A gene delivery system and its application in rapid and long-lasting alteration of animal hair color.

Abstract

This invention relates to a gene delivery system and its application in the rapid and long-lasting alteration of animal hair color. The gene delivery system comprises an exogenous gene that promotes and / or inhibits eumelanin production, and an adeno-associated virus carrying the exogenous gene. Compared with existing technologies, this invention achieves rapid and long-lasting effects on hair color in the target skin tissue of an animal by delivering the gene delivery system for altering hair color to the animal during the early stages of hair growth, thus forming a visually intuitive and stable marker.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a gene delivery system and its application in rapid and long-lasting changes in animal hair color. Background Technology

[0002] Hair color is one of the important phenotypic characteristics of animals, and its formation is closely related to the differentiation state of melanocytes in the hair follicle and the synthesis, secretion, and transport of melanin. Melanin mainly includes eumelanin and pheomelanin, and the synthesis ratio of different types of melanin directly determines the depth and color pattern of hair. During hair follicle development and hair growth, melanocytes continuously transport melanin granules to the forming hair shaft, thereby establishing and maintaining a relatively stable hair color phenotype.

[0003] In existing technologies, altering animal hair color primarily relies on chemical dyes or physical coloring methods. These methods only affect the already formed hair shaft and cannot regulate the differentiation and functional state of melanocytes within the hair follicle. Consequently, they generally suffer from problems such as short-lasting effects, easy fading, the need for repeated treatments, and potential skin irritation or hair follicle damage. Furthermore, these methods struggle to achieve precise, localized control and cannot achieve long-term, stable color changes at the hair growth cycle level. Therefore, there is currently a lack of methods that can intervene in the differentiation trajectory of melanocytes within the hair follicle to achieve rapid and long-lasting changes in animal hair color.

[0004] On the other hand, with the rapid development of the pet economy, the demand for pet management, safety identification, and personalized appearance is constantly increasing. Existing pet identification methods mostly rely on collars, tags, or implanted chips, which have shortcomings such as being easily lost, not intuitive, or requiring specialized equipment for reading. Meanwhile, existing pet dyeing methods generally suffer from poor safety, short duration of effect, and potential damage to the skin and hair follicles, making it difficult to meet the long-term, stable marking or beautification needs. Therefore, developing a technical solution that can achieve rapid onset, long-term maintenance, and regional controllability of hair color without affecting hair growth and skin health has significant research value and application prospects. Summary of the Invention

[0005] To address the shortcomings of existing hair color regulation methods, which primarily target already formed hair shafts and struggle to achieve rapid onset, long-term stability, and regional controllability, this invention aims to provide a gene delivery system and its application in rapidly and effectively altering animal hair color. This method delivers genes to a localized area of ​​the skin during the critical early developmental stage of the hair follicle's growth phase. This regulates the differentiation trajectory of melanocytes and the melanin synthesis pathway within the hair follicle, thereby achieving a change in the color of newly grown hair in a relatively short time and maintaining stability throughout subsequent hair growth cycles.

[0006] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide a gene delivery system for altering the color of animal hair, comprising an exogenous gene that promotes and / or inhibits the production of eumelanin, and an adeno-associated virus loaded with the exogenous gene that promotes and / or inhibits the production of eumelanin. The exogenous gene that promotes the production of eumelanin is selected from any one of MSH, MITF, SOX10, PAX3, TYR, TYRP1, MC1R, and CREB1, and the exogenous gene that inhibits the production of eumelanin is selected from any one of ASIP, FOXD3, SNAI2, PMEL, and GSK3β.

[0007] In some specific embodiments, the adeno-associated virus is selected from any one of AAV-DJ, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAVrh74, AAV-PHP.S, AAV-PHP.B, AAV-PHP.eB, and AAV-retro.

[0008] The second technical solution of the present invention is to provide a gene delivery system as described in one of the above technical solutions for the application in the preparation of products that regulate rapid and long-lasting changes in animal hair color.

[0009] In some specific embodiments, the product is used during the early stage of the animal's hair growth cycle.

[0010] In some specific embodiments, the early stage of the animal hair growth cycle is the stage when the hair follicle first or simultaneously enters the growth phase.

[0011] In some specific embodiments, the product is applied to hair follicle cells in the skin region of an animal.

[0012] In some specific embodiments, the effective quantity of the product is greater than or equal to 1×10⁻⁶. 10 GC / only.

[0013] In some specific embodiments, the product includes injections, dressings, and drugs.

[0014] In some specific embodiments, the injectable also includes pharmaceutically acceptable excipients.

[0015] In some specific embodiments, the pharmaceutically acceptable excipients include fillers, pH adjusters, suspending agents, antioxidants, antibacterial agents, and emulsifiers.

[0016] Compared with the prior art, the present invention has the following advantages: (1) The present invention delivers a gene delivery system that alters the color of animal hair to the target skin tissue of the animal during the early stage of the hair growth period, thereby achieving rapid effect on hair color.

[0017] (2) This invention intervenes in the differentiation trajectory of melanocytes, rather than just changing the color of the already formed hair shaft, so that the hair color remains stable in multiple subsequent hair growth cycles, and has significant long-term effects.

[0018] (3) This invention delivers exogenous genes that promote and / or inhibit the production of eumelanin into the skin tissue of animals, thereby achieving bidirectional controllable regulation of hair color and making it more widely applicable.

[0019] (4) The present invention can use local skin gene delivery to achieve regional and patterned control of hair color change, forming an intuitive and stable visual marker.

[0020] (5) This invention provides a safe, durable and controllable new technology path for animal model research, as well as pet marking, anti-lost identification, and appearance enhancement, while ensuring the integrity of the skin and hair follicle structure. Attached Figure Description

[0021] Figure 1 Example 1: A fluorescence micrograph showing the distribution of relevant signals in the skin and hair follicle tissue in the target skin region after control treatment (CTR) or delivery of AAV-GFP, where the dashed line represents the boundary between the epidermis and dermis.

[0022] Figure 2 Example 2 illustrates the characterization of changes in hair appearance and the expression of related genes in the target skin region after the adeno-associated virus vector AAV-DJ carrying the hair color regulation-related gene ASIP is delivered to the skin on the back of an animal. Figure 2 A is a schematic diagram of the delivery of the adeno-associated virus vector AAV-DJ carrying the hair color regulation-related gene ASIP to the skin on the back of an animal. Figure 2 B is a schematic diagram showing the appearance of the back hair observed on the 12th day after delivery; Figure 2 C is a schematic diagram showing the results of detecting the expression levels of relevant genes in the target skin region on day 12 after delivery.

[0023] Figure 3 Example 2 shows a schematic diagram of the morphological and structural characteristics of different types of hair in the target region after delivery of the hair color regulation-related gene ASIP. The image below is an enlarged view of the area within the box in the image above. Wherein: Figure 3 A is a schematic diagram of Guard type hair in mice; Figure 3 B is a schematic diagram of Zig-zag type hair in mice; Figure 3 C is a schematic diagram of Aw / Auchene type hair in mice.

[0024] Figure 4 Example 5 illustrates how the delivery of the hair color regulation-related gene ASIP along a preset path to the skin on the back of an animal creates a specific shape or marker in the back hair.

[0025] Figure 5 Example 4 illustrates the maintenance of hair color changes in the target area on the back of an animal at different time points (D12, D395, D670) throughout its life cycle by delivering the hair color regulation-related gene ASIP to the skin on the back of the animal along a preset path.

[0026] Figure 6 Example 3 illustrates the characterization of hair appearance changes after the adeno-associated virus vector AAV-DJ, carrying the hair color regulation-related gene MSH, is delivered to the skin on the back of an animal. The area within the dotted circle represents the region of color change. Wherein: Figure 6 A is a schematic diagram of the delivery of the adeno-associated virus vector AAV-DJ carrying the hair color regulation-related gene MSH to the skin on the back of an animal; Figure 6 B is a schematic diagram showing the appearance of the back hair observed on day 135 after delivery. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0029] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.

[0030] Example 1: I. Constructing a gene delivery system carrying a reporter gene Adeno-associated virus (AAV-DJ) was selected as the delivery vector. The Rep and Cap genes on AAV-DJ were replaced with the fluorescent reporter gene GFP using conventional techniques, and the result was denoted as AAV-GFP.

[0031] 2. Healthy experimental animals (C57BL / 6 mice, female) were selected as research subjects. When the animals were in the hair resting phase (P42, which is the 42nd day after the mice were born), the hair in the target skin area was appropriately treated to expose the hair follicles and put them in a state suitable for the delivery of exogenous genes.

[0032] Subsequently, AAV-GFP was injected intradermally (injection volume 4 × 10⁻⁶). 10 GC (per animal) is applied to the target skin area of ​​the animal.

[0033] On the 5th day after delivery, samples were taken from the target skin area. After fixation, embedding and sectioning, the skin tissue and hair follicle structure were observed using a fluorescence microscope.

[0034] like Figure 1 As shown, reporter signals can be detected in the epidermis, dermis, and hair follicle-related regions, indicating that the gene delivery system can effectively enter skin and hair follicle-related cells, providing a basis for subsequent hair color regulation.

[0035] In this embodiment, the gene delivery system was delivered to an animal in the quiescent phase of its hair growth, solely to demonstrate that the AAV-GFP gene delivery system prepared in this embodiment can efficiently infect the skin.

[0036] Example 2: An example of hair color change from dark to light induced by inhibition of melanin production-related genes. I. Constructing a gene delivery system carrying the ASIP gene Adeno-associated virus (AAV-DJ) was selected as the delivery vector. The Rep and Cap genes on AAV-DJ were replaced with the ASIP gene (GeneID: 50518) encoding the guinea pig signal protein using conventional techniques, thus constructing a gene delivery system carrying the ASIP gene, denoted as AAV-ASIP. II. Figure 2 As shown in Figure A, C57BL / 6 mice with dark hair morphology were selected as the research subjects. Target areas were selected on the skin of their backs, and local hair treatment was performed when the hair follicles were in the early stage of hair growth (P23, which is the 23rd day after the mouse was born).

[0037] AAV-ASIP was injected intradermally (injection volume 4 × 10⁻⁶). 10 GC (per dog) is administered to the target skin area. After delivery, no further administration is required; allow hair to grow naturally.

[0038] Subsequent observations revealed that, compared to the untreated area, the color of newly grown hair in the target skin area gradually changed from its original dark color to a lighter color, and remained stable during hair growth. Figure 2Day 12 (D12) as shown in Figure B. Further sampling of the target skin area and detection of relevant gene expression, such as... Figure 2 The results of C indicate that the ASIP gene is continuously expressed in the target region, providing a molecular basis for hair color changes.

[0039] like Figure 3 As shown, the color of all types of fur and the color of different areas of fur in mice have changed.

[0040] Example 3: An example of hair color change from light to dark induced by genes related to promoting melanin production. I. Constructing a gene delivery system carrying the MSH gene Adeno-associated virus (AAV-DJ) was selected as the delivery vector. The Rep and Cap genes on AAV-DJ were replaced with the melanocyte-stimulating hormone-related gene MSH (Gene ID: 18976) using conventional techniques to construct a gene delivery system carrying the MSH gene, denoted as AAV-MSH.

[0041] II. Figure 6 As shown in A, 129 mice with light-colored or brown hair morphology were selected as the research subjects. Local treatment and delivery were performed on the target skin area when the hair follicles were in the early stage of hair growth (P23, which is the 23rd day after the mice were born).

[0042] AAV-MSH was injected (4×10) via intradermal delivery. 10 GC (per animal) is delivered to the target skin area, and hair growth is continuously observed after delivery without repeated treatment.

[0043] like Figure 6 As shown in Figure B, the color of newly grown hair in the target skin area gradually changes to a darker color, forming a clear distinction from the surrounding untreated area, indicating that the method of the present invention can control the hair color from light to dark.

[0044] Example 4: Long-lasting hair color maintenance and its application across the hair growth cycle Following the procedure in Example 2, when the hair follicles of C57BL / 6 mice were in the early stage of hair growth (P23, i.e., day 23 after birth), AAV-ASIP (4 × 10⁻⁶) was injected intradermally. 10 GC / each (the injection volume can be increased appropriately according to the pattern) is injected into the target skin area to form an H-shape, and long-term follow-up observation is conducted. Changes in the appearance of hair in the target skin area are recorded at different time points.

[0045] The results are as follows Figure 5As shown, the hair color change formed in the target area can be maintained throughout the entire life cycle of the experimental animal and remain stable during the hair renewal process without obvious fading or regression. It can be seen that the method of the present invention can achieve a long-term regulation effect on hair color without frequent repeated delivery.

[0046] Example 5: An example of pet identification and appearance management Healthy pet animals are selected as subjects. Target areas are chosen on the skin of the animal, such as its back, neck, or limbs, where observation is easy and does not affect normal activities. Preferably, the target area is in the early stage of hair growth. A gene delivery system carrying exogenous genes that promote and / or inhibit melanin production is injected into the selected target area.

[0047] In this embodiment, when the hair follicles of C57BL / 6 mice were in the early stage of hair growth (P23, i.e., day 23 after birth), AAV-ASIP (4 × 10⁻⁶) was injected intradermally. 10 GC / each (the injection volume can be increased appropriately according to the pattern) is injected into the target skin area, forming J-shaped and S-shaped areas respectively. During the natural hair growth process, areas of hair color change with obvious identifiable characteristics gradually form within the target area, such as... Figure 4 As shown. This color-changing area can serve as a long-term visual identifier for pets and remains stable without affecting hair follicle structure and hair growth ability. It can be used in applications such as pet identification, preventing pets from getting lost, or beautifying their appearance.

[0048] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A gene delivery system for altering the color of animal fur, characterized in that, The invention includes exogenous genes that promote and / or inhibit eumelanin production, and adeno-associated viruses loaded with the exogenous genes that promote and / or inhibit eumelanin production, wherein the exogenous genes that promote eumelanin production are selected from any one of MSH, MITF, SOX10, PAX3, TYR, TYRP1, MC1R, and CREB1, and the exogenous genes that inhibit eumelanin production are selected from any one of ASIP, FOXD3, SNAI2, PMEL, and GSK3β.

2. The gene delivery system according to claim 1, characterized in that, The adeno-associated virus is selected from any one of AAV-DJ, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAVrh74, AAV-PHP.S, AAV-PHP.B, AAV-PHP.eB, and AAV-retro.

3. The use of a gene delivery system as described in any one of claims 1 or 2 in the preparation of products that regulate rapid and long-lasting changes in animal hair color.

4. The application according to claim 3, characterized in that, The product is effective during the early stage of animal hair growth.

5. The application according to claim 4, characterized in that, The early stage of the animal hair growth cycle is the stage when the hair follicle first or simultaneously enters the growth phase.

6. The application according to claim 3, characterized in that, The product is applied to the hair follicle cells in the skin of animals.

7. The application according to claim 3, characterized in that, The effective quantity of the product is greater than or equal to 1×10⁻⁶. 10 GC / only.

8. The application according to claim 3, characterized in that, The products include injections, dressings, and medications.

9. The application according to claim 8, characterized in that, The injectable also includes pharmaceutically acceptable excipients.

10. The application according to claim 9, characterized in that, Pharmaceutically acceptable excipients include fillers, pH adjusters, suspending agents, antioxidants, antibacterial agents, and emulsifiers.

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