Recombinant vector for treating dystrophinopathy and use thereof

Abstract

Provided is a recombinant vector, and specifically provided is a replication-defective recombinant herpes simplex virus vector and the use thereof. Specifically, gene editing is performed on a herpes simplex virus genome to obtain a replication-defective recombinant viral vector, and a gene encoding dystrophin is introduced into the vector genome. The replication-defective recombinant herpes simplex virus vector, as an excellent delivery vector, has a significant therapeutic effect on a mouse model of muscular dystrophy.

Description

Recombinant vectors for the treatment of dystrophinosis and their applications Technical Field

[0001] This application relates to the field of biotechnology, and more specifically, to a recombinant vector, such as a replication-defective recombinant herpes simplex virus vector and its applications. Background Technology

[0002] Dystrophinopathy is a group of X-linked recessive inherited muscle diseases primarily affecting skeletal muscle and / or cardiac muscle, caused by pathogenic mutations in the dystrophin (Dys) gene (OMIM: 300377) located at Xp21.2. These include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), intermediate muscular dystrophy (IMD), and X-linked dilated cardiomyopathy (XLDCM). The Dys gene, approximately 2.2 Mb in length and containing 79 exons, is the largest known human gene and is prone to mutation due to its length. The Dys protein is a structural protein that maintains the integrity of muscle fibers and protects them from damage caused by contraction. In patients with dysproteinemia, the lack of dysprotein mainly leads to defects in skeletal muscle cell membranes, leakage of intracellular creatine kinase and other substances, muscle cell necrosis, proliferation of adipose tissue and fibrous connective tissue, impairing the stability and function of muscle fibers, and ultimately resulting in muscle degeneration.

[0003] Dementia malignancy (DMD) is a fatal disease and one of the most common fatal genetic disorders, primarily affecting males. Globally, the incidence of DMD in male newborns is approximately 1 in 3,500, and the estimated number of symptomatic female carriers is 1 in 100,000–1 in 45,000. Thousands of different gene mutations exist in DMD patients, occurring in almost any part of the Dys gene. DMD patients develop an inflammatory response shortly after birth, leading to muscle fibrosis, atrophy, and degeneration. Muscle damage initially occurs in the proximal muscles of the lower limbs, accompanied by decreased muscle strength and progressive contractures with gait disturbances. Subsequently, as upper limb muscle strength decreases, patients develop arm contractures, and respiratory and cardiac muscle damage, leading to respiratory and / or heart failure, resulting in a life expectancy rarely exceeding 40 years. Despite decades of research, DMD remains incurable. Standardized multidisciplinary comprehensive treatment can slow disease progression, prolong life, and improve quality of life. The most commonly used medication for treating DMD clinically is glucocorticoids. Glucocorticoids are used to reduce inflammation-induced muscle damage, thereby decreasing muscle strength loss and slowing disease progression. However, long-term use of hormones brings many side effects, such as osteoporosis, obesity, hirsutism, and adrenal insufficiency. Moreover, these hormone drugs do not treat the cause and cannot prevent the occurrence and progression of DMD. They only play a role in temporarily prolonging the patient's survival (Chinese Guidelines for the Diagnosis and Treatment of Duchenne Muscular Dystrophy, Chinese Journal of Neurology, 2016, 49(1): 17-20).

[0004] The prevalence of BMD is at least 2.4 per 100,000 men. The clinical course is similar to that of DMD, but the disease progresses slowly and skeletal muscle is less affected. Heart failure is a common cause of onset and death, with an average age of death of around 40 years. The diagnosis of BMD and DMD is roughly the same. There are few studies on the use of corticosteroids to treat BMD, and there is no consensus yet. At present, the emphasis is on the timeliness and regularity of application, so as to achieve the goal of delaying the course of the disease and reducing side effects (Luo Xinlin, Fan Chaomei, Progress in the diagnosis and treatment of Becker type muscular dystrophy cardiomyopathy, Chinese Journal of Heart Failure and Cardiomyopathy, 2021, 05(2): 123-127).

[0005] The severity of IMD falls between that of DMD and BMD. Typical XLDCM patients usually develop the disease between the ages of 10 and 20, and quickly develop congestive heart failure associated with dilated cardiomyopathy, often dying from heart failure within a few years of diagnosis. Patients may have no clinical manifestations of skeletal muscle involvement or very mild skeletal muscle involvement, which is often manifested as hypercreatine kinaseemia, exercise-induced myalgia or muscle spasms, and gastrocnemius hypertrophy (Chinese Guidelines for the Diagnosis of Anti-dystrophin Disease, Chinese Medical Journal, 2024, 104(11): 822-833).

[0006] The development of molecular and cellular medicine has spurred a series of promising cell and gene therapy strategies. These new strategies aim to restore the normal expression of dystrophin, thereby improving muscle function in DMD patients. Cell therapy is still in its early stages; some in vitro results have not yet been replicated in human studies, and cell therapy still awaits clinical trials. Gene therapy aims to restore missing dystrophin by supplementing or repairing the Dys gene at the gene level. Currently, six gene therapy drugs for DMD have been approved for marketing.

[0007] Gene therapy for DMD includes four approaches: exon skipping, stop codon reading through, gene replacement, and gene editing. Currently available exon skipping drugs target patients with mutations in exons 45, 51, and 53, which account for approximately 8%, 13%, and 8% of DMD patients, respectively; they are ineffective for patients with other exon mutations. Stop codon reading through drugs reduce ribosome sensitivity to stop codons and are suitable for patients with nonsense mutations in the Dys gene, accounting for approximately 13% of DMD patients. Gene replacement therapy uses viral vectors such as AAV to deliver the Dys gene. However, due to the small packaging size of AAV viral vectors, it is difficult to package the complete Dys gene; therefore, a truncated Dys gene is used for replacement, but the efficacy is not ideal. Methods for directly editing the Dys gene mutation sites are still under development. Because the Dys gene fragment is large and has many mutation sites, gene editing is only effective for a subset of patients.

[0008] Herpesviruses cause herpes infections, typically manifesting as clusters of small, localized blisters. To date, eight herpesviruses have been identified as associated with human disease: herpes simplex virus type I (HSV-1), herpes simplex virus type II (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpesvirus type 6 (HHV-6), herpesvirus type 7 (HHV-7), and herpesvirus type 8 (HHV-8). The basic structural features of herpesviruses include a core composed of double-stranded DNA and viral DNA-binding proteins, a capsid protein surrounding the core, a mesolayer composed of globular viral proteins surrounding the nucleocapsid, and an envelope containing various lipoproteins surrounding the mesolayer. The complete viral particle has a diameter of 120-300 nm. The genomes of herpesviruses share similarities, all consisting of inverted repeat sequences and unique internal regions, and exhibit high gene homology. For example, the essential gene U for HSV virus replication L 54 is homologous to gene 4 of VZV virus and gene BmLF1 of EBV virus.

[0009] Herpes simplex virus (HSV) belongs to the Herpesviridae family and includes two subtypes: type I (HSV-1) and type II (HSV-2). Currently, HSV-1 viral vectors are used for gene therapy of major diseases such as cancer, neurodegenerative diseases, genetic diseases, and immune system diseases. Compared to other gene therapy viral vectors, HSV-1 viral vectors have many advantages. The HSV-1 genome is relatively large (Figure 1), allowing it to carry large or multiple exogenous genes. The HSV-1 genome is 152 kb long, while the commonly used gene therapy viral vector—AAV—is only about 4.7 kb. Of 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. Its maximum exogenous gene insertion can reach 30-40 kb. This is particularly important for treating many diseases, especially those involving multiple genes. HSV-1 does not integrate with cellular DNA, replication is controllable, and it has high safety. HSV-1 rarely 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 broad host cell range and high safety, the HSV-1 vector is considered a very promising viral vector for gene therapy. Delivering the Dys gene using an HSV-1 gene therapy vector holds promise as a novel gene therapy method for treating dystrophic amyotrophic lateral sclerosis (DLS). Summary of the Invention

[0010] This application provides a recombinant vector, specifically, a replication-defective recombinant herpes simplex virus vector and its applications. Specifically, this application modifies a specific region of the HSV-1 genome (as shown in Figure 2) to obtain an HSV-1 replication-defective viral vector. Using the viral vector obtained in this application to deliver exogenous genes can effectively treat diseases such as dystrophin syndrome.

[0011] Specifically, this application relates to the following technical solutions:

[0012] Item 1. A recombinant vector, wherein the vector comprises a gene encoding dystrophin.

[0013] Item 2. The recombinant vector according to Item 1, wherein the vector comprises plasmids, lipid nanoparticles (LNPs), transposons, granules, bacterial artificial chromosomes, virus-like particles (VLPs), exosomes, and viral vectors; optionally, the viral vector comprises retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, or any hybrid viral vector thereof.

[0014] Item 3. The recombinant vector according to Item 2, wherein the herpesvirus vector includes herpes simplex virus vector, varicella-zoster virus vector, Epstein-Barr virus vector, cytomegalovirus vector, herpesvirus type 6 vector, herpesvirus type 7 vector, and herpesvirus type 8 vector, and any derivative thereof.

[0015] Item 4. The recombinant vector according to Item 3, wherein the herpes simplex virus vector includes a type I herpes simplex virus vector and a type II herpes simplex virus vector.

[0016] Item 5. Use of the recombinant carrier as described in any one of items 1-4 in the preparation of a medicament for treating muscle diseases.

[0017] Item 6. The use according to Item 5, wherein the muscle disease is dystrophinosis, optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy and / or X-linked dilated cardiomyopathy.

[0018] Item 7. A pharmaceutical composition comprising a recombinant vector as described in any one of Items 1-4.

[0019] Item 8. A method of treating a disease, comprising administering to a subject in need an effective amount of the recombinant carrier as described in any one of items 1-4.

[0020] Item 9. The method according to Item 8, wherein the disease is a muscle disease.

[0021] Item 10. The method according to Item 9, wherein the muscle disease is dystrophinosis, optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy, and / or X-linked dilated cardiomyopathy.

[0022] Item 11. A recombinant herpes simplex virus vector comprising a gene encoding dystrophin, wherein the viral vector genome is selected from the ICP0 gene, ICP4 gene, ICP27 gene, ICP34.5 gene, ICP47 gene, ICP22 gene, ICP6 gene, and U. L 41 genes, U L 55 genes, U L 56 genes and IRL / IR S One or more areas in the region have been destroyed or knocked out.

[0023] Item 12. The recombinant herpes simplex virus vector according to Item 11, wherein the herpes simplex virus vector is a type I herpes simplex virus vector or a type II herpes simplex virus vector.

[0024] Item 13. The recombinant herpes simplex virus vector according to Item 12, wherein the viral vector is cultured in compensating cells.

[0025] Item 14. The recombinant herpes simplex virus vector according to Item 13, wherein the compensating cell is a Vero cell.

[0026] Item 15. The recombinant herpes simplex virus vector according to any one of items 11-14, further comprising the ICP34.5 gene and / or IR gene in the genome. L / IR S The area was destroyed or knocked out.

[0027] Item 16. The recombinant herpes simplex virus vector according to Item 11, wherein the gene encoding dystrophin is introduced at the same or different genomic sites that have been disrupted or knocked out as described above.

[0028] Item 17. Use of the recombinant herpes simplex virus vector as described in any one of items 11-16 in the preparation of a medicament for treating muscle diseases.

[0029] Item 18. The use according to Item 17, wherein the muscle disease is dystrophinosis, optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy and / or X-linked dilated cardiomyopathy.

[0030] Item 19. A pharmaceutical composition comprising a recombinant herpes simplex virus vector as described in any one of items 11-16.

[0031] Item 20. A method of treating a disease, comprising administering to a subject in need an effective amount of a recombinant herpes simplex virus vector as described in any one of items 11-16.

[0032] Item 21. The method according to Item 20, wherein the disease is a muscle disease.

[0033] Item 22. The method according to Item 21, wherein the muscle disease is dystrophinosis, optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy, and / or X-linked dilated cardiomyopathy.

[0034] Item 23. A compensatory cell for assisting in the culture of a recombinant herpes simplex virus vector, wherein the recombinant herpes simplex virus vector is any one of items 11-16.

[0035] Item 24. The compensating cell according to Item 23 is a Vero cell.

[0036] Item 25. Use of Vero cells for the assisted culture of recombinant herpes simplex virus vectors, wherein the recombinant herpes simplex virus vector is a recombinant type I herpes simplex virus and type II herpes simplex virus vector as described in any one of Items 12-16.

[0037] Item 26. Use of the recombinant vector as described in any one of Items 1-4 or the recombinant herpes simplex virus vector as described in any one of Items 11-16 in the treatment of muscle diseases.

[0038] Item 27. The use according to Item 26, wherein the muscle disease is dystrophinosis, optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy and / or X-linked dilated cardiomyopathy.

[0039] It should be understood that, within the scope of this application, the above-described technical features and the technical features specifically described below can be combined with each other to form new or preferred technical solutions. Further details are omitted due to space limitations.

[0040] Because different herpesviruses have different genome lengths, the positions of HSV-1 genome bases (nucleotides) described in the various embodiments of this application are determined with reference to the HSV-1 laboratory virus strain 17 genome (NC_001806.2). The positions in different virus strains corresponding to the HSV-1 genome bases (nucleotides) described in the various embodiments of this application can be obtained through sequence alignment or corresponding gene function. Specifically, when describing the mutation, knockout, insertion, or other modifications to the viral genome in this application, the corresponding base positions of the HSV-1 laboratory virus strain 17 genome are listed. These positions are obtained by aligning the genome sequences of any other herpesvirus strain with the genome sequence of laboratory virus strain 17 using sequence alignment tools such as BLAST. Therefore, any technical solutions involving base positions of other herpesvirus strains corresponding to the base positions of laboratory virus strain 17 involved in this application are also included within the scope of the claims of this application.

[0041] The beneficial effects of this application are as follows: This application provides a gene therapy method for treating DMD / BMD based on an HSV vector, which involves inserting the full-length human Dys gene into a replication-defective HSV viral vector to deliver the Dys gene into the body for the treatment of patients with diseases caused by any Dys gene mutation. This invention can solve the problem that current gene therapy drugs for DMD / BMD are only effective for a portion of patients and have unsatisfactory efficacy. Specifically, this application includes:

[0042] 1. Based on the large-capacity exogenous gene carrying capacity of HSV virus vectors (including HSV-1 and HSV-2), HSV viruses were modified into replication-defective vectors, and complete Dys gene expression cassettes were inserted into the vector genome. The expression cassettes can be inserted into ICP0, ICP27, ICP4, ICP34.5, ICP47, ICP22, ICP6, and U of the HSV replication-defective vector. L 41. U L 55 and IR L / IR S Isosites.

[0043] 2. The expression of the Dys gene is initiated using muscle-specific promoters, including the SM22α smooth muscle cell-specific promoter, the MHCK7 skeletal muscle cell-specific promoter, the ACTA1 myogenic cell-specific promoter, and the MyoG myoblast-specific promoter.

[0044] 3. The efficacy was validated in a mouse model of muscular dystrophy, with administration routes including intravenous and intramuscular injection. Attached Figure Description

[0045] Figure 1 is a schematic diagram of the HSV-1 genome.

[0046] Figure 2 shows a schematic diagram of the ICP4 and ICP27 genes of HSV-1.

[0047] Figure 3 is a schematic diagram of the recombinant viral vector A2.

[0048] Figure 4 is a schematic diagram of the recombinant viral vector H.

[0049] Figure 5 is a schematic diagram of recombinant viral vector I.

[0050] Figure 6 shows the results of Western blot analysis of Dys protein. M: protein molecular weight; 1: cell supernatant; 2-3: cell samples transfected with recombinant viral vector H; 4: cell samples transfected with recombinant viral vector I.

[0051] Figure 7 shows the changes in CK concentration in the serum of mice in each experimental group after drug administration.

[0052] Figure 8 shows the changes in the concentration of TNNI2 in the serum of mice in each experimental group after drug administration.

[0053] Figure 9 shows the forelimb grip strength test results of mice in each experimental group at 12 weeks.

[0054] Figure 10 shows the results of immunohistochemical assay of eMHC protein. Detailed Implementation

[0055] The specific embodiments of this application are described in detail below, but it should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of this application. Experimental methods in the examples below that do not specify specific experimental conditions are generally operated under conventional conditions or as recommended by the manufacturer.

[0056] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated ingredients or components without excluding other ingredients or other components.

[0057] As used in this article, dystrophin is a cytoplasmic protein encoded by the Dys gene and is used to link actin filaments of the cytoskeleton to membrane proteins. Typically located primarily in skeletal and cardiac muscle, dystrophin is expressed in lower amounts in the brain. It acts as a shock absorber during muscle fiber contraction by attaching actin, the contractile mechanism, to the connective tissue layer surrounding each muscle fiber. In muscle, dystrophin is located at the cytoplasmic face of the muscle fiber membrane. The Dys gene, first identified in 1987, is the largest known human gene, approximately 2.5 Mb. It is located on the X chromosome at position Xp21 and contains 79 exons. The most common mutations leading to DMD or BMD are large deletions of one or more exons (60-70%), but duplication mutations (5-10%) and single nucleotide variants (including small deletions or insertions, single base changes, and splice site changes, accounting for approximately 25-35% of pathogenic variants in men with Duchenne muscular dystrophy (DMD) and approximately 10-20% of pathogenic variants in men with Becker muscular dystrophy (BMD)) can also cause pathogenic dystrophin variants.

[0058] Dystrophinopathy (DMD) is a genetic skeletal muscle disease caused by a deficiency of dystrophin, resulting from a mutation in the Deschenne muscular dystrophy gene. It is inherited in an X-linked recessive manner, with the main clinical manifestations being progressive weakness and atrophy of the proximal muscles of the limbs and lumbar girdle muscles, hypertrophy of the gastrocnemius muscle, severely affecting daily motor function. In the late stages, respiratory and cardiac muscles are affected, leading to death. Based on clinical manifestations and the degree of dystrophin deficiency, DMD is generally classified into: Duchenne muscular dystrophy, Benedict's muscular dystrophy, intermediate muscular dystrophy, and / or X-linked dilated cardiomyopathy. In DMD, mutations often lead to frameshifts, resulting in premature stop codons and truncated, nonfunctional, or unstable proteins. Nonsense point mutations can also lead to premature stop codons with the same outcome. Although mutations causing DMD can affect any exon, exons 2-20 and 45-55 are common hotspots for large deletions and duplications. Intra-box defects result in less severe BMD, in which patients express truncated, partially functional dystrophin. IMD is an intermediate stage between DMD and BMD.

[0059] As used herein, the term "replication-deficient" refers to a viral vector that cannot replicate completely and efficiently. A replication-deficient virus is a mutant or defective variant of one or more functions essential for viral genome replication or viral particle synthesis and assembly. Replication-deficient viruses can multiply in complementary cell lines expressing the missing gene product. However, in normal target cells, replication-deficient viruses can express the viral gene product but do not replicate to form infectious progeny viral particles. In some embodiments, a replication-deficient virus or viral vector is a mutant or defective virus or vector that functions one or more functions essential for viral genome replication. In some embodiments, a replication-deficient virus or viral vector is a mutant or defective virus or vector that functions one or more functions essential for viral particle synthesis and assembly.

[0060] In one embodiment of this application, the replication-defective viral vector is an HSV virus.

[0061] "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 this application were determined with reference to the HSV-1 laboratory virus strain 17 genome (NC_001806.2). Those skilled in the art will understand that the specific nucleotide positions may differ in the genomes of different herpesvirus strains; however, those skilled in the art can determine the nucleotide positions corresponding to the specific genomic segments disclosed in this application based on conventional techniques. Therefore, this application covers the specific genomic segments determined with reference to the HSV-1 laboratory virus strain 17 genome (NC_001806.2), as well as the genomic segments corresponding to this genomic segment in different herpesvirus strains.

[0062] As used herein, the term "exogenous" refers to a nucleotide sequence that is not derived from a specific cell but is introduced into the cell via DNA delivery methods, such as transfection with a viral vector, electroporation, or transduction. Therefore, exogenous nucleotide sequences are artificial sequences, where the artificiality can originate from, for example, a combination of subsequences from different sources (e.g., a combination of a recombinase recognition sequence with the coding sequences for the SV40 promoter and green fluorescent protein to form an artificial nucleic acid), or deletions of partial sequences (e.g., sequences encoding only the extracellular domain of a membrane-binding receptor or cDNA), or mutations in nucleobases. The term "endogenous" refers to a nucleotide sequence derived from a cell. An "exogenous" nucleotide sequence may have an "endogenous" counterpart with the same base composition, but the sequence (e.g., via recombinant DNA technology) is introduced into the cell and thus becomes an "exogenous" sequence.

[0063] As used in this article, the term "Vero cells," also known as green monkey kidney cells, is an aneuploid kidney cell line from the African green monkey (genus: Chlorocebus). This cell line was obtained in 1962 by Yoshihiro Yasumura of Chiba University, Japan, who isolated kidney epithelial cells from normal adult African green monkeys. Vero cells are a continuous aneuploid cell line, meaning they have an abnormal chromosome number. As a continuous cell line, Vero cells can undergo many division cycles without aging. Vero cells have a defective interferon secretion function; unlike normal mammalian cells, they do not secrete interferon α / β when infected by viruses. However, they still possess interferon α / β receptors, so they can still respond when recombinant interferon is added to their culture medium. Currently, Vero cells are widely used in research on the molecular mechanisms of viral infection, the production of vaccines and recombinant proteins, and are considered an ideal cell model for culturing influenza vaccines and studying the molecular mechanisms of viral infection.

[0064] In some implementations, compensatory cell lines that support the replication of replication-defective viral vectors are obtained by transfecting Vero cells.

[0065] In some embodiments, the compensating cell line is the ZH02 compensating cell line.

[0066] As used herein, the term "coding sequence" refers to a polynucleotide, which directly defines the amino acid sequence of a polypeptide. The boundaries of a coding sequence are typically defined 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. Coding sequences can be DNA, cDNA, synthetic, or recombinant polynucleotides.

[0067] 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.

[0068] As used herein, the term "expression vector" refers to a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operatively linked to additional nucleotides that provide for its expression.

[0069] In a first aspect, this application provides a recombinant vector, wherein the vector includes a gene encoding dystrophin.

[0070] In some embodiments, the vector includes plasmids, lipid nanoparticles (LNPs), transposons, kinases, bacterial artificial chromosomes, virus-like particles (VLPs), exosomes, and viral vectors. Optionally, the viral vector includes retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, or any hybrid viral vectors thereof.

[0071] In some implementations, the herpesvirus vector includes herpes simplex virus vector, varicella-zoster virus vector, Epstein-Barr virus vector, cytomegalovirus vector, herpesvirus type 6 vector, herpesvirus type 7 vector, and herpesvirus type 8 vector, as well as any derivatives thereof.

[0072] In some embodiments, the herpes simplex virus vector includes type I herpes simplex virus vector and type II herpes simplex virus vector. In some embodiments, the herpes simplex virus vector is either a type I herpes simplex virus vector or a type II herpes simplex virus vector.

[0073] Secondly, this application provides the use of the recombinant vector described in the first aspect of this application in the preparation of a medicament for treating muscle diseases.

[0074] In some embodiments, the muscle disease is anti-dystrophin disease, optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy and / or X-linked dilated cardiomyopathy.

[0075] Thirdly, this application provides the use of the recombinant vector described in the first aspect of this application in the treatment of muscle diseases.

[0076] In some embodiments, the muscle disease is anti-dystrophin disease, optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy and / or X-linked dilated cardiomyopathy.

[0077] Fourthly, this application provides a pharmaceutical composition comprising the recombinant vector described in the first aspect of this application.

[0078] Fifthly, this application provides a method for treating a disease, comprising administering an effective amount of the recombinant vector described in the first aspect of this application to a subject in need.

[0079] In some implementations, the disease is a muscle disease.

[0080] In some embodiments, the muscle disease is anti-dystrophin disease; optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy, and / or X-linked dilated cardiomyopathy.

[0081] Sixthly, this application provides a recombinant herpes simplex virus vector, comprising a gene encoding dystrophin, wherein the viral vector genome is selected from ICP0, ICP4, ICP27, ICP34.5, ICP47, ICP22, ICP6, and U... L 41 genes, U L 55 genes, U L 56 genes and IR L / IR S One or more areas in the region have been destroyed or knocked out.

[0082] In some embodiments, the viral vector genome is selected from ICP4, ICP34.5, ICP22, ICP6, and U. L 41. U L 55 and IR L / IR S One or more of the regions are destroyed or knocked out. In some embodiments, the ICP34.5 gene and / or IR gene in the genome are... L / IR S The area was destroyed or knocked out.

[0083] In some implementations, the herpes simplex virus vector is a type I herpes simplex virus vector or a type II herpes simplex virus vector.

[0084] In some embodiments, the viral vector is cultured in compensating cells. In some embodiments, the compensating cells are Vero cells.

[0085] In some embodiments, the Vero cells are genetically modified Vero cells. In some embodiments, the Vero cells stably express one or more genes necessary for the replication and / or packaging of the recombinant herpes simplex virus vector, thereby forming compensatory cells that support the amplification of the recombinant herpes simplex virus vector. In some embodiments, the Vero cells are the ZH02 compensatory cell line.

[0086] In this application, the terms "genetically modified" and "genetically engineered" are used interchangeably and refer to cells that have undergone artificial manipulation, modification, or recombination of DNA or other nucleic acid molecules to alter the characteristics (phenotype) of the cell. Such cells are no longer considered naturally occurring cells. Genetically modified cells can refer to cells with added, deleted, and / or altered genes or gene portions. Genetically modified cells can refer to cells with added nucleic acid sequences (which are not genes or gene portions). Genetic modification includes, for example, transient knock-in or knock-down mechanisms, and mechanisms that result in permanent knock-in, knock-down, or knock-out of a target gene or a portion of a gene or nucleic acid sequence. Genetic modification includes, for example, transient knock-in and mechanisms that result in permanent knock-in of nucleic acid sequences. Genetic modification also includes, for example, decreased or increased transcription, decreased or increased mRNA stability, decreased or increased translation, and decreased or increased protein stability.

[0087] In some embodiments, the genetic modification includes transfecting cells with nucleic acids. "Transfection," or other related terms, refers to the process of transferring or introducing exogenous nucleic acids into host cells (such as the Vero cells described herein). In some embodiments, the Vero cells are genetically modified Vero cells obtained by transfecting cells with one or more genes necessary for the replication and / or packaging of a recombinant herpes simplex virus vector.

[0088] In some embodiments, the ICP34.5 gene and / or IR in the genome L / IR S The region is disrupted or knocked out. In some embodiments, the gene encoding dystrophin is introduced at the same or different genomic sites that are disrupted or knocked out as described above.

[0089] In a seventh aspect, this application provides the use of the recombinant herpes simplex virus vector described in the sixth aspect of this application in the preparation of a medicament for treating muscle diseases.

[0090] In some embodiments, the muscle disease is anti-dystrophin disease. Optionally, the muscle disease is Duchenne muscular dystrophy, Benedict's muscular dystrophy, intermediate muscular dystrophy, and / or X-linked dilated cardiomyopathy.

[0091] Eighthly, this application provides the use of the recombinant herpes simplex virus vector described in the sixth aspect of this application in the treatment of muscle diseases.

[0092] In some embodiments, the muscle disease is anti-dystrophin disease. Optionally, the muscle disease is Duchenne muscular dystrophy, Benedict's muscular dystrophy, intermediate muscular dystrophy, and / or X-linked dilated cardiomyopathy.

[0093] Ninthly, this application provides a pharmaceutical composition comprising the recombinant herpes simplex virus vector described in the sixth aspect of this application.

[0094] In a tenth aspect, this application provides a method for treating a disease, comprising administering to a subject in need an effective amount of the recombinant herpes simplex virus vector as described in the sixth aspect of this application.

[0095] In some implementations, the disease is a muscle disease.

[0096] In some embodiments, the muscle disease is anti-dystrophin disease; optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy, and / or X-linked dilated cardiomyopathy.

[0097] In the eleventh aspect, this application provides a compensatory cell for assisting in the culture of a recombinant herpes simplex virus vector, wherein the recombinant herpes simplex virus vector is the recombinant herpes simplex virus vector described in the sixth aspect of this application.

[0098] In some implementations, the compensating cells are Vero cells.

[0099] In some embodiments, the Vero cells are genetically modified Vero cells. In some embodiments, the Vero cells stably express one or more genes necessary for the replication and / or packaging of the recombinant herpes simplex virus vector, thereby forming compensatory cells that support the amplification of the recombinant herpes simplex virus vector. In some embodiments, the Vero cells are the ZH02 compensatory cell line.

[0100] In a twelfth aspect, this application provides the use of Vero cells for the assisted culture of recombinant herpes simplex virus vectors, wherein the recombinant herpes simplex virus is the recombinant herpes simplex virus vector described in the sixth aspect of this application.

[0101] In some embodiments, the Vero cells are genetically modified Vero cells. In some embodiments, the Vero cells stably express one or more genes necessary for the replication and / or packaging of the recombinant herpes simplex virus vector, thereby forming compensatory cells that support the amplification of the recombinant herpes simplex virus vector. In some embodiments, the Vero cells are the ZH02 compensatory cell line.

[0102] Example

[0103] Preparation Example 1: Preparation of HSV-1 Replication-Defective Recombinant Viral Vector

[0104] The HSV-1 viral genome consists of long terminal repeat (TR) sequences. L ), long unique segment (U L ), long internal repeat (IR) sequences L ), short internal repeat (IR) sequences S ), short unique segment (U S ) and short terminal repeat (TR) sequences S The composition is shown in Figure 1. By modifying specific regions of the HSV-1 genome (as shown in Figure 2), HSV-1 replication-defective viral vectors can be obtained.

[0105] Refer to the method described in patent application publication number CN113046331A for knocking out HSV-1 genomic IR S and TR S The regions 126785-131176bp and 147025-151439bp involve the HSV-1 virus ICP4 gene region, and the GFP gene is inserted at this position to obtain the viral vector A2 (Figure 3).

[0106] Viral vectors containing deletions of the essential HSV-1 replication genes ICP4 and ICP27 need to be prepared and grown on compensatory cell lines capable of compensating for the deletions of 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, and grown according to Lipofectamine... TMFollowing the instructions of Thermo (L3000015), the plasmid was stably transfected into Vero cells using liposomes to obtain the ZH02 compensatory cell line that supports the replication of replication-defective viral vectors.

[0107] Based on recombinant viral vector A2, the ICP34.5 gene was further knocked out, and the GFP gene was replaced at the ICP4 gene knockout position by inserting it into the MHCK7 promoter, the full-length Dys gene (NCBI: NM004006.3), and the polynucleotide to obtain recombinant viral vector H (Figure 4). Based on recombinant viral vector A2, the ICP34.5 gene and IR were further knocked out. L / IR S Region (Figure 5), in IR L / IR S Recombinant virus I was obtained by inserting the MHCK7 promoter, the full-length Dys gene, and polynucleotides into the region.

[0108] In all embodiments and preparation examples of this application, the pA used for the recombinant viral vector is TK pA.

[0109] Example 1: Expression verification of HSV-1 vector carrying the Dys gene

[0110] The expression levels of Dys protein by recombinant viral vectors H and I in human skeletal muscle cells (HSkMC, Yaji Biotechnology) were detected using Western blotting. HSkMC cells were cultured in DMEM medium containing 10% newborn calf serum. Cells were seeded in 6-well plates (1.5 × 10⁻⁶). 6After incubating at 37°C for 24 hours in wells (cells / well), recombinant viral vector H and recombinant viral vector I were added at an MOI of 0.5, and blank cell control wells were set up. Cells were harvested after 72 hours, and whole protein lysis buffer was prepared using a protein extraction kit (DE101, TransGold). The content of the whole lysed protein was detected using a BCA protein detection kit (23227, Thermo). Each protein sample was added to 6× loading buffer (Q20315, TransGold) and boiled for 10 minutes to prepare a loading mixture. 20 μg of each protein sample was loaded onto the wells of a protein precast gel (PG41510, Solarbio). Electrophoresis was performed at 90V for 40 minutes, followed by 120V for 50 minutes. The gel was then transferred to a PVDF membrane (ISEQ0001010, Millipore) at 200mA for 2 hours. Western blotting experiments were performed using the specific antibody Anti-Dystrophin (mouse monoclonal) (MAB1645, Millipore) and HRP-labeled goat anti-mouse IgG (H+L) (A0216, Beyotime). Chemiluminescence detection was then performed using Pierce ECL Western Blotting Substrate (32109, Thermo). The results showed that both recombinant viral vector H and recombinant viral vector I significantly expressed the Dys protein (as shown in Figure 6).

[0111] Example 2: Pharmacodynamic study of HSV-1 vector carrying the Dys gene

[0112] The recombinant vector H was tested in 4-week-old mdx (C57BL / 10ScSn-Dmdmdx / J) muscular dystrophy model mice (WuXi AppTec). The pharmacodynamic effects of the recombinant vector H and the influence of different administration routes on pharmacodynamics were evaluated by detecting serum creatine kinase (CK) and skeletal muscle fast muscle troponin I (TNNI2), behavioral tests, and histopathological observation.

[0113] The experiment was conducted with two groups: an intramuscular injection group and a tail vein injection group, with four animals in each group. The drug volume was 200 μl, and the drug titer was 3.6 × 10⁻⁶. 8The pfu / ml concentration was administered to mice starting at week 4, for 3 consecutive weeks, with observation continuing until week 10. A control group of model mice administered saline and a wild-type control group were also included. Before and weekly after administration, 150 μl of whole blood was collected from each group via submandibular sampling. At the end of the experiment (EOL), two mice from each of the tail vein injection group, intramuscular injection group, and model control group were selected, and 150 μl of whole blood was collected from each group via submandibular sampling. The whole blood was processed as follows: after standing at 4°C for 30 minutes, the serum was collected by centrifugation at 12000 rpm for 15 minutes at 4°C. The serum was divided into two portions, used for CK and TNNI2 detection, respectively. CK was detected using a Hitachi 3500 fully automated analyzer. TNNI2 was detected according to the instructions of the TNNI2 ELISA kit (MBS2022948, Mybiosource). Mice were observed twice weekly. At 12 weeks, forelimb grip strength was tested using a grip strength tester to assess forelimb function. At the experimental endpoint, two mice from each group were euthanized, and tissue samples were collected from the heart, liver, quadriceps femoris muscle, tibialis anterior muscle, and gastrocnemius muscle. These samples were then soaked in formalin, fixed at 4°C for at least 24 hours, dehydrated, embedded in paraffin, and sectioned. Immunohistochemical staining of embryonic myosin heavy chain (eMHC, antibody from abcam, ab264038) was performed using the Bond Rx Platform (Leica) to assess the level of regenerated muscle fibers.

[0114] No abnormalities were observed in mice during the experiment, either at the cage edge or outside the cage. Drug administration, behavioral testing, and blood sampling did not cause a decrease in mouse body weight.

[0115] The results of CK and TNNI2 detection (Figures 7 and 8) showed that the serum concentrations of CK and TNNI2 in mice in both the tail vein injection group and the intramuscular injection group decreased to varying degrees throughout the experiment after drug administration, with a more significant decrease in serum CK and TNNI2 concentrations in the tail vein injection group. CK and TNNI2 are key biomarkers of muscle injury, and the decrease in serum CK and TNNI2 concentrations indicates that the recombinant viral vector H helps reduce muscle damage and prevent the progression of muscular dystrophy.

[0116] The forelimb grip strength test results showed that the relative grip strength of mice in the tail vein injection group and the intramuscular injection group was significantly higher than that of the model control group at 12 weeks (Figure 9).

[0117] Immunohistochemical results (Figure 10) showed that no eMHC-positive fibers were detected in the heart, quadriceps femoris, gastrocnemius, tibialis anterior, and liver of wild-type control mice, while eMHC-positive fiber aggregation was observed in model control mice. Compared with the model control group, mice in the tail vein injection and intramuscular injection groups showed a significant increase in eMHC-positive fibers. eMHC is a structural protein specific to newly formed muscle fibers and can serve as a marker for muscle fiber regeneration. The experimental results showed that recombinant viral vector H increased the level of muscle regeneration in experimental mice, whether administered via tail vein injection or intramuscular injection.

[0118] The above experimental results show that recombinant vector H has shown good effects in muscular dystrophy model mice, demonstrating great therapeutic potential in the field of muscular dystrophy treatment.

[0119] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application, without departing from the scope of the technical solution of this application, shall still fall within the protection scope of this application.

Claims

1. A recombinant vector, wherein the vector comprises a gene encoding dystrophin.

2. The recombinant vector according to claim 1, wherein the vector comprises plasmids, lipid nanoparticles (LNPs), transposons, granules, bacterial artificial chromosomes, virus-like particles (VLPs), exosomes, and viral vectors; optionally, the viral vector comprises retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, or any hybrid viral vector thereof.

3. The recombinant vector according to claim 2, wherein the herpesvirus vector includes herpes simplex virus vector, varicella-zoster virus vector, Epstein-Barr virus vector, cytomegalovirus vector, herpesvirus type 6 vector, herpesvirus type 7 vector, and herpesvirus type 8 vector, and any derivative thereof.

4. The recombinant vector according to claim 3, wherein the herpes simplex virus vector includes a type I herpes simplex virus vector and a type II herpes simplex virus vector.

5. Use of the recombinant carrier as described in any one of claims 1-4 in the preparation of a medicament for treating muscle diseases.

6. The use according to claim 5, wherein the muscle disease is dystrophinosis, optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy, and / or X-linked dilated cardiomyopathy.

7. A pharmaceutical composition comprising the recombinant vector as described in any one of claims 1-4.

8. A method of treating a disease, comprising administering to a subject in need an effective amount of the recombinant vector as described in any one of claims 1-4.

9. The method of claim 8, wherein the disease is a muscle disease.

10. The method of claim 9, wherein the muscle disease is dystrophinosis, optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy, and / or X-linked dilated cardiomyopathy.

11. A recombinant herpes simplex virus vector comprising a gene encoding dystrophin, wherein the viral vector genome is selected from ICP0, ICP4, ICP27, ICP34.5, ICP47, ICP22, ICP6, and U... L 41 genes, U L 55 genes, U L 56 genes and IR L / IR S One or more areas in the region have been destroyed or knocked out.

12. The recombinant herpes simplex virus vector according to claim 11, wherein the herpes simplex virus vector is a type I herpes simplex virus vector or a type II herpes simplex virus vector.

13. The recombinant herpes simplex virus vector according to claim 12, wherein the viral vector is cultured in compensating cells.

14. The recombinant herpes simplex virus vector according to claim 13, wherein the compensating cell is a Vero cell.

15. The recombinant herpes simplex virus vector according to any one of claims 11-14, further comprising the ICP34.5 gene and / or IR gene in the genome. L / IR S The area was destroyed or knocked out.

16. The recombinant herpes simplex virus vector of claim 11, wherein the gene encoding dystrophin is introduced at the same or different genomic sites that have been disrupted or knocked out as described above.

17. Use of the recombinant herpes simplex virus vector as described in any one of claims 11-16 in the preparation of a medicament for treating muscle diseases.

18. The use according to claim 17, wherein the muscle disease is dystrophinosis, optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy, and / or X-linked dilated cardiomyopathy.

19. A pharmaceutical composition comprising a recombinant herpes simplex virus vector as described in any one of claims 11-16.

20. A method of treating a disease, comprising administering to a subject in need an effective amount of the recombinant herpes simplex virus vector as described in any one of claims 11-16.

21. The method of claim 20, wherein the disease is a muscle disease.

22. The method of claim 21, wherein the muscle disease is dystrophinosis, optionally, the muscle disease is Duchenne muscular dystrophy, Behringer's muscular dystrophy, intermediate muscular dystrophy, and / or X-linked dilated cardiomyopathy.

23. A compensatory cell for assisting in the culture of a recombinant herpes simplex virus vector, wherein the recombinant herpes simplex virus vector is the recombinant herpes simplex virus vector as described in any one of claims 11-16.

24. The compensating cell according to claim 23, wherein it is a Vero cell.

25. Use of Vero cells for the auxiliary culture of recombinant herpes simplex virus vectors, wherein the recombinant herpes simplex virus vector is a recombinant type I herpes simplex virus or type II herpes simplex virus vector as described in any one of claims 12-16.

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