by spreading on the ulcer surface, intradermal injection or subcutaneous injection.

33. A method for treating or preventing a muscle disease, a muscle disorder or a decrease in muscle function in a subject in need of such treatment or prevention, wherein the method include: The steroid is allowed to act systemically or locally on the subject in advance, simultaneously or early after the introduction, and the gene transfer vector is administered to the subject by intramuscular injection.

34. A method for treating or preventing osteochondral disease, osteochondral disorder or osteochondral function reduction in a subject in need of such treatment or prevention, the method comprising: include: The steroid is allowed to act systemically or locally on the subject in advance, simultaneously or early after the introduction, and the gene transfer vector is administered to the subject by local injection.

35. A method for making an upper limb amputation model animal, It is characterized in that After the upper limb of the non-human mammal is displaced, the upper limb is amputated at the upper arm / forearm / hand / finger plane.

36. A method for making an upper limb amputation model animal, It is characterized in that After the upper limb of the non-human mammal is displaced, the upper limb is amputated at the forearm plane.

37. The method of claim 35 or 36, wherein the non-human mammal is a rabbit, dog, cat, guinea pig, hamster, rat or mouse.

38. The method according to any one of claims 35 to 37, wherein the upper limb displacement is performed while maintaining the main trunk nerves, arteries and veins of the upper limb.

39. The method according to any one of claims 35 to 38, wherein the upper limb displacement is performed at the shoulder joint.

40. The method according to any one of claims 35 to 39, wherein the upper limb displacement is accompanied by resection of the scapula.

41. An upper limb amputation model animal produced by the method for producing an upper limb amputation model animal according to any one of claims 35 to 40.

42. A method for screening a pharmaceutical substance, It is characterized in that The test substance is applied to the upper limb amputation model animal as claimed in claim 41 to study the effect on the amputation site.

43. A method for screening a pharmaceutical substance, wherein the pharmaceutical substance according to claim 42 is a gene transfer vector for the purpose of gene transfer.

44. A method for screening a medicinal substance, wherein the medicinal substance according to claim 42 or 43 is a substance for regeneration of severed tissue.

45. A research and development reagent or pharmaceutical product, the research and development reagent being used to stimulate the regeneration of a missing limb or stimulating the regeneration of a missing tissue of a limb in a mammal, the pharmaceutical product being used as a medicament, the research and development reagent or pharmaceutical product comprising a vector for introducing or causing one or more of the following (1) to (3) to act, or the following (4), (1) at least one of the genes that is expressed relatively strongly in cells having tissue regeneration ability, (2) at least one gene that functions as a master regulatory gene in the differentiation / production process of cells, (3) at least one of the cytokine genes that exerts a strong effect in the environment where tissue regeneration occurs, (4) At least one of the proteins encoded by the genes included in (1) to (3) above.

46. ​​The research and development reagent or pharmaceutical according to claim 45, wherein the gene is a gene encoding FGF10, FGF20, FGF2, FGF8, SHH and OCT4, or the protein is FGF10, FGF20, FGF2, FGF8, SHH and OCT4.

47. The research and development reagent or pharmaceutical according to claim 45, wherein the gene is a gene encoding FGF10, FGF20, FGF2, FGF8, SHH, OCT4 and SOX2, or the protein is FGF10, FGF20, FGF2, FGF8, SHH, OCT4 and SOX2.

48. The research and development reagent or pharmaceutical according to claim 45, wherein the gene is a gene encoding FGF10, FGF20, FGF2, FGF8, SHH, OCT4, SOX2 and TBX6, or the protein is FGF10, FGF20, FGF2, FGF8, SHH, OCT4, SOX2 and TBX6.

49. The research and development reagent or pharmaceutical according to claim 45, wherein the gene comprises a gene encoding at least one selected from FGF10, FGF20, FGF2, FGF8, SHH, OCT4, SOX2 and TBX6, or the protein comprises at least one selected from FGF10, FGF20, FGF2, FGF8, SHH, OCT4, SOX2 and TBX6.

50. The research and development reagent or pharmaceutical according to claim 45, wherein the gene is a gene encoding FGF10, FGF20, FGF2, FGF8, SHH, LEF1, NFIB, PPARD, POLE4, PRRX1 and HDAC2, or the protein is FGF10, FGF20, FGF2, FGF8, SHH, LEF1, NFIB, PPARD, POLE4, PRRX1 and HDAC2.

51. The research and development reagent or pharmaceutical according to claim 45, wherein the gene comprises a gene encoding at least one selected from FGF10, FGF20, FGF2, FGF8, SHH, LEF1, NFIB, PPARD, POLE4, PRRX1 and HDAC2, or the protein comprises at least one selected from FGF10, FGF20, FGF2, FGF8, SHH, LEF1, NFIB, PPARD, POLE4, PRRX1 and HDAC2.

52. The research and development reagent or pharmaceutical according to claim 45, wherein the gene is a gene encoding FGF10, FGF20, FGF2, FGF8, SHH, LEF1, NFIB, PPARD, POLE4, PRRX1, HDAC2 and BMP5, or the protein is FGF10, FGF20, FGF2, FGF8, SHH, LEF1, NFIB, PPARD, POLE4, PRRX1, HDAC2 and BMP5.

53. The research and development reagent or pharmaceutical according to claim 45, wherein the gene comprises a gene encoding at least one selected from FGF10, FGF20, FGF2, FGF8, SHH, LEF1, NFIB, PPARD, POLE4, PRRX1, HDAC2 and BMP5, or the protein comprises at least one selected from FGF10, FGF20, FGF2, FGF8, SHH, LEF1, NFIB, PPARD, POLE4, PRRX1, HDAC2 and BMP5.

54. The research and development reagent or pharmaceutical according to claim 45, wherein the gene is a gene encoding FGF10, FGF20, FGF2, FGF8, SHH, NFIB, PPARD and POLE4, or the protein is FGF10, FGF20, FGF2, FGF8, SHH, NFIB, PPARD and POLE4.

55. The research and development reagent or pharmaceutical according to claim 45, wherein the gene comprises a gene encoding at least one selected from FGF10, FGF20, FGF2, FGF8, SHH, NFIB, PPARD and POLE4, or the protein comprises at least one selected from FGF10, FGF20, FGF2, FGF8, SHH, NFIB, PPARD and POLE4.

56. A research and development reagent or pharmaceutical product for use as a medicine for stimulating the regeneration of cartilage tissue in a mammal, the research and development reagent or pharmaceutical product comprising a carrier for introducing or causing one or more of the following (1) to (3) to act, or the following (4), (1) at least one of the genes that is expressed relatively strongly in cells having tissue regeneration ability, (2) at least one gene that functions as a master regulatory gene in the differentiation / production process of cells, (3) at least one of the cytokine genes that exerts a strong effect in the environment where tissue regeneration occurs, (4) At least one of the proteins encoded by the genes included in (1) to (3) above.

57. The research and development reagent or pharmaceutical according to claim 56, wherein the gene is a gene encoding FGF10, FGF2, SHH, HMGB3, DMNT1, HDAC2 and PRDX2, or the protein is FGF10, FGF2, SHH, HMGB3, DMNT1, HDAC2 and PRDX2.

58. The research and development reagent or pharmaceutical according to claim 56, wherein the gene is a gene encoding FGF10, FGF2, SHH, HMGB3, DMNT1 and HDAC2, or the protein is FGF10, FGF2, SHH, HMGB3, DMNT1 and HDAC2.

59. The research and development reagent or pharmaceutical according to claim 56, wherein the gene comprises a gene encoding at least one selected from FGF10, FGF2, SHH, HMGB3, DMNT1, HDAC2 and PRDX2, and the protein comprises at least one selected from FGF10, FGF2, SHH, HMGB3, DMNT1, HDAC2 and PRDX2.

60. The research and development reagent or pharmaceutical according to claim 56, wherein the gene comprises a gene encoding at least one selected from FGF10, FGF2, SHH, HMGB3, DMNT1 and HDAC2, or the protein comprises at least one selected from FGF10, FGF2, SHH, HMGB3, DMNT1 and HDAC2.

61. The research and development reagent or pharmaceutical according to claim 56, wherein the gene is a gene encoding FGF10, FGF2, FGF8, LEF1, SHH, MSX1, MSX2, LIN28A and MEIS1, or the protein is FGF10, FGF2, FGF8, LEF1, SHH, MSX1, MSX2, LIN28A and MEIS1.

62. The research and development reagent or pharmaceutical according to claim 56, wherein the gene comprises at least one gene encoding at least one selected from FGF10, FGF2, FGF8, LEF1, SHH, MSX1, MSX2, LIN28A and MEIS1, or the protein comprises at least one selected from FGF10, FGF2, FGF8, LEF1, SHH, MSX1, MSX2, LIN28A and MEIS1.

63. The research and development reagent or pharmaceutical according to any one of claims 57 to 62, wherein the pharmaceutical is a pharmaceutical for promoting endochondral ossification from articular cartilage or for promoting long bone growth.

64. The research and development reagent or pharmaceutical according to claim 56, wherein the gene is a gene encoding FGF10 and SHH, or the protein is FGF10 and SHH.

65. The research and development reagent or pharmaceutical according to claim 56, wherein the gene is a gene encoding FGF10 or SHH, or the protein is FGF10 or SHH.

66. A research and development reagent or pharmaceutical product for use as a medicine for stimulating the regeneration of muscle tissue in a mammal, the research and development reagent or pharmaceutical product comprising a vector for introducing or causing one or more of the following (1) to (3) to act, or the following (4), (1) at least one of the genes that is expressed relatively strongly in cells having tissue regeneration ability, (2) at least one gene that functions as a master regulatory gene in the differentiation / production process of cells, (3) at least one of the cytokine genes that exerts a strong effect in the environment where tissue regeneration occurs, (4) At least one of the proteins encoded by the genes included in (1) to (3) above.

67. The research and development reagent or pharmaceutical according to claim 66, wherein the gene is a gene encoding FGF10, FGF2, LEF1, SHH, MSX1, MSX2, LIN28A and WNT7A, or the protein is FGF10, FGF2, LEF1, SHH, MSX1, MSX2, LIN28A and WNT7A.

68. The research and development reagent or pharmaceutical according to claim 66, wherein the gene comprises a gene encoding at least one selected from FGF10, FGF2, LEF1, SHH, MSX1, MSX2, LIN28A and WNT7A, or the protein comprises at least one selected from FGF10, FGF2, LEF1, SHH, MSX1, MSX2, LIN28A and WNT7A.

69. The research and development reagent or pharmaceutical according to claim 66, wherein the gene is a gene encoding FGF10, FGF2, LEF1, SHH, HDAC2, PRDX2 and WNT7A, or the protein is FGF10, FGF2, LEF1, SHH, HDAC2, PRDX2 and WNT7A.

70. The research and development reagent or pharmaceutical according to claim 66, wherein the gene comprises a gene encoding at least one selected from FGF10, FGF2, LEF1, SHH, HDAC2, PRDX2 and WNT7A, or the protein comprises at least one selected from FGF10, FGF2, LEF1, SHH, HDAC2, PRDX2 and WNT7A.

71. A research and development reagent or pharmaceutical product for use as a medicine for stimulating regeneration of adipose tissue in mammals, the research and development reagent or pharmaceutical product comprising a vector for introducing or causing one or more of the following (1) to (3) to act, or the following (4), (1) at least one of the genes that is expressed relatively strongly in cells having tissue regeneration ability, (2) at least one gene that functions as a master regulatory gene in the differentiation / production process of cells, (3) at least one of the cytokine genes that exerts a strong effect in the environment where tissue regeneration occurs, (4) At least one of the proteins encoded by the genes included in (1) to (3) above.

72. According to the research and development reagent or pharmaceutical product according to claim 71, the gene is a gene encoding FGF10, FGF20, FGF2, BMP5, LEF1, SHH, PRRX1, HDAC2, PRDX2 and PPARD, or the protein is FGF10, FGF20, FGF2, BMP5, LEF1, SHH, PRRX1, HDAC2, PRDX2 and PPARD.

73. The research and development reagent or pharmaceutical product according to claim 71, wherein the gene comprises a gene encoding at least one selected from FGF10, FGF20, FGF2, BMP5, LEF1, SHH, PRRX1, HDAC2, PRDX2 and PPARD, or the protein comprises at least one selected from FGF10, FGF20, FGF2, BMP5, LEF1, SHH, PRRX1, HDAC2, PRDX2 and PPARD.

74. The research and development reagent or pharmaceutical according to any one of claims 45 to 73, wherein the mammal is a human, a monkey, a rat or a mouse.

75. The research and development reagent or pharmaceutical according to claim 74, wherein the mammal is a mouse.

Images