Engineered herpes simplex virus vectors for improving skin structure and function and uses thereof

Abstract

The invention discloses an HSV-1 replication-deficient recombinant virus vector and an application of the HSV-1 replication-deficient recombinant virus vector. Specifically, the method comprises the following steps: performing gene editing on a specific genome segment in an HSV-1 genome to obtain an HSV-1 replication-defective recombinant virus vector; wherein compared with the existing HSV-1 replication-deficient recombinant virus vector, the HSV-1 replication-deficient recombinant virus vector can express exogenous genes more efficiently, and the HSV-1 replication-deficient recombinant virus vector, as an excellent delivery vector, can effectively deliver collagen genes in a mouse model.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and particularly relates to a replication-defective recombinant virus vector of HSV-1 and its applications in skin diseases and cosmetology. Background Art

[0002] Herpes Simplex Virus (HSV) belongs to the Herpesviridae family and is an enveloped double-stranded DNA virus, including two subtypes: type I (HSV-1) and type II (HSV-2).

[0003] Herpes Simplex Virus consists of four parts: the core, the capsid, the tegument protein, and the envelope. The virus core is composed of dense double-stranded DNA, coiled into a filamentous scroll. The capsid surrounding the DNA has an icosahedral structure with a diameter of 100 - 110 nm and is composed of 162 capsomeres. The capsid is covered by an irregularly arranged and amorphous tegument protein. The outermost layer of the virus is a lipid bilayer envelope with short protrusions, and the diameter of the virus enclosed by the envelope is 150 - 200 nm. The virus envelope is composed of 11 glycoproteins and at least two non-glycosylated proteins, including viral glycoprotein B (gB), viral glycoprotein C (gC), viral glycoprotein D (gD), viral glycoprotein G (gG), and viral glycoprotein M (gM), etc. The three-dimensional structure of the virus envelope glycoprotein is related to the cell infection ability of the virus, which determines whether the virus can enter the host cell and the amount of virus entering the host cell.

[0004] At present, HSV-1 virus vectors have been used in gene therapy for major diseases such as tumors, neurodegenerative diseases, genetic diseases, and immune system diseases, as well as in cosmetology. Compared with other gene therapy virus vectors, HSV-1 virus vectors have many advantages. The genome of HSV-1 is relatively large and can carry large or multiple foreign genes. The genome of HSV-1 is up to 152 kb long, while the genome of the commonly used gene therapy vector, adeno-associated virus vector (AAV), is only 4.7 kb. Among the more than 80 known genes in the HSV-1 genome, about half are non-essential genes in in vitro culture and can be replaced by multiple exogenous therapeutic genes. The maximum exogenous gene insertion amount can reach 30-40 kb. This is particularly important for treating many diseases, especially those related to multiple genes. HSV-1 does not integrate with cellular DNA, its replication can be controlled, and it has high safety. HSV-1 hardly causes life-threatening diseases in immunocompetent adults [Shen Y, Nemunaitis J. Herpes simplex virus 1 (HSV-1) for cancer treatment[J]. Cancer Gene Therapy, 2006, 13(11): 975-992]. Due to its wide host cell range and high safety, the HSV-1 vector is considered a very promising virus vector in gene therapy.

[0005] The HSV-1 virus genome consists of long terminal repeats (TR L ), long unique segments (U L ), long internal repeats (IR L ), short internal repeats (IR S ), short unique segments (U S ), and short terminal repeats (TR S ) ([[]] Figure 1 ). The proteins encoded by the HSV-1 genome are involved in various functions such as cell adhesion, cell invasion, replication, assembly, cell interaction, and immune interaction, and have relatively complex gene regulatory elements. Affected by the viral regulatory elements, the position of exogenous gene insertion greatly affects the expression level of exogenous genes. How to obtain an HSV-1 virus vector with high efficiency in expressing exogenous genes has become an urgent problem to be solved at present.

[0006] ​The skin is the largest organ of the human body, and collagen is the main component in the skin, containing 85 - 90% type I collagen and 10 - 15% type III collagen. The skin is mainly composed of the epidermis and the dermis. The dermis contains fibroblasts, which are responsible for producing collagen and elastin. Collagen maintains the physiological state of the skin by providing mechanical integrity and other functions. Skin aging is caused by a decrease in collagen density, a reduction in the thickness of the dermis, and a decline in the synthesis and renewal of important structural proteins. The metabolic changes that occur with age and the visible internal characteristics brought about by growth are inevitable, such as dry skin, the appearance of wrinkles, etc. Smoking and long-term stress are external factors that affect collagen. In particular, smoking degrades immature collagen in the skin, accelerating the growth of wrinkles caused by collagen loss. Existing research has proven that damaged collagen fibers can be repaired by replacement. Supplementing collagen can improve the integrity of the skin and delay skin aging, and has obvious effects on removing wrinkles and rejuvenating the skin. Among all types of collagen, type I collagen and type III collagen are the most widely used collagens in the field of medical aesthetics due to their high biocompatibility.

[0007] At present, the sources of collagen in the market mainly include natural extraction, recombinant collagen, and synthetic collagen. The raw materials for natural extraction of collagen mainly come from tissues such as the skin, tendons, and cartilage of terrestrial animals such as pigs, cows, sheep, and chickens. In addition, some also come from the skin and fins of fish. Despite the wide sources, collagen from animal sources has the risks of immunogenicity and the transmission of interspecies diseases. Recombinant collagen and synthetic collagen can avoid the above risks of natural extracted collagen, and the production is stable between batches, which is conducive to large-scale production. At present, the production of recombinant collagen mainly uses expression systems such as Escherichia coli and yeast. However, the yield of recombinant proteins is usually relatively low. At the same time, the modification of recombinant collagen in lower cells is lacking. For example, collagen cannot be prolyl hydroxylated in Escherichia coli, and although it can be glycosylated in eukaryotic cells such as yeast, the glycosylation characteristics may be different. In addition, due to the usually large size of collagen genes, modified genes with a smaller molecular weight that only have the core triple helix structure are usually used in recombinant systems. These above factors result in differences in the structure and characteristics between recombinant collagen and natural collagen, having problems such as lower thermal stability and being more easily degraded by enzymes. To improve the above situation, more complex expression systems such as transgenic animals and plants are applied to express recombinant collagen, but the low yield and technological maturity still need to be improved. With the development of synthetic technology, synthetic collagen has become an important source of collagen. Synthetic collagen has the advantages of clear source, no background pollution, and no immunogenicity. The synthesized collagen is called collagen-like protein because it only has the recombinant structure of glycine-X-Y, and its length is usually less than 10 nm, much shorter than the 300 nm length of the α-helix of type I collagen. Therefore, synthetic collagen is generally used as nanospheres, nanolayers, and other nanostructures. Improved synthetic technology has been able to extend the length of collagen-like protein to the micron level, but this technology is limited by the high production cost.

[0008] The silent information regulator (SIRT) gene family, also known as the deacetylase gene family, mainly includes multiple members such as SIRT1 to SIRT7. These genes play important roles in regulating cell metabolism, growth, aging, and gene expression. In recent years, the application of the SIRT gene family in the fields of beauty and anti-aging has attracted extensive attention. The SIRT genes encode deacetylases, which can remove acetyl groups from target proteins. This process affects the functions of various bioactive proteins, including transcription factors and DNA repair enzymes, thus playing a role in gene expression and cell metabolism. The SIRT gene family enhances the resistance of cells to free radicals and other oxidative damages by regulating antioxidant enzymes and stress response proteins, reducing the risks of aging and related diseases. The SIRT genes can inhibit the expression of pro-inflammatory factors and regulate the inflammatory response, which has a positive effect on alleviating skin inflammation-related diseases such as acne and eczema. The SIRT genes play a regulatory role in cell energy metabolism, promoting mitochondrial biogenesis and improving the energy level of cells, thereby improving the health status of the skin. Summary of the Invention

[0009] This application provides a replication-defective recombinant HSV-1 viral vector and its applications in the fields of disease treatment and beauty. Specifically, in this application, specific regions of the HSV-1 genome (such as Figure 2 shown) are modified to obtain a replication-defective HSV-1 viral vector. This vector highly expresses the carried foreign gene, thereby efficiently, precisely, and more safely supplementing the missing components in cells, such as collagen, to achieve the treatment of diseases or the improvement of skin conditions, such as improving skin dryness, improving skin vitality, removing wrinkles, and / or delaying skin aging.

[0010] Specifically, this application relates to the following technical solutions:

[0011] Item 1. A replication-defective recombinant herpes simplex virus type 1 (HSV-1) vector, in which the nucleotide sequences of the following genomic segments in its genome are disrupted or knocked out:

[0012] 1) The nucleotide sequence from 1 - 100 nucleotides upstream and downstream of the 126,785th nucleotide to 1 - 100 nucleotides upstream and downstream of the 131,176th nucleotide, and the nucleotide sequence from 1 - 823 nucleotides upstream and downstream of the 147,025th nucleotide to 1 - 93 nucleotides upstream and downstream of the 151,439th nucleotide; and / or

[0013] 2) The nucleotide sequence from 1 - 257 nucleotides upstream and downstream of the 113,494th nucleotide to 1 - 223 nucleotides upstream and downstream of the 115,314th nucleotide;

[0014] Among them, the nucleotide positions of the above HSV-1 genome are determined with reference to the HSV-1 laboratory virus strain 17 genome (NC_001806.2).

[0015] Moreover, the disrupted or knocked-out genomic loci described above contain one or more exogenous protein-coding genes introduced from the group consisting of: collagen, elastin, and growth factors.

[0016] Item 2. The recombinant herpes simplex virus type I vector according to Item 1, further, one or more selected from the ICP34.5 gene, ICP6 gene, ICP0 gene, ICP47 gene, ICP22 gene, ICP4 gene, ICP27 gene, US3 gene, UL56 gene, functional VP16 gene, VHS gene, UNG gene, IRL / IRS region, glycoprotein H gene, thymidine kinase gene in the genome are disrupted or knocked out.

[0017] Item 3. The recombinant herpes simplex virus type I (HSV-1) vector according to Item 1, wherein the viral vector is cultured in complementing cells.

[0018] Item 4. The recombinant herpes simplex virus type I (HSV-1) vector according to Item 3, wherein the complementing cells are Vero cells.

[0019] Item 5. The recombinant herpes simplex virus type I vector according to any one of Items 1-2, further, ICP0 and / or ICP22 in the genome are disrupted or knocked out.

[0020] Item 6. The recombinant herpes simplex virus type I vector according to any one of Items 1-5, wherein the exogenous protein-coding gene is introduced at the same or different genomic loci that are disrupted or knocked out as described above.

[0021] Item 7. The recombinant herpes simplex virus type I vector according to Item 6, wherein the exogenous protein is selected from one or more of the following proteins: epidermal growth factor (EGF), fibroblast growth factor (FGF), type I collagen (COL1), type II collagen (COL2), type III collagen (COL3), type IV collagen (COL4), type VII collagen (COL7), type IX collagen (COL9), type XVII collagen (COL17), elastin (ELN), sirtuin 1 (SIRT1), sirtuin 3 (SIRT3), sirtuin 6 (SIRT6), and sirtuin 7 (SIRT7).

[0022] Item 8. The recombinant herpes simplex virus type I according to any one of Items 1-7, for beauty treatment. Optionally, the beauty treatment includes one or more of the following: improving skin elasticity, improving skin dryness, improving skin vitality, removing wrinkles, and / or delaying skin aging.

[0023] Item 9. The recombinant herpes simplex virus type I according to any one of Items 1-7, for treating epidermolysis bullosa.

[0024] Item 10. A composition comprising the recombinant herpes simplex virus type I vector according to any one of Items 1-7 and a pharmaceutically acceptable solvent or excipient.

[0025] Item 11. The composition according to Item 10, which is a pharmaceutical composition or a composition for beauty treatment. Optionally, the composition for beauty treatment is a composition for topical application, subcutaneous injection, intradermal injection, or microneedle injection for skin improvement.

[0026] Item 12. A beauty treatment method comprising administering to a subject in need an effective amount of the composition for beauty treatment according to Item 10 or 11. Optionally, the beauty treatment includes one or more of the following: improving skin dryness, improving skin vitality, removing wrinkles, and / or delaying skin aging. Optionally, the composition for beauty treatment is a composition for topical application, subcutaneous injection, intradermal injection, or microneedle injection for skin improvement.

[0027] Item 13. A disease treatment method comprising administering to a subject in need an effective amount of the composition according to Item 10 or 11, wherein the disease is epidermolysis bullosa

[0028] Item 14. A complementing cell for culturing a herpes simplex virus vector, wherein the herpes simplex virus vector is the recombinant herpes simplex virus type I (HSV-1) vector according to any one of Items 1-7.

[0029] Item 15. The complementing cell according to Item 14, which is a Vero cell.

[0030] Item 16. Use of Vero cells for culturing a herpes simplex virus vector, wherein the herpes simplex virus vector is the recombinant herpes simplex virus type I (HSV-1) vector according to any one of Items 1-7.

[0031] It should be understood that within the scope of this application, each of the above technical features of this application and the technical features specifically described hereinafter can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one.

[0032] Due to the different genome lengths of different HSV-1 virus strains, the positions of the HSV-1 genomic bases (nucleotides) described in the embodiments of the present application are determined with reference to the genome of the HSV-1 laboratory virus strain 17 (NC_001806.2). The positions corresponding to the HSV-1 genomic base (nucleotide) positions described in the embodiments of the present application in different virus strains can be obtained through sequence alignment. Specifically, when describing the positions of modifications such as mutations, knockouts, and insertions performed on the viral genome in the present application, the base positions of the HSV-1 laboratory virus strain 17 genome are listed. Any other HSV-1 virus strain genome sequence is aligned with the laboratory virus strain 17 genome sequence using sequence alignment tools such as BLAST, Vector NTI, DNAstar, etc. Therefore, technical solutions regarding the base positions of any other HSV-1 virus strain corresponding to the base positions of the laboratory virus strain 17 involved in the present application are also included within the scope of the claims of the present application.

[0033] The beneficial effects of the present application are as follows: The present application provides a novel HSV-1 replication-deficient recombinant virus vector, which can express foreign genes more efficiently compared to the existing HSV-1 replication-deficient recombinant virus vectors. This vector has good application prospects in the beauty field. It can highly express the beauty-related genes carried, and can supplement collagen or rejuvenate cells efficiently, precisely, and more safely. Brief Description of the Drawings

[0034] Figure 1 It is a schematic diagram of the HSV-1 genome.

[0035] Figure 2 It is a schematic diagram of the ICP4 gene and ICP27 gene of HSV-1.

[0036] Figure 3 It is a schematic diagram of the recombinant virus vector A1.

[0037] Figure 4 It is a schematic diagram of the recombinant virus vector B1.

[0038] Figure 5 It is a schematic diagram of the recombinant virus vector A2.

[0039] Figure 6 It is a schematic diagram of the recombinant virus vector B2.

[0040] Figure 7 They are fluorescence microscopy pictures of different recombinant virus vectors expressing green fluorescent protein after infecting Vero and CCC-ESF-1 cells respectively.

[0041] Figure 8 It is a schematic diagram of the recombinant virus vector C.

[0042] Figure 9 It is a schematic diagram of recombinant viral vector D.

[0043] Figure 10 It is a schematic diagram of recombinant viral vector E.

[0044] Figure 11 It is a schematic diagram of recombinant viral vector F.

[0045] Figure 12 It is a schematic diagram of recombinant viral vector G.

[0046] Figure 13 It is the result of the expression verification of the gene carried by the HSV-1 replication-defective recombinant viral vector.

[0047] Figure 14 It is the detection result of type VII collagen in the supernatant of human primary keratinocytes infected with recombinant viral vector D at different time points. M: Protein molecular weight (kD); 1: 24 hours; 2: 48 hours; 3: 72 hours; 4: Negative control; 5: Positive control.

[0048] Figure 15 It is the detection result of type VII collagen in the supernatant of human immortalized dermal fibroblasts HSF(SV40) infected with recombinant viral vector D at different time points. M: Protein molecular weight (kD); 1: 24 hours; 2: 48 hours; 3: 72 hours; 4: Negative control; 5: Positive control.

[0049] Figure 16 It is the NC1 immunofluorescence staining of the skin tissue at the drug administration site in mice.

[0050] Figure 17 It is the NC2 immunofluorescence staining of the skin tissue at the drug administration site in mice.

[0051] Figure 18 It is the NC1 immunohistochemical staining of the skin tissue at the drug administration site in mice.

[0052] Figure 19 It is the NC2 immunohistochemical staining of the skin tissue at the drug administration site in mice.

[0053] Figure 20 It is the phenotypic change diagram of mice before and after the administration of the HSV-1 replication-defective recombinant virus.

[0054] Figure 21 It is a schematic diagram of recombinant viral vector H.

[0055] Figure 22 It is a schematic diagram of recombinant viral vector I.

[0056] Figure 23 It is a schematic diagram of recombinant viral vector J.

[0057] Figure 24 For the detection of the expression product of recombinant viral vector H. M: Protein molecular weight standard; 1: Type I collagen, the expression product of recombinant viral vector H; 2: Cellular protein control.

[0058] Figure 25 For the detection of the expression product of recombinant viral vector I. M: Protein molecular weight standard; 1: Type III collagen, the expression product of recombinant viral vector I; 2: Cellular protein control.

[0059] Figure 26 For the detection of the expression product of recombinant viral vector J. M: Protein molecular weight standard; 1: Type XVII collagen, the expression product of recombinant viral vector J; 2: Cellular protein control.

[0060] Figure 27 Schematic diagram of recombinant viral vector K.

[0061] Figure 28 Schematic diagram of recombinant viral vector L.

[0062] Figure 29 Schematic diagram of recombinant viral vector M.

[0063] Figure 30 For the detection of the expression product of recombinant viral vector K. M: Protein molecular weight standard; 1: Cellular protein control; 2: Blank control; 3 - 5: SIRT1 protein, the expression product of recombinant viral vector K.

[0064] Figure 31 For the detection of the expression product of recombinant viral vector L. M: Protein molecular weight standard; 1 - 3: SIRT3 protein, the expression product of recombinant viral vector L; 2: Cellular protein control.

[0065] Figure 32 For the detection of the expression product of recombinant viral vector M. M: Protein molecular weight standard; 1 - 4: SIRT6 protein, the expression product of recombinant viral vector M; 2: Cellular protein control. Detailed implementation manners

[0066] The following describes in detail the specific implementation manners of the present application. However, it should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific experimental conditions indicated in the following examples, they are generally operated according to conventional conditions or the conditions recommended by the manufacturer.

[0067] Unless otherwise clearly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "having" etc. will be understood to include the stated components or constituent parts, without excluding other components or other constituent parts.

[0068] As used herein, the term "replication-defective" refers to a viral vector that cannot replicate fully and effectively. A replication-defective viral vector is a mutant or defective form with respect to one or more functions essential for viral genome replication or viral particle synthesis and assembly. A replication-defective viral vector can be propagated in a complementary cell line that expresses the missing gene product. However, in normal target cells, a replication-defective viral vector can express viral gene products but will not replicate to form infectious progeny viral particles. In some embodiments, the replication-defective virus or viral vector is a mutant or defective form of the virus or vector with respect to one or more functions essential for viral genome replication. In some embodiments, the replication-defective virus or viral vector is a mutant or defective form of the virus or vector with respect to one or more functions essential for viral particle synthesis and assembly.

[0069] In one embodiment of the present application, the replication-defective viral vector is an HSV-1 viral vector.

[0070] As used herein, the term "foreign" means that a nucleotide sequence does not originate from a particular cell but is introduced into the cell by a DNA delivery method, such as by transfection, electroporation, or transduction with a viral vector. Thus, a foreign nucleotide sequence is an artificial sequence, where the artifact can be derived, for example, from a combination of subsequences from different sources (such as a combination of a recombinase recognition sequence, an SV40 promoter, and a coding sequence for green fluorescent protein as an artificial nucleic acid), or from a deletion of a partial sequence (such as a sequence encoding only the extracellular domain of a membrane-bound receptor or cDNA), or from a mutation of a nucleobase. The term "endogenous" means a nucleotide sequence that originates from a cell. A "foreign" nucleotide sequence can have an "endogenous" counterpart that is identical in base composition, but where the sequence is introduced into the cell (such as by recombinant DNA technology) to become a "foreign" sequence.

[0071] As used herein, the term "Vero cell", also known as green monkey kidney cells, is an aneuploid kidney cell line of African green monkeys (genus: Chlorocebus). In 1962, Yoshihiro Yasamura of Chiba University in Japan isolated the kidney epithelial cells of normal adult African green monkeys to obtain this cell line. Vero cells are a continuous aneuploid cell line, which means that their chromosome number is abnormal. As a continuous cell line, Vero cells can undergo many division cycles without aging. Vero cells have a defect in interferon secretion function. Different from normal mammalian cells, they do not secrete interferon α / β when infected with a virus. However, they still have receptors for interferon-α / β, so they can still respond when recombinant interferon is added to their culture medium. Currently, Vero cells are widely used in the research on the molecular mechanism of viral infection, the production of vaccines and recombinant proteins, and are regarded as an ideal cell model for culturing influenza vaccines and studying the molecular mechanism of viral infection.

[0072] In some embodiments, a complementing cell line that supports the replication of replication-defective viral vectors is obtained by transfecting Vero cells.

[0073] Preferably, the complementing cell line is the ZH02 complementing cell line and / or the ZH06 complementing cell line.

[0074] As used herein, the term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are typically determined by an open reading frame, usually beginning with an ATG start codon or alternative start codons such as GTG and TTG, and ending with a stop codon such as TAA, TAG, and TGA. The coding sequence can be DNA, cDNA, synthetic, or recombinant polynucleotide.

[0075] As used herein, the term "expression" includes any step involved in the preparation of a polypeptide, including but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0076] As used herein, the term "expression vector" refers to a linear or circular DNA molecule that includes a polynucleotide encoding a polypeptide and is operably linked to additional nucleotides that provide for its expression.

[0077] As used herein, the term "epidermolysis bullosa" (EB) is a rare genetic skin disease that affects 1 in 20,000 newborns in the United States (approximately 200 children are born with EB each year). There are many genetic and symptomatic variants of epidermolysis bullosa, but all variants share a common prominent symptom, namely, extremely fragile skin that blisters and tears due to minor friction or trauma. The list of complications and secondary diseases can be long and requires multiple interventions by a range of medical specialists. EB affects all genders, races, and ethnic groups equally.

[0078] As used herein, the term "type VII collagen (COL7)" consists of three identical alpha collagen chains and is confined to the basal region beneath the stratified squamous epithelium. COL7 is associated with all forms of dystrophic epidermolysis bullosa.

[0079] As used herein, the term "type XVII collagen (COL17)" is a transmembrane protein. Collagen XVII is a structural component of hemidesmosomes, which are multi-protein complexes located in the dermal-epidermal basement membrane zone and mediate the adhesion of keratinocytes to the underlying membrane. COL17 has functions of promoting cell adhesion, improving skin elasticity, antioxidation, and protecting skin health, and plays a key regulatory role in skin aging, hair loss, and gray hair.

[0080] As used herein, the term "epidermal growth factor (EGF)" is the prototype member of the EGF superfamily of peptide growth factors and consists of a single-chain polypeptide containing 53 amino acid residues. EGF plays an important role in the growth, proliferation, and differentiation of many types of cells, especially fibroblasts and epithelial cells, and has strong skin cell repair ability.

[0081] As used herein, the term "fibroblast growth factors (FGFs)" is a class of structurally related polypeptide growth factors that play diverse roles in regulating cell proliferation, migration, differentiation, and apoptosis, and have the effects of promoting cell repair and improving skin condition.

[0082] As used herein, the term "type I collagen (COL1)" is a fibril-forming collagen that is present in most connective tissues and is abundant in bone, cornea, dermis, and tendon. COL1 is the most common type of collagen in the body, with a thick diameter, tightly arranged, and no elasticity, mainly playing a supporting role. Supplementing COL1 can improve aging states such as skin laxity, wrinkles, and dryness, making the skin plump and firm.

[0083] As used herein, the term "type III collagen (COL3)" is a fibrous collagen that is present in extensible connective tissues such as skin, lung, uterus, intestine, and vascular system and is usually associated with type I collagen. COL3 is one of the main structural proteins of the skin and plays an important role in the elasticity and firmness of the skin.

[0084] As used herein, the term "elastin (ELN)" is one of the two components of elastic fibers. Elastic fibers form part of the extracellular matrix and endow organs and tissues with elasticity. Elastin can maintain and support skin elasticity and has an anti-aging effect.

[0085] As used herein, the term "sirtuin 1 (SIRT1)" is a NAD + -dependent deacetylase and is regarded as a sensor of cellular energy and metabolism. SIRT1 is considered to be one of the members most related to anti-aging. Studies have found that SIRT1 regulates multiple signaling pathways related to aging through deacetylation, promotes cell survival and resistance to oxidative stress, and slows down the aging process.

[0086] As used herein, the term "sirtuin 3 (SIRT3)" is a NAD +-dependent deacetylase, which can regulate the functions of many mitochondrial proteins in metabolism, oxidative stress and cell survival. It is an instrumental regulator of mitochondrial adaptive responses, including metabolic reprogramming and enhanced antioxidant defense mechanisms. SIRT3 is involved in the regulation of fat metabolism and can help control body weight by promoting fatty acid oxidation and inhibiting fat synthesis, which is of great significance in body management and shaping.

[0087] As used herein, the term "sirtuin 6 (SIRT6)" is an NAD + -dependent deacetylase that plays a role in chromatin signaling and genome maintenance. Through these functions, SIRT6 can prevent aging-related diseases, and SIRT6 and SIRT1 play roles in the metabolism and inflammatory responses of skin cells. By activating these genes, skin hydration, elasticity and luster can be improved, and wrinkles can be reduced.

[0088] As used herein, the term "sirtuin 7 (SIRT7)" is a recently identified member of the SIRT family. SIRT7 plays an important role in DNA repair and maintaining genome stability, helping cells maintain physiological homeostasis and providing anti-aging protection.

[0089] In a first aspect, the present application provides a replication-defective recombinant herpes simplex virus type 1 (HSV-1) vector. The nucleotide sequence of a specific genomic segment in the viral vector genome is disrupted or knocked out.

[0090] As is well known in the art, a "genomic segment" refers to a specific nucleotide position in the genome. The nucleotide positions of the specific genomic segments of the HSV-1 genome in the present application are determined with reference to the genome of the HSV-1 laboratory virus strain 17 (NC_001806.2). Those skilled in the art can understand that in the genomes of different HSV-1 virus strains, the specific nucleotide positions may vary, but those skilled in the art can determine the nucleotide positions corresponding to the specific genomic segments disclosed in the present application based on conventional techniques. Therefore, the present application covers the specific genomic segments determined with reference to the genome of the HSV-1 laboratory virus strain 17 (NC_001806.2), and also covers the genomic segments corresponding to this genomic segment in different HSV-1 virus strains.

[0091] In some embodiments, the genome is selected from the ICP34.5 gene, ICP6 gene, ICP0 gene, ICP47 gene, ICP22 gene, ICP4 gene, ICP27 gene, U S 3 gene, U L 56 gene, functional VP16 gene, VHS gene, UNG gene, IR L / IR SOne or more of the regions, glycoprotein H gene, and thymidine kinase gene are disrupted or knocked out.

[0092] In some embodiments, ICP0 and / or ICP22 in the genome are disrupted or knocked out.

[0093] In some embodiments, the genomic segment of the nucleotide sequence from 1 - 100 nucleotides upstream and downstream of nucleotide position 126785 to 1 - 100 nucleotides upstream and downstream of nucleotide position 131176 in the genome of a replication-defective recombinant herpes simplex virus type 1 (HSV-1) vector is disrupted or knocked out. In this application, the numerical range of 1 - 100 covers any positive integer within this range. In this application, the term "genomic segment of the nucleotide sequence from 1 - 100 nucleotides upstream and downstream of nucleotide position 126785 to 1 - 100 nucleotides upstream and downstream of nucleotide position 131176" covers the genomic segment of the nucleotide sequence from any positive integer position nucleotide within the range of 1 - 100 nucleotides upstream and downstream of nucleotide position 126785 to any positive integer position nucleotide within the range of 1 - 100 nucleotides upstream and downstream of nucleotide position 131176. For example, the genomic segments of the nucleotide sequences from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides upstream and downstream of nucleotide position 126785 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides upstream and downstream of nucleotide position 131176 are disrupted or knocked out (any combination of the specific nucleotide sites upstream and downstream of nucleotide position 126785 and the specific nucleotide sites upstream and downstream of nucleotide position 131176 described here is possible).

[0094] In some embodiments, the genomic segment of the nucleotide sequence from nucleotide positions 147025 plus or minus 1 - 823 nucleotides to nucleotide positions 151439 plus or minus 1 - 93 nucleotides in the genome of a replication-defective recombinant herpes simplex virus type 1 (HSV-1) vector is disrupted or knocked out. In this application, the numerical ranges of 1 - 823 and 1 - 93 cover any positive integer within the above numerical ranges. In this application, the term "genomic segment of the nucleotide sequence from nucleotide positions 147025 plus or minus 1 - 823 nucleotides to nucleotide positions 151439 plus or minus 1 - 93 nucleotides" covers the genomic segment of the nucleotide sequence from any positive integer nucleotide position within the range of nucleotide positions 147025 plus or minus 1 - 823 nucleotides to any positive integer nucleotide position within the range of nucleotide positions 151439 plus or minus 1 - 93 nucleotides. For example, the genomic segment of the nucleotide sequence from nucleotide positions 147025 plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 805, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823 nucleotides to nucleotide positions 151439 plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93 nucleotides is disrupted or knocked out (any combination of the specific nucleotide sites upstream and downstream of nucleotide position 147025 and the specific nucleotide sites upstream and downstream of nucleotide position 151439 described herein is possible).

[0095] In some embodiments, the genomic segment of the nucleotide sequence from nucleotide 113494 plus or minus 1 to 257 nucleotides upstream and downstream to nucleotide 115314 plus or minus 1 to 223 nucleotides upstream and downstream in the genome of a replication-deficient recombinant herpes simplex virus type 1 (HSV-1) vector is disrupted or knocked out. In this application, the numerical ranges of 1-257 and 1-223 cover any positive integer within the above numerical ranges. In this application, the term "genomic segment of the nucleotide sequence from nucleotide 113494 plus or minus 1 to 257 nucleotides upstream and downstream to nucleotide 115314 plus or minus 1 to 223 nucleotides upstream and downstream" covers the genomic segment of the nucleotide sequence from any positive integer nucleotide within the range of nucleotide 113494 plus or minus 1 to 257 nucleotides upstream and downstream to any positive integer nucleotide within the range of nucleotide 115314 plus or minus 1 to 223 nucleotides upstream and downstream. For example, the genomic segment of the nucleotide sequence from nucleotide 113494 plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 251, 252, 253, 254, 255, 256, 257 nucleotides upstream and downstream to nucleotide 115314 plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 205, 210, 215, 220, 221, 222, 223 nucleotides upstream and downstream is disrupted or knocked out (any combination of the specific nucleotide sites upstream and downstream of nucleotide 113494 and the specific nucleotide sites upstream and downstream of nucleotide 115314 described herein is possible).

[0096] In some embodiments, the viral vector is cultured in complementing cells.

[0097] In some embodiments, the complementing cells are Vero cells.

[0098] In some embodiments, the viral vector contains one or more introduced exogenous protein-coding genes.

[0099] In some embodiments, the one or more exogenous protein-coding genes are introduced at the same or different genomic loci that are disrupted or knocked out as described above.

[0100] In some embodiments, the exogenous protein is selected from one or more of the following proteins: epidermal growth factor (EGF), fibroblast growth factor (FGF), type I collagen (COL1), type II collagen (COL2), type III collagen (COL3), type IV collagen (COL4), type VII collagen (COL7), type IX collagen (COL9), type XVII collagen (COL17), and elastin (ELN).

[0101] In a second aspect, the present application provides the recombinant herpes simplex virus type I vector described in the first aspect of the present application for beauty treatment. Optionally, the beauty treatment includes one or more of the following: improving skin elasticity, improving skin dryness, improving skin vitality, removing wrinkles, and / or delaying skin aging.

[0102] In a third aspect, the present application provides the recombinant herpes simplex virus type I vector described in the first aspect of the present application for treating epidermolysis bullosa.

[0103] In a fourth aspect, the present application provides a composition comprising the recombinant herpes simplex virus type I vector described in the first aspect of the present application and a pharmaceutically acceptable solvent or excipient.

[0104] In some embodiments, the composition is a pharmaceutical composition or a composition for beauty treatment. Optionally, the composition for beauty treatment is a composition for topical application, subcutaneous injection, intradermal injection, or microneedle injection for skin improvement.

[0105] In some embodiments, the beauty composition is used to maintain and / or increase the gene and / or protein expression of collagen XVII, preferably protein expression, in the following: skin and / or mucosa and / or scalp and / or skin appendages, particularly skin glands, especially sweat glands and sebaceous glands and hair follicles, particularly the basement membrane of the skin and / or mucosa and / or scalp and / or sebaceous glands, and / or adipocytes, endothelial cells, the basement membrane of sweat glands and / or hair follicles; preferably used to increase the thickness of the dermal-epidermal junction on the skin and / or mucosa and / or scalp; used to maintain and / or increase the firmness and / or elasticity of the skin and / or mucosa and / or scalp; and / or used to reduce the visibility of skin pores and / or make the skin smoother and / or used to limit and / or reduce sweating and / or used to limit and / or reduce hair and / or body hair loss and / or increase hair and / or body hair growth and / or reduce or limit sebum production.

[0106] The cosmetic composition may further comprise one or more cosmetically acceptable excipients selected from surfactants and / or emulsifiers, preservatives, buffers, chelating agents, denaturing agents, opacifying agents, pH regulators, reducing agents, stabilizers, thickeners, gelling agents, film-forming polymers, fillers, mattifying agents, brightening agents, pigments, dyes, fragrances, and mixtures thereof. CTFA (Cosmetic Ingredient Handbook, 2nd Edition (1992)) describes various cosmetic excipients suitable for use in the present invention.

[0107] Advantageously, the excipients are selected from polyglycerols, esters, cellulose polymers and derivatives, lanolin derivatives, phospholipids, lactoferrin, lactoperoxidase, sucrose-based stabilizers, vitamin E and its derivatives, xanthan gum, natural and synthetic waxes, vegetable oils, triglycerides, unsaponifiable substances, phytosterols, silicones, protein hydrolysates, betaines, amine oxides, plant extracts, sucrose esters, titanium dioxide, glycine, and parabens, more preferably selected from stearyl polyoxyethylene (2) ether, stearyl polyoxyethylene (21) ether, ethylene glycol-15 stearyl ether, cetearyl alcohol, phenoxyethanol, methylparaben, ethylparaben, propylparaben, butylparaben, butylene glycol, octylene glycol, natural tocopherol, glycerin, sodium dihydroxycetyl phosphate, isopropyl hydroxycetyl ether, ethylene glycol stearate, triisononanoin, octyl cocoate, polyacrylamide, isoparaffin, laureth-7, carbomer, propylene glycol, hexylene glycol, glycerin, bisabolol, dimethicone, sodium hydroxide, PEG 30-dipolyhydroxystearate, caprylic / capric triglyceride, cetearyl octanoate, dibutyl adipate, grape seed oil, jojoba oil, magnesium sulfate, EDTA, cyclomethicone, xanthan gum, citric acid, sodium lauryl sulfate, mineral oil and wax, isostearyl isostearate, propylene glycol dinonanoate, propylene glycol isostearate, PEG 8, beeswax, hydrogenated palm kernel oil glyceride, lanolin oil, sesame oil, cetyl lactate, lanolin alcohol, castor oil, titanium dioxide, lactose, sucrose, low density polyethylene, isotonic saline solution, and mixtures thereof.

[0108] The cosmetic composition of the present invention may be selected from aqueous or oily solutions, creams or hydrogels or oleogels, in particular bath gels, milks, lotions, microemulsions or nanoemulsions, which are especially of the oil-in-water or water-in-oil or multiple or silicone type, face masks, serums, lotions, liquid soaps, dermatological sticks, ointments, foams, patches, anhydrous products, preferably in the form of a liquid or a paste or a solid, such as a cosmetic powder, stick or lipstick, especially in the form of a lipstick. Advantageously, it is a cream or a serum.

[0109] In a fifth aspect, the present application provides a cosmetic method, comprising administering to a subject in need an effective amount of the cosmetic composition according to the fourth aspect of the present application. Optionally, the cosmetic includes one or more of the following: improving skin dryness, improving skin vitality, removing wrinkles, and / or delaying skin aging. Optionally, the cosmetic composition is a composition for topical application, subcutaneous injection, intradermal injection, or microneedle injection for skin improvement.

[0110] In a sixth aspect, the present application provides a method for treating a disease, comprising administering to a subject in need an effective amount of the composition according to the fourth aspect of the present application, wherein the disease is epidermolysis bullosa.

[0111] In a seventh aspect, the present application provides a complementing cell for culturing a herpes simplex virus vector, wherein the herpes simplex virus vector is the recombinant type I herpes simplex virus (HSV-1) vector according to the first aspect of the present application.

[0112] In some embodiments, the complementing cell is a Vero cell.

[0113] In an eighth aspect, the present application provides the use of Vero cells for culturing a herpes simplex virus vector, wherein the herpes simplex virus vector is the recombinant type I herpes simplex virus (HSV-1) vector according to the first aspect of the present application.

[0114] Examples

[0115] Preparation Example 1 Preparation of an HSV-1 replication-defective recombinant virus vector

[0116] Using genetic engineering methods, IR was knocked out on the HSV-1 virus genome according to the method in the literature (Stuart C R, Seymour L D, James F N. Mutant simplexviruses and uses thereof. WO9938955A3) S and TR SPositions 126785 - 131735 bp and 146471 - 151439 bp in the region, which involve the ICP4 region of the HSV-1 virus. At the same time, the ICP4 promoter and the ICP4 gene were deleted, and a green fluorescent protein (GFP) - labeled gene was inserted at this position to obtain the recombinant virus vector A1( Figure 3 ).

[0117] According to the method in the literature (Howard, M., Kershaw, T., Gibb, B. et al. High efficiency gene transfer to the central nervous system of rodents and primates using herpesvirus vectors lacking functional ICP27 and ICP34.5. Gene Ther 5, 1137–1147 (1998)), the U L region at positions 113280 - 116878 bp on the HSV-1 virus genome was knocked out, and ICP27, U L 55 and U L 56 genes were deleted, and a GFP gene was inserted at this position to obtain the recombinant virus vector B1( Figure 4 ).

[0118] Optimization of the HSV-1 replication - defective virus vector in Preparation Example 2

[0119] Knock out the IR S and TR S regions at positions 126785 - 131176 bp and 147025 - 151439 bp on the HSV-1 virus genome according to the method described in the patent application with the publication number CN113046331A, which involves the ICP4 gene region of the HSV-1 virus, and insert a GFP gene at this position to obtain the virus vector A2( Figure 5 ).

[0120] Knock out the U L region at positions 113494 - 115314 bp on the HSV-1 virus genome according to the method described in the patent application with the publication number CN113046331A, which involves the ICP27 gene region of the HSV-1 virus, and insert a GFP gene at this position to obtain the virus vector B2( Figure 6 ).

[0121] Viral vectors with deletions of the HSV-1 replication-essential genes ICP4 and ICP27 need to be prepared and grown on complementing cell lines that can compensate for the ICP4 and ICP27 genes. The ICP4 gene (Gene ID: 2703392) and the ICP27 gene (Gene ID: 24271474) were simultaneously cloned into a plasmid (Takara, 3244) carrying a puromycin selection gene. According to the instructions of Lipofectamine TM 3000 (Thermo, L3000015), the plasmid was stably transfected into Vero cells by the liposome method to obtain the ZH02 complementing cell line that supports the replication of the replication-defective viral vector. The ICP0 gene (Gene ID: 2703390) is a non-essential gene for HSV-1 replication, but its knockout affects the expression of foreign genes. The ICP4, ICP27, and ICP0 genes were simultaneously cloned into a plasmid carrying a puromycin selection gene, and the plasmid was stably transfected into Vero cells by the liposome method to obtain the ZH06 complementing cell line that supports the replication of the replication-defective virus.

[0122] Example 1 Optimized HSV-1 Replication-Defective Recombinant Viral Vectors Express Higher Levels of Foreign Genes

[0123] Vero African green monkey kidney cells (ATCC) and human embryonic skin fibroblasts CCC-ESF-1 (Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences) were cultured separately in DMEM medium containing 10% serum at 37 °C and 5% CO 2 conditions. The two types of cells were respectively seeded onto six-well plates, with 2 experimental groups set for each type of cell, 2 wells in each group, and blank cell control wells were also set simultaneously. After 24 hours, the recombinant viral vectors A1, recombinant viral vector B1, recombinant viral vector A2, and recombinant viral vector B2 were respectively inoculated into each experimental group at an MOI of 1. After 72 hours, the green fluorescence was observed under a fluorescence microscope and photographed. The results showed that the green fluorescence expressed by recombinant viral vectors A2 and B2 after infecting the two types of cells was brighter than that of A1 and B1; the green fluorescence expressed by recombinant viral vector A2 after infecting the two types of cells was brighter than that of recombinant viral vector B2 ( Figure 7 ).

[0124] The experimental results indicate that in the ICP4 and ICP27 regions, the recombinant viral vectors obtained by inserting foreign genes into specific positions in the above regions of the HSV-1 genome according to the present application express higher amounts of foreign genes compared to the recombinant viral vectors obtained by the methods in the literature. In particular, the recombinant viral vectors obtained by inserting foreign genes into specific positions in the ICP4 region express the highest amounts of foreign genes.

[0125] HSV-1 has a genome of 152 kb. After the virus genome infects cells, there will be epigenetic modifications, and different genomic regions have different modifications, which results in different expression levels of foreign genes at different sites. After knocking out different viral fragments at the same site (such as ICP4 or ICP27) and inserting foreign genes at the same time, the expression levels of foreign genes will also be different, mainly because the knocked-out viral sequences may contain regulatory elements of gene expression (such as promoters, enhancers, cis- or trans-regulatory elements). In this application, the position of the viral knockout fragment in the foreign gene insertion region is optimized. The recombinant virus vector obtained by inserting the foreign gene at a specific position in the above-mentioned region of the HSV-1 genome expresses a higher amount of foreign gene than the recombinant virus vector obtained by the method in the literature.

[0126] Preparation Example 3 Preparation of HSV-1 replication-defective recombinant virus vector carrying foreign gene

[0127] According to the method described in the patent application with the application publication number CN113046331A, replace the GFP gene at the knockout position of recombinant virus vector A2 and insert the COL7A1 gene (NM_000094.4) encoding type VII collagen to obtain recombinant virus vector C( Figure 8 ). On the basis of recombinant virus vector A2, further knock out the ICP34.5 gene, replace the GFP gene and insert the COL7A1 gene to obtain recombinant virus vector D( Figure 9 ). On the basis of recombinant virus vector A2, knock out ICP22 and ICP27, replace the GFP gene and insert the COL7A1 gene to obtain recombinant virus vector E( Figure 10 ).

[0128] According to the method described in the patent application with the application publication number CN113046331A, replace the GFP gene at the knockout position of recombinant virus vector B2 and insert the COL7A1 gene to obtain recombinant virus vector F( Figure 11 ). According to the method in the literature (Miyagawa Y, Marino P, et al., Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity. Proc Natl Acad Sci U SA. 2015, 112(13):E1632-41.), on the basis of recombinant virus vector B2, knock out ICP0, ICP22, ICP4 and the JOINT region, replace the GFP gene and insert the COL7A1 gene to obtain recombinant virus vector G( Figure 12 ).

[0129] Example 2: Verification of the expression of genes carried by HSV-1 replication-defective recombinant virus vectors

[0130] Inoculate the ZH02 and ZH06 complement cell lines into six-well plates and proliferate and culture them under the conditions of 37 °C and 5% CO 2 2. After culturing the cells for 2 days, add recombinant virus vectors C, D, and E to the ZH02 cells at an MOI of 0.01, and add recombinant virus vectors F and G to the ZH06 cells. Set up cell negative control wells. The transfected cells continue to be cultured under the conditions of 37 °C and 5% CO 2 After 3 days of culture, take the culture supernatant and detect the expression level of type VII collagen using an ELISA kit (Abclonal, RK01162). The results show that there is no significant difference in the expression level of the target protein after transfection of the recombinant virus vectors C, D, and E into the cells, and there is no significant difference in the expression level of the target protein after transfection of the recombinant virus vectors F and G into the cells.

[0131] The results indicate that the recombinant virus vectors obtained by specifically modifying the ICP4 and ICP27 regions of the HSV-1 genome in this application and inserting the functional gene COL7A1, and further modifying them did not significantly affect the expression level of the COL7A1 gene ( Figure 13 ).

[0132] Example 3: Functional verification of HSV-1 vectors carrying the type VII collagen gene (COL7A1)

[0133] Use Western blotting to detect the expression level of the COL7A1 gene on human keratinocytes and fibroblasts by the recombinant virus vector D. Human primary keratinocytes and human immortalized dermal fibroblasts HSF (SV40) (Hefei Wanwu) are cultured in DMEM medium containing 15% fetal bovine serum. Inoculate the cells in 6-well plates (1.5×10 6In (number of cells per well), recombinant virus vector D was added at an MOI of 2, and the supernatant was harvested at 24, 48, and 72 hours respectively. 50 μl of each supernatant sample was added to 6× loading buffer (DL101 - 02, TransGen Biotech) and boiled for 10 minutes to prepare the loading mixture, which was then added to the wells of a precast denaturing protein gel (PG41510 - S, Solarbio). Electrophoresis was carried out at 150 V for 50 minutes, and then the proteins on the gel were transferred to a PVDF membrane (ISEQ0001010, Millipore) by electroblotting at 200 mA for 2 hours. Protein immunoblotting experiments were performed using specific antibodies, anti - COL7A1 antibody (including goat anti - rabbit secondary antibody) (D161588 - 0100, Sangon Biotech) and β - actin antibody (PA1 - 183, Thermo). Finally, chemiluminescence detection was carried out using the reagent Pierce ECL Western Blotting Substrate (32109, Thermo). The results showed that recombinant virus vector D could express the target gene COL7A1 in human keratinocytes and fibroblasts, produce type VII collagen, and it could be detected at 24 hours ( Figure 14 and Figure 15 ).

[0134] Type VII collagen is a structural protein that anchors the epidermis and dermis. Immunofluorescence technique (IF) and immunohistochemistry (IHC) were used to detect the expression level of type VII collagen in mouse skin tissues. C57BL / 6J mice aged 6 - 8 weeks were selected for the experiment. A recombinant virus vector D administration group and a PBS control group were set up, with 1 mouse in each group. The hair on the back was shaved in a 1 cm × 1 cm square, and there were 3 injection sites, with a total volume of 100 μl and a drug titer of 1E8 pfu / ml. Drugs were administered on the 1st and 3rd days, and the administered part was sampled on the 5th day. After the tissues were fixed overnight with 4% paraformaldehyde, they were dehydrated and sedimented in a gradient of 20% - 30% sucrose solution. They were embedded with OCT embedding medium and quickly frozen in a cryogenic refrigerator. Sections were cut using a cryostat (CM1950, Leica) at a thickness of 10 μm, and the sections were stored at - 20°C. IF detection: After the frozen slides were rewarmed at room temperature, they were incubated with 0.1% TritonX - 100 and blocked with serum. They were incubated with an antibody against the NC1 domain at the N - terminus of type VII collagen (NBP2 - 37900, NOVUS - BIO) and an antibody against the NC2 domain at the C - terminus of type VII collagen (D161588, Sangon Biotech) respectively. The secondary antibody used was goat anti - rabbit IgG (H + L) (A - 11008, Invitrogen). After incubation with DAPI, the slides were sealed with 50% glycerol for detection. A negative control without primary antibody was set up in the experiment. IHC detection: After the frozen slides were rewarmed at room temperature, they were stained and prepared using an immunohistochemistry kit (rabbit) (KIT - 9707, Maixin Biotech). A negative control without primary antibody was set up in the experiment.

[0135] IF staining ( Figure 16 , Figure 17 ) and IHC staining ( Figure 18 , Figure 19 ) results showed that after administration of recombinant viral vector D twice, staining of NC1 and NC2 was visible between the epidermis and dermis of mice and in the blood vessel walls, indicating that recombinant viral vector D could correctly express intact type VII collagen in mice, and that type VII collagen could be correctly distributed between the epidermis and dermis, with good physiological activity.

[0136] Example 4 Efficacy of HSV-1 Vector Carrying Type VII Collagen Gene (COL7A1) in DEB Mice

[0137] Deletion or mutation of the gene encoding COL7 protein can lead to the occurrence of dystrophic epidermolysis bullosa (DEB). Delivery of the COL7A1 gene encoding COL7 protein into skin cells for gene therapy is a very promising therapy. A pharmacodynamic experiment of HSV-1 vector carrying COL7A1 gene (NM_000094.4) was carried out on a DEB mouse model (from Jicui Yaokang). A PBS control group, a recombinant viral vector control group without the therapeutic gene, and a recombinant viral vector D treatment group were set up, with 3 mice in each group. The viral vector titer was 1E8 pfu / ml, and the drug was administered once every 2 days. The drug was administered according to the wound size, and the wound healing situation was observed. The results were as Figure 20 shown. By observing typical mouse healing pictures, it was found that the wound healed around 3 days after treatment with recombinant viral vector D, while the wound healing in the PBS and recombinant viral vector control groups was not obvious.

[0138] Example 5 Expression Verification of HSV-1 Vectors Carrying Other Collagen Genes

[0139] On the basis of recombinant viral vector A2, the ICP34.5 gene was further knocked out, and the GFP gene was replaced at the position where the ICP4 gene was knocked out and the type I collagen gene (COL1A1) (NM_000088.4) was inserted to obtain recombinant viral vector H ( Figure 21 ), the type III collagen gene (COL3A1) (NM_000090.4) was inserted to obtain recombinant viral vector I ( Figure 22 ), and the type XVII collagen gene (COL17A1) (NM_000494.4) was inserted to obtain recombinant viral vector J ( Figure 23 ).

[0140] The levels of collagen expression by recombinant viral vectors H, I, and J were detected by Western blotting in human keratinocyte HaCat cells. HaCat cells (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in DMEM medium containing 15% fetal bovine serum. The cells were seeded in 6-well plates (1.5×10 6 cells / well), and recombinant viral vectors H, I, and J were added respectively at an MOI of 0.5, with blank cell controls set. After 72 hours, the cells were harvested, and whole protein lysates were prepared using a protein extraction kit (DE101, TransGen Biotech). The protein content of the whole lysate was detected using a BCA protein assay kit (23227, Thermo). 30 μg of each protein sample was loaded into the wells of a precast denaturing protein gel, and electrophoresis was performed at 100 V for 90 minutes, followed by transfer of the proteins from the gel to a PVDF membrane at 200 mA for 2 hours. Corresponding detections were carried out using specific antibodies, namely COL1A1 antibody (PA5-29569, Thermo), COL3A1 antibody (B-10) (sc-271249, Santa Cruz), and Collagen XVII antibody (NBP1-91800, novus biologicals), and the secondary antibody used was HRP-labeled goat anti-mouse IgG (H+L) (A0216, Beyotime). Finally, chemiluminescence detection was performed using the reagent Pierce ECL Western Blotting Substrate.

[0141] The results showed that recombinant viral vector H could significantly express type I collagen ( Figure 24 ) on HaCat cells, recombinant viral vector I could significantly express type III collagen ( Figure 25 ) on HaCat cells, and recombinant viral vector J could significantly express type XVII collagen ( Figure 26 ) on HaCat cells.

[0142] Example 6 Expression verification of HSV-1 vectors carrying SIRT genes

[0143] Based on recombinant viral vector A2, the ICP34.5 gene was further knocked out, and at the position where the ICP4 gene was knocked out, the GFP gene was replaced and the SIRT1 gene (NM_012238.5) was inserted to obtain recombinant viral vector K ( Figure 27 ), the SIRT3 gene (NM_001370322.1) was inserted to obtain recombinant viral vector L ( Figure 28 ), and the SIRT6 gene (NM_016539.4) was inserted to obtain recombinant viral vector M ( Figure 29 ).

[0144] The protein immunoblotting method was used to detect the levels of the target proteins expressed by recombinant viral vectors K, L, and M on ZH02 cells. The cells were seeded in 6-well plates (1.5×10 6 cells / well), and recombinant viral vectors K, L, and M were added respectively at an MOI of 0.5, and a cell control was set. After 72 hours, the cells were harvested, and whole protein lysates were prepared using a protein extraction kit (DE101, TransGen Biotech). The content of the whole lysate protein was detected using a BCA protein assay kit (23227, Thermo). 30 μg of each protein sample was loaded into the wells of a precast denaturing protein gel, electrophoresed at 90 V for 40 minutes and then continued at 110 V for 60 minutes, and then transferred to a PVDF membrane by electroblotting at 200 mA for 2 hours. Specific antibodies SIRT1 (B-10) mouse monoclonal IgG2b (sc-74504, Santa), Mouse anti-Sirtuin3 / SIRT3 (6B2A1)-BSA Free mA (NBP2-52563, novus biologicals), and SIRT6 (2G1H1) mouse monoclonal IgG1 (sc-517196, Santa) were used for corresponding detections, and the secondary antibody was HRP-labeled goat anti-mouse IgG (H+L) (A0216, Beyotime). Finally, the reagent Pierce ECL Western Blotting Substrate was used for chemiluminescence detection. The results showed that recombinant viral vector K could significantly express SIRT1 protein on the cells ( Figure 30 ), recombinant viral vector L could significantly express SIRT3 protein on the cells ( Figure 31 ), and recombinant viral vector M could significantly express SIRT6 protein on the cells ( Figure 32 ).

[0145] The above are only the preferred embodiments of the present application and do not limit the present application in any form. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present application without departing from the technical solution content of the present application still fall within the protection scope of the technical solution of the present application.

Claims

1. A replication-deficient recombinant herpes simplex virus type I (HSV-1) vector, wherein the nucleotide sequence of the following genomic segments in the genome is destroyed or knocked out: 1) the nucleotide sequence from 1-100 nucleotides upstream and downstream of nucleotide 126785 to 1-100 nucleotides upstream and downstream of nucleotide 131176, and the nucleotide sequence from 1-823 nucleotides upstream and downstream of nucleotide 147025 to 1-93 nucleotides upstream and downstream of nucleotide 151439; and / or 2) the nucleotide sequence from nucleotides 1-257 upstream and downstream of nucleotide 113494 to nucleotides 1-223 upstream and downstream of nucleotide 115314; in, The nucleotide positions of the above HSV-1 genome were determined with reference to the HSV-1 laboratory strain 17 genome (NC_001806.2). Furthermore, the disrupted or knocked-out genomic site contains one or more exogenous protein encoding genes selected from the group consisting of: collagen, elastin and growth factor.

2. The recombinant herpes simplex virus type I vector according to claim 1, further, the genome is selected from ICP34.5 gene, ICP6 gene, ICP0 gene, ICP47 gene, ICP22 gene, ICP4 gene, ICP27 gene, U S 3 genes, U L 56 genes, functional VP16 gene, VHS gene, UNG gene, IR L / IR S One or more of the glycoprotein H gene, thymidine kinase gene, or region is disrupted or knocked out.

3. The recombinant herpes simplex virus type 1 (HSV-1) vector according to claim 1, wherein the viral vector is cultured in compensatory cells. The recombinant herpes simplex virus type 1 (HSV-1) vector according to claim 3 , wherein the compensating cells are Vero cells.

5. The recombinant herpes simplex virus type I vector according to any one of claims 1-2, further, ICP0 and / or ICP22 in the genome are destroyed or knocked out.

6. The recombinant herpes simplex virus type I vector according to any one of claims 1 to 5, wherein the exogenous protein encoding gene is introduced into the same or different genomic site that is destroyed or knocked out.

7. A recombinant herpes simplex virus type I vector according to claim 6, wherein the exogenous protein is selected from one or more of the following proteins: epidermal growth factor (EGF), fibroblast growth factor (FGF), type I collagen (COL1), type II collagen (COL2), type III collagen (COL3), type IV collagen (COL4), type VII collagen (COL7), type IX collagen (COL9), type XVII collagen (COL17), elastin (ELN), sirtuin 1 (SIRT1), sirtuin 3 (SIRT3), sirtuin 6 (SIRT6), and sirtuin 7 (SIRT7).

8. The recombinant herpes simplex virus type I according to any one of claims 1 to 7, for use in cosmetic treatment, wherein the cosmetic treatment comprises one or more of the following: improving skin elasticity, improving skin dryness, improving skin vitality, removing wrinkles, and / or delaying skin aging.

9. The recombinant herpes simplex virus type I according to any one of claims 1 to 7, for use in treating epidermolysis bullosa.

10. A composition comprising the recombinant herpes simplex virus type I vector according to any one of claims 1 to 7 and a solvent or excipient acceptable to humans.

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