Agent for treating hereditary bullous dermatoses

Abstract

The invention relates to the field of pharmacology, and more particularly to a gene therapy agent for treating hereditary bullous dermatoses related to a deficiency of and / or structural and / or functional defects in the COL7A1 gene and its transcriptional regulatory genes. The invention further relates to a DNA sequence containing herpes simplex virus 1 (HSV-1) DNA and one or two polynucleotides independently containing a combination of regulatory sequences that provide for the most effective expression of type VII collagen, as well as to an HSV-1 or to an HSV-1-based vector and to a pharmaceutical composition containing said DNA, said virus or said vector, and can be used for the treatment (gene therapy) of hereditary bullous dermatoses, such as epidermolysis bullosa.

Description

[0001] A REMEDY FOR THE TREATMENT OF BULLOUS GENODERMATOSIS

[0002] DESCRIPTION

[0003] Field of technology

[0004] The invention relates to the field of pharmacology and biotechnology, namely, to a gene therapy agent for the treatment of genetically determined bullous genodermatoses, which is a herpes simplex virus type I (Bill -1) containing a recombinant HSV genome including one or two expression cassettes for expressing the y-chain of human collagen type VII, as well as to a pharmaceutical composition intended for gene therapy of bullous genodermatosis, containing a therapeutically effective amount of such a virus, to a vector containing one or two polynucleotides independently encoding the y-chain of human collagen type VII, to a host cell containing such a vector or modified by such a vector, to a method for the prophylactic, palliative or therapeutic treatment of wounds of the skin and mucous membranes of a patient suffering from epidermolysis bullosa, using such a virus, such a pharmaceutical composition or such a vector, as well as to the use of the said virus,said pharmaceutical composition or said host cell for the manufacture of a medicinal product for the prophylactic, palliative or therapeutic treatment of wounds of the skin and mucous membranes of a patient suffering from epidermolysis bullosa, and for the prophylactic, palliative or therapeutic treatment of wounds of the skin and mucous membranes of a patient suffering from epidermolysis bullosa.,

[0005] The invention can be used for prophylactic, palliative or therapeutic treatment, i.e. gene (gene replacement) therapy of bullous genodermatoses, such as epidermolysis bullosa and its various clinical forms, including, without limitation, dominant dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa and other forms and varieties of epidermolysis bullosa known to those skilled in the art.

[0006] State of the art

[0007] Bullous genodermatosis (epidermolysis bullosa) is a group of congenital (hereditary) skin diseases caused by mutations in the genes encoding structural proteins of the epidermis and dermal-epidermal junction, which are responsible for the strength of the epidermis-dermis junction. Epidermolysis bullosa typically manifests at birth or within the first days (sometimes months) of life. The clinical presentation of epidermolysis bullosa is varied and depends on the clinical subtype, but the main clinical sign of bullous genodermatoses is the formation of blisters and / or erosions on the skin and mucous membranes in response to minor mechanical trauma (Fine et al., 2014), while the most severe clinical course of subtypes of epidermolysis bullosa, such as severe generalized recessive dystrophic epidermolysis bullosa (RDEB) or severe generalized borderline epidermolysis bullosa, are accompanied by widespread lesions of the skin, mucous membranes of internal organs and the presence of multiple erosive and ulcerative defects.

[0008] Bullous genodermatosis is classified as an orphan disease. According to the National Institutes of Health (NIH, USA), an orphan disease is defined as one with an incidence of fewer than 200,000 cases in the United States (Cuatrecasas P., 2006; Wastfelt M. et al., 2006). In the Russian Federation, Federal Law No. 323-F3 (as amended on June 25, 2012) "On the Fundamentals of Health Protection of Citizens in the Russian Federation" legislatively introduced the concept of rare (orphan) diseases: according to this law, diseases with an incidence of no more than 10 cases per 100,000 population are considered orphan.

[0009] To study the incidence and prevalence of bullous genodermatosis and to facilitate data collection, registries of patients suffering from this disease have been established in many countries (Pavicic et al., 1990; McKenna et al., 1992; Hom et al., 1997; Kho et al., 2010; Danescu et al., 2015; Fine, 2016).

[0010] In the Russian Federation, the prevalence and incidence of bullous genodermatosis have been studied since 2014 (Kubanov et al., 2015). As of the end of 2016, 417 cases of bullous genodermatosis were registered in 60 constituent entities of the Russian Federation. The corresponding prevalence rate for this disease is 3.9 per 1 million population. The incidence of bullous genodermatosis in the entire population of the Russian Federation in 2016 was 13.90 per 1 million population (Kubanov et al., 2018). According to the 2014 classification of bullous genodermatosis, depending on the layer of the epidermis in which the defective protein is predominantly localized and blisters are formed, 4 main types of the disease are distinguished: simple epidermolysis bullosa, junctional epidermolysis bullosa (JEB), dystrophic epidermolysis bullosa, and Kindler syndrome (Fine et al., 2014).

[0011] Bullous genodermatosis is a clinically and genetically heterogeneous group of diseases that arise from mutations in genes encoding structural proteins of the epidermis and dermal-epidermal junction (Has et al., 2018). Currently, specialists know more than 20 genes responsible for the development of bullous genodermatosis (Has C., Fischer J., 2018). Thus, recessive and dominant dystrophic epidermolysis bullosa occurs as a result of mutations in the COL7A1 gene encoding the alpha-1 chain of type VII collagen (Dang N., Murrell DF, 2008), and a mutation in the KLHL24 protein gene (Kelch-like protein 24) is responsible for the development of a new clinical subtype of EBD (Lin Z., Li S., Feng C. et al., 2016; Lee JYW, Liu L., Hsu C.-K. et al., 2017; He Y., Maier

[0012] K., Leppert J. et al., 2016; Kim S., Coulombe PA, 2007). Furthermore, new clinical phenotypes of bullous genodermatosis have been identified in recent years due to the discovery of mutations in the CD151, PLEC1a, DST, EXPH5, ​​and ITGA3 genes (Vahidnezhad H., Youssefian L., Saeidian A. H. et al., 2018; Gostynska K. V., Nijenhuis M., Lemmink H. et al., 2015; Liu L., Dopping-Hepenstal P. J., Lovell P. A. et al., 2012; Takeichi T., Nanda A., Liu L. et al., Br J Dermatol 2015; 172 (2): 527–31; Turcan I., Pasmooij A. M. G., Gostynski A. et al., 2017; He Y., Leppert J., Steinke H. et al., 2017; Cappuccio G., Pinelli M., Torella A. et al., 2017; McGrath JA, Stone KL, Begum R. et al., 2012; Pigors M., Schwieger-Briel A., Leppert J. et al., 2014; Malchin N., Sarig O., Grafi-Cohen M. et al., 2016; Rashidghamat E., Ozoemena L., Liu

[0013] L. et al., 2016; Has J., Sparta G., Kiritsi D. et al., 2012; Nicolaou N., Margadant C., Kevelam SH et al., 2012; Yalcin EG, He Y, Orhan D, 2015; Lovric S., Fang H., Vega-Warner V. et al., 2014 and Shukrun R., Vivante A., Pleniceanu O. et al., 2014).

[0014] The involvement of a large number of genes in the pathogenesis of bullous genodermatosis determines the diversity of clinical manifestations of this disease. The prevalence of lesions varies from localized rashes limited to the skin of the palms and soles to generalized rashes involving the mucous membranes of internal organs (Kubanov A. A. et al., 2020). According to the current classification of various forms of epidermolysis bullosa, the forms of this disease are classified according to skin morphology (according to the level of blister formation in the tissues); Kindler syndrome, associated with an autosomal recessive mutation of the kindlin-1 protein, is separately distinguished (Anwar MI, Rashid A., Ghafoor R. et al., 2014; Gadzhimuradova KM, Zhukova O. V. et al., 2022).

[0015] According to this classification, the following forms of the disease are distinguished:

[0016] • simple epidermolysis bullosa (SEB),

[0017] • junctional epidermolysis bullosa (JEB),

[0018] • dystrophic epidermolysis bullosa (DEB),

[0019] • Kindler syndrome.

[0020] In turn, simple epidermolysis bullosa can be represented by two subtypes: suprabasal simple epidermolysis bullosa, which, as a rule, has a mutation in the plakophilin-1 or desmoplaktin genes, and basal simple epidermolysis bullosa, which has a mutation in the keratin-5, keratin-14 or abβ4 integrin genes; junctional epidermolysis bullosa can be represented by the Herlitz subtype, which has a mutation in laminin-332 (laminin-5), or other subtypes, which have a mutation in laminin-332; collagen type XVII or abβ4 integrin;Dystrophic epidermolysis bullosa can be represented by a dominant subtype (dominant dystrophic epidermolysis bullosa), in which there is a dominant mutation of type VII collagen - the main component of the anchor fibrils of the dermo-epidermal zone of the basement membrane of the skin, and a recessive subtype (recessive dystrophic epidermolysis bullosa), in which there is a recessive mutation of the gene encoding this protein.

[0021] Type VII collagen, previously also called long-chain collagen, has an unusually long triple-helix region, approximately 450 nm in length. This collagen has only one type of alpha chain, type VII, and its triple-helix collagen domain is joined by a large nonhelical (noncollagenous) domain (NC-1) at the amino terminal group and a shorter nonhelical domain at the carboxyl group (NC-2). Type VII collagen molecules extend from the lower part of the lamina densa to the papillary dermis, forming antiparallel dimers linked at their carboxyl termini. The large amino-terminal noncollagenous domains of type VII collagen interact with type IV collagens and components of laminin-332 of the dermal-epidermal basement membrane; It is proposed that most of the anchoring fibrils form U-shaped loops that trap wide dermal collagen fibers composed of collagen types I and III.Thus, changes in the expression, structure, or molecular interactions of type VII collagen with other basement membrane components can lead to skin fragility. An example of such a situation is dystrophic epidermolysis bullosa (DEB), a group of mechanobullous diseases characterized by the formation of blisters on the skin as a result of minor trauma. Characteristic mutations in the collagen VII gene (COL7A1) have been identified in 500 families with various forms of DEB, and none of these families have yet been found to have mutations in genes other than those encoding the oc(VII) polypeptide of type VII collagen. Furthermore, type VII collagen serves as an autoantigen in the acquired form of epidermolysis bullosa, an autoimmune disease characterized by the presence of circulating antibodies to the collagen type VII antigen.

[0022] One of the most prognostically unfavorable clinical subtypes of bullous genodermatosis is severe generalized recessive dystrophic epidermolysis bullosa (RDEB) (Fine, 2010).

[0023] RDBE is characterized by multiple blisters and erosive-ulcerative defects that constantly arise spontaneously and / or as a result of trauma and lead to progressive scarring and the development of widespread foci of cicatricial atrophy, pseudosyndactyly, mitten-type hand deformities, joint contractures (Fine et al., 2002), while such defects can persist on the skin from 1 month to several years even with rational external therapy.

[0024] The occurrence of such erosive and ulcerative defects in patients with severe generalized RDBE is associated with a number of pathogenetic factors that impede normal healing: the formation of biofilms on the surface of erosions / ulcers by bacteria; increased expression of proinflammatory cytokines (IL-6, CXCL13, MCP-1, HMGB-1, etc.); the absence of sebaceous hair follicles, which are the source of regional stem cells, in the foci of cicatricial atrophy; a deficiency of type VII collagen, leading to a decrease in the rate of keratinocyte migration from the wound edges and the maturation of granulation tissue.

[0025] Severe generalized RDBE is accompanied by failure to thrive, anemia, nutritional or protein-energy deficiency, early development of complications and early mortality (Fine et al., 2008; Zidorio et al., 2015).

[0026] Excessive scar tissue formation and the presence of long-term non-healing skin defects lead to the development of squamous cell carcinoma (SCC) (Fine et al., 2009; Mellerio et al., 2016). SCC in bullous genodermatosis is characterized by a high risk of manifestation at an early age - as early as 20-25 years, rapid tumor growth, rapid metastasis, and the development of multiple foci of primary tumor (Mellerio et al., 2016). SCC is the leading cause of mortality among patients with recessive dystrophic epidermolysis bullosa.

[0027] The cumulative risk of developing PPH in patients with RDBE with severe generalized subtype is 7.5% at the age of 20 years; 26.7% at the age of 25 years; 51.7% at the age of 30 years; 90.1% at the age of 55 years (Mellerio et al., 2016). The 5-year survival rate among patients with RDBE-associated PPH is almost 0% (Fine et al., 2009; Mellerio et al., 2016).

[0028] Other severe complications of epidermolysis bullosa, which worsen quality of life and prognosis and shorten life expectancy, include damage to the heart, gastrointestinal tract, and blood. Sepsis is a severe and life-threatening complication of epidermolysis bullosa.

[0029] Treatment of bullous genodermatoses currently includes both therapeutic methods for treating the symptoms of the disease, aimed at accelerating the healing of erosive and ulcerative defects of the skin and mucous membranes of internal organs, preventing the development of secondary infections and correcting complications and concomitant pathology (Denyer et al., 2017), in particular, antibiotic therapy aimed at suppressing infections that occur in open wounds of the skin and mucous membranes, as well as methods aimed at relieving the pain and itching experienced by patients, as well as surgical methods for the treatment of foci of cicatricial atrophy, pseudosyndactyly and joint contractures. Among the most promising and actively developing approaches to the treatment of bullous genodermatoses, cell therapy, protein replacement therapy and gene therapy (Rashidghamat, McGrath, 2017) should be separately noted.Cell therapy typically involves intradermal injections of allogeneic fibroblasts containing type VII collagen, while protein replacement therapy involves injections of purified recombinant type VII collagen into the patient.

[0030] The prior art has shown that a single intradermal injection of allogeneic fibroblasts in the amount of 5 * 10 6 cells in an area of ​​the superficial layer of the dermis of approximately 1 cm 2provided enhanced expression of the COL7A1 gene in most patients, while the study noted low immunogenicity of these fibroblasts (Wong T., Gammon L. et al., 2008). Clinical studies have shown that a single intradermal injection of allogeneic fibroblasts increases the expression of the COL7A1 gene for 3-6 months, and the expression of type VII collagen protein for 9-12 months (Nagy N., Almaani N. et al., 2011). During phase II clinical trials, a double-blind, placebo-controlled study showed a significant decrease in the area and number of erosions in patients with RDBE who received a single intradermal injection of allogeneic fibroblasts, with this effect lasting more than 28 days (Petrof G., Martinez-Queipo M., et al., 2013).

[0031] However, for the effective implementation of cell therapy, such as the above-mentioned therapy with allogeneic fibroblasts, it is necessary to have a constantly accessible source of cells, use a safe and cost-effective method of their isolation and processing, exclude or, in extreme cases, minimize the possibility of developing an immune conflict, ensure complete biological safety for the patient, realistically assess the technical complexity of the methods used, material costs, and also take into account the legislative and ethical restrictions provided by national legislation for the use of such methods.

[0032] The solution to the above-mentioned problems is personalized cell therapy (PCT) technologies, which utilize the patient's own (autologous) cells, obtained and stored in advance or obtained immediately before treatment (Khabriev R.U., Kovalchuk L.V. et al., 2011; Patel S.A., King S.S. et al., 2010). However, each specific cell technology is characterized by a unique algorithm for obtaining, storing, processing, and using autologous cells or cellular products, which significantly increases the labor intensity and cost of such therapy.

[0033] Furthermore, patients suffering from bullous genodermatosis typically already have extensive wounds in areas of the skin that are subject to constant mechanical stress, and therefore any intradermal injection for such patients would be a highly invasive and high-risk intervention, increasing the likelihood of severe, including life-threatening, complications.

[0034] Regarding protein replacement therapy, which involves administering purified recombinant collagen type VII to patients, when administered topically, recombinant collagen cannot penetrate intact skin, and therefore its effect is limited to lesions. Intradermal injections of purified recombinant collagen type VII for patients with RDBE represent another alternative; however, limited diffusion and uncontrolled penetration depth with a standard needle require the use of sterile microneedle kits, which are not yet widely available for clinical use (Nagarkar R. et al., 2020).

[0035] On the other hand, it has been shown in the literature that systemic administration of recombinant type VII collagen to a patient can cause systemic toxicity (Hou Y., Guey LT et al., 2015), which significantly limits the possibilities of protein replacement therapy for bullous genodermatoses.

[0036] Among the most modern approaches to the treatment of bullous genodermatoses currently being studied are allogeneic bone marrow transplantation with or without infusion of mesenchymal stem cells, the use of agents that facilitate reading through a premature termination or stop codon, systemic administration of recombinant collagen type VII to patients, and gene therapy (Prodinger C., Reichelt J. et al., 2019).

[0037] However, allogeneic bone marrow transplantation, the effectiveness of which was shown by different authors on a small number of patients with recessive-dystrophic epidermolysis bullosa (Boull CL, Hylwa SA, et al., 2016; Ebens CL, McGrath JA, Tamai K. et al., 2019; Geyer MB, Radhakrishnan K. et al., 2015; Wagner JE, Ishida-Yamamoto A. et al., 2010), was ineffective for the treatment of other clinical forms of epidermolysis bullosa, for example, junctional epidermolysis bullosa (Hammersen J., Has C. et al., 2016).

[0038] Particularly noteworthy are methods using autologous transplantation of the patient's keratinocytes.Such methods, which are currently being intensively investigated, typically involve the isolation of a patient's own keratinocytes in the form of a small biopsy, followed by their expansion ex vivo and subsequent transplantation into the patient, and therefore, for their successful application, it is necessary that the biopsy contains a sufficient number of healthy epidermal stem cells for their subsequent transfection and subsequent growth into epidermal equivalents that can be used for transplantation into the patient and that can provide for the continuous regeneration of both a healthy, functional and renewing epidermis and the extracellular environment, which in the case of patients with bullous genodermatosis is a significant problem, since such patients have a very limited number of epidermal stem cells, and their number is constantly decreasing with age as a result of the constant occurrence of wounds and subsequent scarring of the wounds.

[0039] One of the most convenient and flexible tools for gene replacement therapy are herpesviruses, particularly the herpes simplex virus (HSV), such as herpes simplex virus type 1 (HSV-1). HSV is a complex, non-integrative DNA virus capable of infecting a wide range of human and animal cells. The viral genome contains over 80 genes and consists of two unique segments, UL and US, each flanked by inverted repeats encoding critical diploid genes. A key feature of viral replication is the wave-like expression of its genes, a process known as cascade regulation (Rajcani, 2004). Deletion or inactivation of the essential immediate-early (IE) genes UL54(ICP27) and RS1(ICP4) renders the virus completely defective and incapable of expressing early (E) genes involved in viral genome replication and late (L) genes functioning in the assembly of progeny virions.These replication-deficient (replication-incompetent) viruses can be grown in complementing cells that express (complement) the missing RS1(ICP4) and UL54(ICP27) gene products and can then be used to infect non-complementing cells where the viral genome exists as a stable nuclear episome.

[0040] The widespread use of HSV-based vectors is due to the natural properties of this virus, which make it attractive for the creation of vectors based on it. Among these properties, it is worth noting the latent nature of herpesvirus infection and long-term expression of HSV genes, due to which HSV vectors can be administered to a patient in a latent state, as well as the episomal (non-integrating) DNA genome of the virus, which avoids unwanted insertional mutations in the host genome (Garber, DA et al., 1993, Efstathiou, S. et al., 1986, Jackson, SA, DeLuca, NA, 2003), and the presence of HSV genes unnecessary for replication that can be deleted, which gives the researcher the opportunity, using HSV vectors, to clone large transgenes along with their regulatory sequences (Roizman B., 1996).(The term “regulatory sequence” or “regulatory element” hereinafter in the present description includes promoters, enhancers, insulators, polyadenylation signals, as well as other expression control elements well known to those skilled in the art, including regulatory elements capable of increasing transgene expression (Powell et al., 2015; Maksimenko et al., 2015; Ermekova S. A., 2017).

[0041] Also among the features of HSV that make it particularly attractive for the creation of vectors, it is necessary to note its broad cellular tropism, high capacity for the cloned gene (about 30 thousand base pairs), as well as its ability to transduce both dividing and dormant cells (Spaete RR, Frenkel N., 1982).

[0042] Attempts to create various HSV-based vectors for the treatment of various genetic diseases have been undertaken in the prior art by various authors. For example, US Patent No. US 6,319,703 B1, November 20, 2001 (SPECK PETER G [US]) discloses a herpesvirus mutant containing inactivating mutations of one or more vital viral genes necessary for its replication, in particular, the g / 7 gene, as well as containing inactivating mutations of genes necessary for the lysis of virus-infected cells, in particular, the VP16 gene.The herpesvirus mutant proposed in the aforementioned patent, according to the description of patent US6319703, can be used to create vectors for gene therapy of such severe genetic diseases as cystic fibrosis (mucoviscidosis), Tay-Sachs disease (GM2 gangliosidosis), β-thalassemia, and Hurler disease (mucopolysaccharidosis type IH). However, the possibility of creating vectors for gene therapy of genodermatoses, in particular, epidermolysis bullosa, based on the aforementioned mutant is not considered in this patent. Further attempts to create vectors for gene therapy of genodermatoses, such as epidermolysis bullosa, based on this herpesvirus mutant are also unknown.

[0043] The prior art also includes several attempts to create a vector based on replication-deficient (replication-incompetent) HSV intended for gene therapy of genodermatoses such as epidermolysis bullosa. For example, the prior art includes information from international application WO 2017 / 176336 A1, filed October 12, 2017, by KRYSTAL BIOTECH, INC. (US), a replication-defective (replication-incompetent) HSV-1 virus is known, containing a vector including one or more transgenes encoding a polypeptide selected from the polypeptide chain of type VII collagen, the polypeptide chain of lysyl hydroxylase 3 and a chimeric polypeptide of the α-chain of type VII collagen and lysyl hydroxylase 3, optionally containing inactivating mutations of one or more immediate-early, early and late genes of HSV-1 selected from RL2(ICP0), RS 1(ICP4), US 1(ICP22), UL54(ICP27), US 12(ICP47), UL23(tk), UL41 and UL55.The immediate-early genes (also referred to as immediate-early regulatory genes) of the herpes simplex virus are genes encoding proteins that regulate the expression of early and late genes of the virus and are expressed first after the virus enters the cell (Honess RW, Roizman B., 1974).

[0044] Also known from WO 2017 / 176336 A1 is a pharmaceutical composition, optionally in the form of an ointment, paste, cream, suspension, emulsion, fatty ointment, gel, powder, lotion, solution, spray, patch, set of pre-filled microneedles or inhalation form, containing such a HSV-1 virus together with a pharmaceutically acceptable carrier, intended to increase, strengthen or maintain the level of type VII collagen in cells, for example, in skin cells, including in epidermal or dermal cells, especially in patients suffering from epidermolysis bullosa.

[0045] The said virus or vector and the pharmaceutical composition containing such virus or vector according to WO 2017 / 176336 A1 can be used for the prophylactic, palliative or therapeutic treatment of a skin disease or disorder, such as epidermolysis bullosa, skin cancer, psoriasis, lichen planus, lupus, rosacea, eczema, cutaneous candidiasis, cellulitis, impetigo (superficial pyoderma), bedsores, erysipelas, ichthyosis vulgaris, dermatomyositis, acrodermatitis, stasis dermatitis, Netherton syndrome, simple epidermolysis bullosa associated with a mutation in the LAMB3 gene, autosomal recessive congenital ichthyosis, xeroderma pigmentosum and pemphigoid.

[0046] WO 2017 / 176336 A1 discloses an illustrative example of such a replication-defective (replication-incompetent) HSV-1 virus, KB 103, proposed as the closest analogue of the virus according to the present invention and containing an expression cassette including the immediate-early promoter of human cytomegalovirus (HCMV 1Ep), a transgene encoding the single chain of human collagen type VII (COL7A1) and a bovine growth hormone polyadenylation signal (BGHpA), and it is shown that infection of experimental animals (Balb / c and SKH1 mice) with this virus ensured the production of a high number of collagen type VII transcripts in mouse tissues, and also led to the achievement of a high level of collagen type VII in isolated mouse tissue samples and to the onset of collagen type VII deposition in the basal membrane region.

[0047] However, there is still a need to select an optimal combination of regulatory elements, as well as expression cassettes containing such a combination of regulatory elements together with a transgene encoding the α-chain of human collagen type VII, which, when included in a recombinant HSV genome and in a virus (HSV-1) containing such a genome, would provide even more efficient expression of the transgene and, as a consequence, more effective gene therapy for bullous genodermatosis, for example, epidermolysis bullosa.

[0048] In addition, the tasks of ensuring longer-term stable expression of the transgene (collagen type VII), as well as a higher yield of the virus during its production and obtaining a drug with improved therapeutic efficacy and fewer side effects, remain relevant.

[0049] However, neither WO 2017 / 176336 A1 nor any other prior art sources provide detailed information on the optimal combination of regulatory elements that could ensure the most efficient expression of the transgene (the COL7A1 gene), nor on the efficiency of transgene expression and the level of collagen type VII production from it. At the same time, it is well known from the prior art that transgene expression depends on the type and combination of regulatory sequences used (Powell et al., 2015; Maksimenko et al., 2015).

[0050] In addition, according to published data from partially completed clinical trials of the drug developed by KRYSTAL BIOTECH, INC. (US), described in documents WO 2017 / 176336 A1 and WO 2019 / 210219 A1, which received the international nonproprietary name beremagene geperpavec, the use of this drug in a patient, such as a human, may be accompanied by a number of side effects, including chills, cough, runny nose, itching, redness of the skin, or rashes. Moreover, publications covering the results of clinical trials of this drug also reported three cases of squamous cell skin cancer in patients treated with this drug (Guide SV, Gonzalez ME et al., 2022).

[0051] Therefore, there remains a need for new, more advanced, and safer viral vectors for transducing epidermal and dermal cells to express collagen and for gene therapy of bullous genodermatoses. Furthermore, as is well known to those skilled in the art, universal regulatory elements with a clear mechanism of action have not yet been identified (Powell et al., 2015; Maksimenko et al., 2015). Neither WO 2019 / 210219 A1 nor WO 2017 / 176336 A1 disclose in detail which regulatory sequences and in what combination were used in the aforementioned viruses (HSV-1) and vectors based on them, intended for gene therapy of bullous genodermatosis. At the same time, it is well known that the choice of regulatory elements is very important for efficient transgene expression (Maksimenko O.G. et al., 2015).

[0052] Therefore, a specialist in this field faces the challenge of finding the optimal combination of regulatory sequences that would increase the expression level of the transgene (the type VII collagen U chain gene), which would, in turn, improve the effectiveness of gene therapy for bullous genodermatosis. Furthermore, a specialist in this field faces the challenges of ensuring longer-term stable expression of the transgene, ensuring higher viral yields during its production, and producing a drug with improved therapeutic efficacy and fewer side effects.

[0053] Finally, despite the currently used therapies for bullous genodermatosis approved for clinical use, there remains a need to develop new, more effective treatments for this disease, including gene therapy agents that would improve the quality of life and increase the life expectancy of patients with bullous genodermatosis, as well as delay the development of squamous cell skin cancer and other life-threatening complications of bullous genodermatosis.

[0054] Thus, the objective of the present invention is to create gene therapy agents (gene therapy agents) based on the herpes simplex virus, which would ensure, in comparison with known analogues, the achievement of such technical results as more effective and more stable expression of type VII collagen in the patient's target tissues and, as a consequence, more effective therapy of bullous genodermatosis, as well as ensuring longer-term stable expression of the transgene, ensuring a higher yield of the virus during its production and obtaining a drug with improved therapeutic efficacy, with fewer side effects.

[0055] Finally, the task of expanding the arsenal of effective and safe gene therapy agents for the treatment (gene therapy) of bullous genodermatosis, such as epidermolysis bullosa, remains relevant, including for therapeutic, prophylactic or palliative treatment of this disease.

[0056] The essence of the invention

[0057] The problem posed in the present invention is solved, and the technical results are achieved in the present invention by the fact that a herpes virus type I (Bill-1) is proposed, containing a recombinant HSV genome, wherein said genome contains one or two expression cassettes for expressing the s-chain of human collagen type VII, wherein each of said expression cassettes independently includes a transgene encoding the y-chain of human collagen type VII, and a combination of regulatory sequences selected from: - the immediate-early promoter and enhancer of human cytomegalovirus (I);

[0058] - bovine growth hormone polyadenylation signal (II);

[0059] - Kozak sequences (III);

[0060] - SV40 intron (IV);

[0061] - UCOE intron (V), wherein said transgene together with said combination of regulatory sequences forms one of the following combinations:

[0062] (A) immediate-early promoter and enhancer of human cytomegalovirus - Kozak sequence - transgene encoding the single chain of human collagen type VII - bovine growth hormone polyadenylation signal;

[0063] (B) immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - transgene encoding the single chain of human type VII collagen - bovine growth hormone polyadenylation signal;

[0064] (C) immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - Kozak sequence - transgene encoding the single chain of human collagen type VII - bovine growth hormone polyadenylation signal;

[0065] (D) UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - transgene encoding the single chain of human collagen type VII - bovine growth hormone polyadenylation signal;

[0066] (E) UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - Kozak sequence - transgene encoding the single chain of human collagen type VII - bovine growth hormone polyadenylation signal;

[0067] (F) UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - transgene encoding the single chain of human collagen type VII - bovine growth hormone polyadenylation signal;

[0068] (G) UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - Kozak sequence - transgene encoding the α-chain of human collagen type VII - bovine growth hormone polyadenylation signal; (H) SV40 intron - immediate-early promoter and enhancer of human cytomegalovirus - UCOE intron - transgene encoding the β-chain of human collagen type VII - bovine growth hormone polyadenylation signal;

[0069] (I) the SV40 intron - the immediate-early promoter and enhancer of human cytomegalovirus - the UCOE intron - the Kozak sequence - the transgene encoding the single chain of human type VII collagen - the bovine growth hormone polyadenylation signal, and wherein said recombinant VIG genome contains inactivating mutations of one or more immediate-early, early, or late genes of HSV-1.

[0070] Inactivating mutations of one or more immediate-early, early or late genes of the said virus (HSV-1) are preferably selected from inactivating mutations of the genes RS1(ICP4), UL54(ICP27), UL41 and US1(ICP22); at the same time, the HSV-1 proposed in the present invention preferably does not contain inactivating mutations of other genes.

[0071] Also, the above-mentioned problem set forth in the present invention is solved, and the above-mentioned technical results of the present invention are achieved by the fact that a herpes virus type I (HSV1) is proposed, containing a recombinant HSV genome, where said genome contains one or two expression cassettes for expressing the α-chain of human collagen type VII, where each of said expression cassettes independently includes a transgene encoding the α-chain of human collagen type VII, and the following combination of regulatory sequences:

[0072] (J) the immediate-early promoter and enhancer of human cytomegalovirus - a transgene encoding the single chain of human collagen type VII - a bovine growth hormone polyadenylation signal, wherein said recombinant HSV genome contains inactivating mutations of the genes RS1(ICP4), UL54(ICP27), US1(ICP22), UL41 and does not contain inactivating mutations of the genes RL2(ICP0) and / or US12(ICP47).

[0073] In the proposed combinations (A) - (J), the immediate-early promoter and enhancer of human cytomegalovirus are preferably represented by the sequence SEQ ID NO: 10 (Schmidt et al., 1990; Xia et al., 2006); the bovine growth hormone polyadenylation signal is preferably represented by the sequence SEQ ID NO: 11 (Iwakuma et al., 1999); the Kozak sequence is represented by the sequence SEQ ID NO: 12 (Kozak, 1984); the SV40 intron is preferably represented by the sequence SEQ ID NO: 13 (Xu et al., 2018); the UCOE intron is preferably represented by the sequence SEQ ID NO: 14 (Neville et al., 2017); Finally, the COL7A1 transgene encoding the single chain of type VII collagen is preferably represented by the sequence SEQ ID NO: 15 (Christiano AM et al., 1994).

[0074] Preferably, but not limited to this embodiment of the invention, said recombinant HSV genome, comprising one or two expression cassettes for expressing the s-chain of human collagen type VII, wherein each of said expression cassettes independently comprises a transgene encoding the y-chain of human collagen type VII, and a combination of regulatory sequences, wherein said combination of regulatory sequences together with said transgene forms one of the combinations (A) - (I), contains inactivating mutations of one or more immediate-early, early or late genes of HSV-1, selected from RS1 (ICP4), UL54 (ICP27), UL41 and US1 (ICP22) and does not contain inactivating mutations of the genes RL2 (ICP0) and / or US12 (ICP47).

[0075] Furthermore, in certain preferred, but also non-limiting embodiments of the invention, said recombinant HSV genome further comprises an inactivating mutation of the UL55 gene.

[0076] Furthermore, in certain preferred, but also non-limiting embodiments of the invention, said recombinant HSV genome does not contain the coding sequence of one or more HSV genes selected from RS1(ICP4), UL54(ICP27), US1(ICP22) and UL41.

[0077] Also, in certain preferred, but non-limiting embodiments of the invention, the recombinant HSV genome proposed in the present invention does not contain regulatory sequences of the HSV US1(ICP22) gene, which ensure its immediate-early expression.

[0078] Also, in certain preferred, but non-limiting embodiments of the invention, the recombinant HSV genome of the present invention further comprises up to two single nucleotide substitutions in the UL27 gene.

[0079] In more preferred, but also non-limiting embodiments of the invention, said single nucleotide substitutions in the UL27 gene result in the substitution of amino acid 285 and amino acid 549 of the protein product of said gene, envelope glycoprotein B.

[0080] In the most preferred, but also non-limiting embodiments of the invention, said single nucleotide substitutions in the UL27 gene are selected arbitrarily from the list of possible substitutions: GAC <AAC, GAC<TAC, GAC<CAC, GAC<GTC, GAC<GGC, GAC<GCA, GAC<GAA, GAC<GAG - для аминокислоты 285, и GCC<ACC, GCC<TCC, GCC<GAC, GCC<GTC, GCC<GGC - для аминокислоты 549, при этом указанные однонуклеотидные замены в указанном гене ВПГ-1 приводят к замене аминокислоты 285 и аминокислоты 549 белкового продукта указанного гена - оболочечного гликопротеина В (с аспарагиновой кислоты на аспарагин, тирозин, гистидин, валин, глицин, аланин, глутаминовую кислоту, и с аланина на треонин, серин, аспарагиновую кислоту, валин, глицин, соответственно).

[0081] Also in preferred, but also non-limiting embodiments of the invention, the DNA sequence of the present invention does not contain the coding sequence of the UL41 gene.

[0082] In alternative embodiments of the invention, the proposed mutant genome of viral DNA based on herpes simplex virus type I (HSV-1) differs from the wild type Bill-1 in that it contains inactivating mutations of one or more immediate-early, early and late genes of HSV-1 selected from RS1(ICP4), UL54(ICP27), UL41 and does not contain inactivating mutations of the UL55 gene.

[0083] It is also preferred, but not limited to this embodiment of the invention, that the DNA sequence according to the present invention further comprises up to two single nucleotide substitutions in the HSV-1 UL27 gene encoding the envelope glycoprotein B of the virus. In the most preferred, but also non-limiting embodiment of the invention, said single nucleotide substitutions in the HSV-1 UL27 gene result in the substitution of amino acid 285 and amino acid 549 of the protein product of said gene, the envelope glycoprotein B (from aspartic acid to asparagine, tyrosine, histidine, valine, glycine, alanine, glutamic acid, and from alanine to threonine, serine, aspartic acid, valine, glycine, respectively), wherein the combination of these substitutions is selected arbitrarily from the list of possible substitutions:

[0084] GAC <AAC, GAC<TAC, GAC<CAC, GAC<GTC, GAC<GGC, GAC<GCA, GAC<GAA, GAC<GAG - для аминокислоты 285, и GCC<ACC, GCC<TCC, GCC<GAC, GCC<GTC, GCC<GGC - для аминокислоты 549.

[0085] The present invention also addresses the stated objective and achieves technical results by providing a pharmaceutical composition for gene therapy of bullous genodermatosis, comprising a therapeutically effective amount of the virus (herpesvirus) described above and at least one pharmaceutically acceptable excipient. In a preferred, but non-limiting, embodiment of the invention, said at least one pharmaceutically acceptable excipient is adapted for cutaneous, transdermal, subcutaneous, and / or intradermal administration.

[0086] In a most preferred (non-limiting) embodiment of the invention, the herpes simplex virus is herpes simplex virus type I (HSV-1).

[0087] In alternative (but also non-limiting) embodiments of the invention, the herpes simplex virus may also be herpes simplex virus type II (HSV-2), or herpes virus type III (varicella zoster virus), or human cytomegalovirus, or herpesvirus type 6A, or herpesvirus type 6B, or herpesvirus type 7, or herpesvirus associated with Kaposi's sarcoma (herpesvirus type 8).

[0088] Most preferably, said pharmaceutical composition is intended for the treatment of bullous genodermatoses associated with a deficiency in the formation and / or structural and / or functional defects of the COL7A1 gene, as well as genes regulating its transcription.

[0089] The inventors of the present invention unexpectedly discovered that a replication-incompetent herpes simplex virus type I (HSV-1) comprising a mutant HSV-1 DNA genome containing inactivating mutations of one or more immediate-early, early and late genes of the virus selected from RS1 (ICP4), UL54 (ICP27), UL41, US1 (ICP22) and UL27, and one or two polynucleotides providing expression of the COL7A1 gene, wherein said polynucleotide contains or said two polynucleotides contain the COL7A1 gene of the sequence SEQ ID NO: 15, encoding the α-chain of type VII collagen, together with combinations of regulatory sequences including one or more elements selected from the immediate-early promoter and enhancer of human cytomegalovirus (SEQ ID NO: 10), the bovine growth hormone polyadenylation signal (SEQ ID NO: 11), the sequence Kozak (SEQ ID NO: 12), SV40 intron (SEQ ID NO: 13) and UCOE intron (SEQ ID NO: 14), provides, in comparison with known analogues,The most effective expression of the singlet chain of type VII collagen. This effect was not described in the prior art and could not have been predicted given the prior art, and suggests that this virus (HSV-1), as well as the pharmaceutical composition containing it, will be most effective as a gene therapy for bullous genodermatosis.

[0090] Brief description of the figures

[0091] Fig. 1 schematically depicts the structure of cassettes (A) - (J). Designations of the structural elements of the cassettes: I - immediate-early promoter and enhancer of human cytomegalovirus; II - bovine growth hormone polyadenylation signal; III - Kozak sequence; IV - SV40 intron; V - UCOE intron; COL7A1 - transgene.

[0092] Fig. 2 shows the level of human collagen type VII (Co17) protein production in HEK293T cells after transfection of a library of plasmids expressing the COL7A1 gene, assessed by immunoblotting. Panel A is the level of human collagen type VII protein production. Panel B is the level of pan-actin protein production. Panel C is the level of green fluorescent protein (GFP) production. Preferred embodiments of the invention

[0093] In a preferred but non-limiting embodiment of the present invention, the proposed DNA sequence does not contain the coding sequence of one or more immediate-early genes of HSV-1 selected from RS1(ICP4), UL54(ICP27), US1(ICP22).

[0094] Also, in a preferred, but non-limiting embodiment of the present invention, the DNA sequence proposed in the present invention does not contain the regulatory sequences of the US1(ICP22) gene, which ensure its immediate-early expression.

[0095] In some embodiments, the virus (HSV-1) is attenuated. Also, in some embodiments, the virus has reduced cytotoxicity compared to wild-type HSV-1. Also, in some embodiments, the HSV-1 virus is non-oncolytic.

[0096] In a preferred but non-limiting embodiment of the present invention, the DNA sequence of the present invention further comprises up to two single nucleotide substitutions in the UL27 gene. Most preferably, the DNA sequence of the present invention further comprises up to two single nucleotide substitutions in the UL27 gene, wherein said single nucleotide substitutions in the UL27 gene result in the substitution of amino acid 285 and amino acid 549 of the protein product of said gene - envelope glycoprotein B (from aspartic acid to asparagine, tyrosine, histidine, valine, glycine, alanine, glutamic acid, and from alanine to threonine, serine, aspartic acid, valine, glycine, respectively), and, most preferably, the combination of these substitutions is selected arbitrarily from the list of possible substitutions:

[0097] GAC <AAC, GAC<TAC, GAC<CAC, GAC<GTC, GAC<GGC, GAC<GCA, GAC<GAA, GAC<GAG - для аминокислоты 285, и GCC<ACC, GCC<TCC, GCC<GAC, GCC<GTC, GCC<GGC - для аминокислоты 549.

[0098] It is also preferred that the DNA sequence according to the present invention does not contain the coding sequence of the UL41 gene. The production of the above-mentioned HSV-1 virus containing the said DNA sequence in accordance with the present invention is carried out using host cell lines (cell lines) conventionally used in this field, which are modified for this purpose to complement the deleted genes, as demonstrated below in the examples of the present description.

[0099] Non-limiting (illustrative) examples of such mammalian cell lines include, but are not limited to, the SV40-transformed C VI monkey kidney cell line (COS-7, ATCC CRL 1651), the human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli+ cells (TM4), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO, ATCC CCL-81; VERO-76, clone E6, ATCC CRL-1586; VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), Buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse breast tumor cells (MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma cell line (Hep G2), Chinese hamster ovary cells (CHO), including DHFR(-) cells,and myeloma cell lines such as NSO and Sp2 / 0. In some embodiments, the host cell may be a human cell or a non-human primate cell. Examples of suitable host cells or cell lines include, but are not limited to, 293, HeLa, SH-Sy5y, Hep G2, CACO-2, A549, L929, 3T3, K562, CHO-KI, MDCK, HUVEC, Vero, N20, COS-7, PSN1, VCaP, CHO cells and the like. One of skill in the art will appreciate that other mammalian cell lines known in the art may be used, particularly primate cell lines, including human cell lines, as well as non-human primate cell lines.

[0100] In some embodiments, the host cell is a target cell, such as a barrier cell. Non-limiting examples of such target cells include epithelial cells, keratinocytes, and fibroblasts.

[0101] The problem posed by the present invention is solved, and the technical results are also achieved, by providing a pharmaceutical composition comprising the above-described DNA sequence or the above-described virus and at least one pharmaceutically acceptable excipient. (The term "pharmaceutically acceptable excipient" in this application refers to an inactive substance traditionally used in the art as an excipient for medicinal products, non-toxic to the recipient at the doses and concentrations used.)Non-limiting (illustrative) examples of such pharmaceutically acceptable excipients include: buffers (buffer solutions); antioxidants; preservatives; low molecular weight peptides; proteins; hydrophilic polymers; amino acids; monosaccharides, disaccharides and other carbohydrates; chelating agents; sugars; polyols; salt-forming counterions; metal complexes; non-ionic surfactants and other auxiliary components well known to those skilled in the art and conventionally used in the art for the preparation of pharmaceutical compositions, including compositions for topical and / or transdermal and / or subdermal and / or intradermal administration. A more detailed, but also non-limiting list of pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention is given in the publication "Remington: The Science and Practice of Pharmacy" - Academic Press Publishing House; 23.rd edition, edited by Adeboye Adejare, November 13, 2020, the contents of which are incorporated herein by reference.

[0102] The pharmaceutical composition may, without limitation, be formulated in a dosage form suitable for topical, transdermal, intradermal, and / or subdermal administration. In certain non-limiting embodiments of the present invention, the composition may be formulated in a dosage form suitable for non-invasive or minimally invasive administration to a patient. The most preferred dosage forms in which the pharmaceutical composition according to the present invention may be formulated include, without limitation, dosage forms for subcutaneous (subdermal) administration, or dosage forms for intradermal (intradermal) administration, or dosage forms for topical use.Non-limiting (illustrative) examples of such dosage forms include ointments, pastes, creams, suspensions, emulsions, fatty ointments, gels, powders, lotions, solutions, sprays, patches, prefilled microneedle kits, injections, and inhalation forms. Those skilled in the art will recognize that this list of dosage forms is not exhaustive and that the pharmaceutical compositions of the present invention may also be formulated in other dosage forms known in the art.

[0103] In alternative, also illustrative (non-limiting) embodiments of the invention, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier, i.e., an inert carrier that is non-toxic to the recipient at the dosages used.

[0104] In some non-limiting embodiments of the invention, such a pharmaceutically acceptable carrier is a carrier suitable for the preparation of compositions for topical and / or transdermal administration (application). In some other, also non-limiting, embodiments of the invention, the pharmaceutically acceptable carrier is a carrier suitable for the preparation of compositions for cutaneous, subcutaneous (subdermal), transdermal, and / or intradermal administration (application).

[0105] In particularly preferred, but also non-limiting, embodiments of the invention, the pharmaceutically acceptable carrier is one conventionally used in the art for the preparation of ointments, pastes, creams, suspensions, emulsions, fatty ointments, gels, powders, lotions, solutions, sprays, patches, prefilled microneedle assemblies, injections, and inhalation forms. In some more preferred embodiments, such a pharmaceutically acceptable carrier is minimally invasive or non-invasive and contains one or more of the excipients listed above.

[0106] In some embodiments of the invention, a pharmaceutically acceptable carrier is suitable for producing dosage forms for topical and / or transdermal and / or intradermal use. Non-limiting (illustrative) examples of such forms include ointments, pastes, creams, suspensions, emulsions, fatty ointments, gels, powders, lotions, solutions, sprays, patches, and inhalation forms. In some non-limiting (illustrative) embodiments of the invention, such a pharmaceutically acceptable carrier is a carrier suitable for producing an ointment, paste, cream, suspension, emulsion, gel, powder, lotion, solution, spray, patch, injection form, or inhalation form.

[0107] Non-limiting (illustrative) examples of such pharmaceutically acceptable carriers include binders (e.g., pregelatinized starch, including pregelatinized corn or potato starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrating agents (e.g., starch, sodium starch glycolate, etc.); humectants (e.g., sodium lauryl sulfate, etc.); salt solutions; alcohols; polyethyleneglycols; gelatin; lactose; amylase; magnesium stearate; talc; silicic acid; viscous paraffin; hydroxymethylcellulose; polyvinylpyrrolidone; sweeteners; flavoring agents; flavorings; dyes; moisturizing agents; sunscreens; antibacterial agents; agents that stabilize polynucleotides and prevent their degradation, etc.

[0108] Other non-limiting (illustrative) examples of such pharmaceutically acceptable carriers can be found in the above-mentioned monograph "Remington: The Science and Practice of Pharmacy" (Academic Press Publishing House; 23 rd edition, edited by Adeboye Adejare, November 13, 2020), as well as in other similar publications well known to specialists in this field, for example, in the monograph "Handbook of Pharmaceutical Excipients", 6 thedition, Edited by Raymond C Rowe, Paul J Sheskey and Marianne E Quinn, Pharmaceutical Press and American Pharmacists Association, 2009, the contents of which are incorporated herein by reference.

[0109] In an alternative embodiment of the invention, the carrier may be a set of microneedles containing the pharmaceutical composition of the invention, wherein methods for making and using such microneedles are generally known in the art, for example from the work of Kim Y.-C. et al., 2012, the contents of which are incorporated herein by reference.

[0110] In some embodiments of the invention, the pharmaceutical composition may contain a combination of two, three, four or more pharmaceutically acceptable carriers suitable for producing dosage forms for topical and / or transdermal and / or intradermal use.

[0111] Also, in some non-limiting embodiments of the invention, the pharmaceutically acceptable carrier may optionally further include, without limitation, one or more of: a binder, fillers, lubricants, disintegrants, humectants, electrolyte solutions, alcohols, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxypropyl cellulose, polyvinylpyrrolidone, dyes, sunscreens, antibacterial agents, agents capable of stabilizing polynucleotides and preventing their degradation, etc. In this case, binders, fillers, lubricants, disintegrants, humectants and other above-mentioned auxiliary components conventionally used in this field of technology and well known to those skilled in the art can be used.

[0112] It is clear to the person skilled in the art that the pharmaceutically acceptable carrier may also contain other excipients not directly mentioned in the present description, but used in the art and well known to the person skilled in the art, and that pharmaceutical compositions containing such a pharmaceutically acceptable carrier or such pharmaceutically acceptable carriers will also be part of the present invention.

[0113] The following examples, demonstrating the implementation of the present invention and the achievement of the claimed technical results during its implementation, are intended to illustrate the invention, but not to limit the scope of claims.

[0114] The present invention is further illustrated, but not limited, by the following examples, which illustrate the present invention.

[0115] Example 1. Obtaining a viral vector according to the present invention

[0116] To create the vector of the present invention, a replication-incompetent viral vector based on the genome of the herpes simplex virus type I was used. The herpes simplex virus genome was modified by introducing a bacterial artificial chromosome (BAC) into the intergenic region UL37-UL38 at the loxP sites of Cre-recombinase according to the method proposed by Tanaka et al., 2003. The AC cassette included gene sequences for selective selection, including the chloramphenicol resistance gene and the EGFP fluorescent marker protein gene, as well as loxP / Cre sequences, which were used to insert into the viral genome.

[0117] To suppress replicative capacity and reduce toxicity in target human cells, mutations of immediate-early genes, including RS1 / ICP4, US1 / ICP22, and UL54 / ICP27, including inactivating mutations, were sequentially introduced into the BAC-modified herpes simplex virus type 1 genome. Inactivating mutations were also introduced into the UL41 and / or UL27 genes. Plasmids expressing lambda phage RED proteins and the restriction enzyme I-Scel were used for mutagenesis.

[0118] The transgene expression cassette was inserted into the vacated region of the genome following the RS1 / ICP4 deletion.

[0119] To obtain a cassette expressing the COL7A1 gene, a library of cassettes was created that included a different set of regulatory elements selected from UCOE (an insulator sequence consisting of the SVX3 and HNRPA2B1 promoters with introns), a heterologous CMV promoter together with its enhancer, a chimeric SV40 intron, a Kozak sequence, a COL7A1 gene sequence, and a bovine growth hormone polyadenylation site (bGHpA).

[0120] The specified library included: cassette (A) containing the immediate-early promoter and enhancer of human cytomegalovirus - Kozak sequence - transgene encoding the single chain of human collagen type VII - bovine growth hormone polyadenylation signal; cassette (B) containing the immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - transgene encoding the al-chain of human collagen type VII - bovine growth hormone polyadenylation signal; cassette (C) containing the immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - Kozak sequence - transgene encoding the single chain of human collagen type VII - bovine growth hormone polyadenylation signal; cassette (D) containing the UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - a transgene encoding the U chain of human collagen type VII - a bovine growth hormone polyadenylation signal;cassette (E) containing the UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - Kozak sequence - transgene encoding the single chain of human collagen type VII - bovine growth hormone polyadenylation signal; cassette (F) containing the UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - transgene encoding the alpha chain of human collagen type VII - bovine growth hormone polyadenylation signal; cassette (G) containing the UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - Kozak sequence - transgene encoding the alpha chain of human collagen type VII - bovine growth hormone polyadenylation signal; and a cassette (J) containing the immediate-early promoter and enhancer of human cytomegalovirus - a transgene encoding the al-chain of human type VII collagen - a bovine growth hormone polyadenylation signal.

[0121] The most effective variant of the composition of regulatory elements of the expression cassette was determined experimentally based on the relative level of COL7A1 mRNA synthesis and production of the ai-chain of type VII collagen upon transfection of the cassettes into HEK293. The level of COL7A1 transcription was assessed by real-time PCR with reverse transcription using specific primers for COL7A1 and SYBR Green with normalization for the housekeeping genes B2M and TFRC using the AACt method. The level of collagen type VII expression was determined by immunoblotting using antibodies to type VII collagen with correction for transfection efficiency using green fluorescent protein and normalization of samples using pan-actin (Fig. 2). The level of protein expression was determined by the intensity of chemiluminescent staining of bands on the membrane using a gel documentation system and calculated in the GelQuant software.The cassette (F) showed the highest efficiency in producing type VII collagen according to the results of the cell culture experiment.

[0122] After insertion of COL7A1 expression cassettes into the vacated regions of the genome due to the RS1 / ICP4 deletion, gene therapy replication-incompetent viral vectors based on the genome of the herpes simplex virus type I, capable of efficient production of type VII collagen, were obtained.

[0123] Example 2. Creation of a cell line for the production of replication-incompetent herpes simplex viruses type I according to the present invention

[0124] To produce replication-incompetent herpes simplex virus type 1, a modified Vero cell line complementary to the synthesis of proteins excluded from the viral vector was used. To obtain a complementing Vero-based cell line, the cells were modified using lentiviruses with an expression cassette that ensures the expression of immediate-early genes of herpes simplex virus type 1, including RS1 / ICP4 and UL54 / ICP27.

[0125] Lentiviruses carrying an expression cassette of the immediate-early genes of herpes simplex virus type 1 (RS1 / ICP4, UL54 / ICP27), as well as the mCherry fluorescent protein, a gene for selective selection, were produced in the HEK293T cell line. Alternatively, other fluorescent proteins, such as the BFP (blue fluorescent protein), as well as antibiotic resistance genes, can be used. The resulting lentiviruses were then used to infect the Vero cell line, and the cells were selected using the selective marker. Insertion of the expression cassette and viral protein production from it were confirmed using PCR and immunoblotting.

[0126] Example 3. Production of replication-incompetent viruses according to the present invention

[0127] A modified replication-incompetent viral vector of the present invention based on the herpes simplex virus type I genome, containing cassettes expressing type VII collagen, was transfected into complementing Vero cells (ICP4+, ICP27+), capable of replacing the synthesis of gene products mutated in the vector and ensuring the reproduction and secretion of the replication-incompetent virus. To eliminate the BAC cassette from the DNA of replication-incompetent viruses, the modified cell lines, together with the replication-incompetent viral vector, were additionally transfected with a plasmid expressing the Cre-recombinase gene (Gierasch WW et al., 2006).

[0128] Viruses were harvested 120–192 hours after transfection. Cells and medium were collected and centrifuged at 300 g for 5 min. The medium was then separated and stored at -70°C. Cells in the pellet were lysed with lysis buffer (0.05 M Tris-HCl pH 8, 0.15 M NaCl), followed by three freeze-thaw cycles and centrifugation at 6300 g for 10 minutes. The supernatant was collected and stored at -70°C.

[0129] Purification of the resulting replication-incompetent viruses was performed using standard techniques well known to those skilled in the art, including gradient centrifugation and filtration.

[0130] Example 4. Study of transgene expression in target cells in vitro

[0131] To evaluate transgene production in target cells in vitro, HaCaT keratinocytes and HDF dermal fibroblasts were infected with the viruses obtained in Example 3 according to the present invention at a multiplicity of infection (MOI) ranging from 0.3 to 50. The HT-29 cell line, which does not express type VII collagen, was also used in the experiment, as previously confirmed by a semiquantitative assessment of the COL7A1 transcript level. The COL7A1 transcript level was assessed by real-time PCR with reverse transcription using specific primers for COL7A1 and housekeeping genes according to the AACt method, with normalization to the mRNA level in uninfected cells. Expression of type VII collagen was determined by immunoblotting using antibodies to type VII collagen and normalization to pan-actin.In infected keratinocytes, fibroblasts, and HT-29 cells, a multiplicity of infection-dependent significant increase in COL7A1 mRNA levels and type VII collagen protein content was observed compared to controls.

[0132] Thus, the presented examples confirm the industrial applicability of the proposed group of inventions. References:

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Claims

Invention formula:

1. Herpes virus type I (HSV-1) containing a recombinant HSV genome, wherein said genome contains one or two expression cassettes for expressing the g-chain of human collagen type VII, wherein each of said expression cassettes independently includes a transgene encoding the g-chain of human collagen type VII, and a combination of regulatory sequences selected from: - immediate-early promoter and enhancer of human cytomegalovirus (I); - bovine growth hormone polyadenylation signal (II); - Kozak sequences (III); - SV40 intron (IV); - UCOE intron (V), wherein said transgene together with said combination of regulatory sequences forms one of the following combinations: (A) immediate-early promoter and enhancer of human cytomegalovirus - Kozak sequence - transgene encoding the α-chain of human collagen type VII - bovine growth hormone polyadenylation signal; (B) immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - transgene encoding the α-chain of human collagen type VII - bovine growth hormone polyadenylation signal; (C) immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - Kozak sequence - transgene encoding the α-chain of human collagen type VII - bovine growth hormone polyadenylation signal; (D) UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - transgene encoding the U chain of human collagen type VII - bovine growth hormone polyadenylation signal; (E) UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - Kozak sequence - transgene encoding the α-chain of human collagen type VII - bovine growth hormone polyadenylation signal; (F) UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - transgene encoding the single chain of human collagen type VII - bovine growth hormone polyadenylation signal; (G) UCOE intron - immediate-early promoter and enhancer of human cytomegalovirus - SV40 intron - Kozak sequence - transgene encoding the single chain of human collagen type VII - bovine growth hormone polyadenylation signal; (H) SV40 intron - immediate-early promoter and enhancer of human cytomegalovirus - UCOE intron - transgene encoding the single chain of human collagen type VII - bovine growth hormone polyadenylation signal; (I) the SV40 intron - the immediate-early promoter and enhancer of human cytomegalovirus - the UCOE intron - the Kozak sequence - the transgene encoding the single chain of human type VII collagen - the bovine growth hormone polyadenylation signal, and wherein said recombinant HSV genome contains inactivating mutations of one or more immediate-early, early, or late genes of HIV-1.

2. Herpes simplex virus type I (HSV-1) containing a recombinant HSV genome, wherein said genome contains one or two expression cassettes for expressing the al-chain of human collagen type VII, comprising the following combination of a transgene encoding the al-chain of human collagen type VII and regulatory sequences: (J) the immediate-early promoter and enhancer of human cytomegalovirus - a transgene encoding the al-chain of human collagen type VII - a bovine growth hormone polyadenylation signal, wherein said recombinant HSV genome contains inactivating mutations of the genes RS1(ICP4), UL54(ICP27), US1(ICP22), UL41 and does not contain inactivating mutations of the genes RL2(ICP0) and / or US12(ICP47).

3. The virus according to any of paragraphs 1-2, characterized in that said transgene encoding the ai chain of human collagen type VII has the sequence SEQ ID NO:

15.

4. The virus according to any one of paragraphs 1-2, characterized in that the immediate-early promoter and enhancer of human cytomegalovirus is represented by the sequence SEQ ID NO:

10.

5. The virus according to any one of paragraphs 1-2, characterized in that the bovine growth hormone polyadenylation signal is represented by the sequence SEQ ID NO:

11.

6. The virus according to any one of paragraphs 1-2, characterized in that the Kozak sequence is represented by the sequence SEQ ID NO:

12.

7. The virus according to any one of paragraphs 1-2, characterized in that the SV40 intron is represented by the sequence SEQ ID NO:

13.

8. The virus according to any one of paragraphs 1-2, characterized in that the UCOE intron is represented by the sequence SEQ ID NO:

14.

9. The virus according to paragraph 1, characterized in that the inactivating mutations of one or more immediate-early, early or late genes of HSV-1 are selected from inactivating mutations of the genes RS1(ICP4), UL54(ICP27), UL41 and US1(ICP22).

10. The virus according to any of paragraphs 1 to 9, characterized in that said recombinant HSV genome additionally contains an inactivating mutation of the UL55 gene.

11. The virus according to paragraph 1, characterized in that the said recombinant Bill genome contains inactivating mutations of the genes RS1(ICP4), UL54(ICP27), US1(ICP22), UL41 and does not contain inactivating mutations of the genes RL2(ICP0) and / or US12(ICP47).

12. The virus according to any of paragraphs 1 to 11, characterized in that said recombinant HSV genome does not contain inactivating mutations of other genes.

13. The virus according to any one of paragraphs 1 to 12, characterized in that said recombinant HSV genome does not contain the coding sequence of one or more HSV-1 genes from RS1(ICP4), UL54(ICP27) and US1(ICP22).

14. The virus according to any of paragraphs 1 to 13, characterized in that said recombinant HSV genome does not contain regulatory sequences of the US1(ICP22) gene, which ensure its immediate-early expression.

15. The virus according to any one of paragraphs 1 to 13, characterized in that said recombinant HSV genome additionally contains up to two single nucleotide substitutions in the UL27 gene.

16. The virus according to paragraph 15, characterized in that the said single nucleotide substitutions in the UL27 gene result in the substitution of amino acid 285 and amino acid 549 of the protein product of the said gene, and the combination of these substitutions is selected arbitrarily from the list of possible substitutions: GAC <AAC, GAC<TAC, GAC<CAC, GAC<GTC, GAC<GGC, GAC<GCA, GAC<GAA, GAC<GAG - для аминокислоты 285, и GCC<ACC, GCC<TCC, GCC<GAC, GCC<GTC, GCC<GGC - для аминокислоты 549.

17. The virus according to any one of paragraphs 1 to 16, characterized in that said recombinant HSV genome does not contain the coding sequence of the UL41 gene.

18. A pharmaceutical composition for gene therapy of bullous genodermatosis, comprising a therapeutically effective amount of a virus according to any one of paragraphs 1 to 17 and at least one pharmaceutically acceptable excipient.

19. The pharmaceutical composition according to paragraph 18, characterized in that it is made in a form for topical application.

20. The pharmaceutical composition according to paragraph 18, characterized in that it is made in a form for transdermal use.

21. The pharmaceutical composition according to paragraph 18, characterized in that it is made in a form for intradermal use.

22. A vector comprising one or two polynucleotides encoding a polypeptide of the α chain of type VII collagen, wherein said polynucleotide comprises or said two polynucleotides each independently comprise a cassette for expressing the α chain of human type VII collagen, comprising a transgene encoding the α chain of human type VII collagen and a combination of regulatory sequences selected from the immediate-early promoter and enhancer of human cytomegalovirus, the bovine growth hormone polyadenylation signal, the Kozak sequence, the SV40 intron and the UCOE intron, wherein said transgene together with said regulatory sequences forms one of the combinations (A) to (I), as specified in paragraph 1, or the combination (J), as specified in paragraph 2, and wherein said vector is a recombinant HSV genome, and wherein said recombinant HSV genome comprises inactivating mutations of one or more immediate-early, early, or late genes of HSV-1.

23. The vector according to paragraph 22, characterized in that said recombinant Bill genome additionally contains an inactivating mutation of the UL55 gene.

24. The vector according to paragraph 22, characterized in that said recombinant HSV genome contains inactivating mutations of the genes RS1(ICP4), UL54(ICP27), US1(ICP22), UL41 and does not contain inactivating mutations of the genes RL2(ICP0) and / or US1(ICP47).

25. The vector according to paragraph 22, characterized in that said recombinant Bill genome does not contain inactivating mutations of other genes.

26. The vector according to paragraph 22, characterized in that said recombinant Bill genome does not contain the coding sequence of one or more HSV-1 genes from RS1(ICP4), UL54(ICP27) and US1(ICP22).

27. The vector according to paragraph 22, characterized in that said recombinant HSV genome does not contain regulatory sequences of the US1(ICP22) gene, which ensure its immediate-early expression.

28. The vector according to paragraph 22, characterized in that said recombinant HSV genome additionally contains up to two single nucleotide substitutions in the UL27 gene.

29. The vector according to paragraph 28, characterized in that the said single nucleotide substitutions in the UL27 gene result in the substitution of amino acid 285 and amino acid 549 of the protein product of the said gene, and the combination of these substitutions is selected arbitrarily from the list of possible substitutions: GAC <AAC, GAC<TAC, GAC<CAC, GAC<GTC, GAC<GGC, GAC<GCA, GAC<GAA, GAC<GAG - для аминокислоты 285, и GCC<ACC, GCC<TCC, GCC<GAC, GCC<GTC, GCC<GGC - для аминокислоты 549.

30. The vector according to paragraph 22, characterized in that said recombinant HSV genome does not contain the coding sequence of the TL G gene 31. The vector according to paragraph 22, characterized in that said transgene encoding the U chain of human collagen type VII has the sequence SEQ ID NO:

15.

32. The vector according to paragraph 22, characterized in that the immediate-early promoter and enhancer of human cytomegalovirus are represented by the sequence SEQ ID NO:

10.

33. The vector according to paragraph 22, characterized in that the bovine growth hormone polyadenylation signal is represented by the sequence SEQ ID NO:

11.

34. The vector according to paragraph 22, characterized in that the Kozak sequence is represented by the sequence SEQ ID NO:

12.

35. The vector according to paragraph 22, characterized in that the SV40 intron is represented by the sequence SEQ ID NO:

13.

36. The vector according to paragraph 22, characterized in that the UCOE intron is represented by the sequence SEQ ID NO:

14.

37. The vector according to paragraph 22, characterized in that the inactivating mutations of one or more immediate-early, early or late genes of HSV-1 are selected from inactivating mutations of the genes RS1(ICP4), UL54(ICP27), UL41 and US1(ICP22).

38. A host cell comprising a vector according to any of paragraphs 22 to 37 or modified with a vector according to any of paragraphs 22 to 37.

39. The cell according to paragraph 38, characterized in that said cell is a mammalian cell.

40. The cell of claim 39, wherein said host cell is selected from a monkey kidney cell transformed with SV40, a human embryonic kidney cell, a baby hamster kidney cell, a mouse Sertoli+ cell, an African green monkey kidney cell, a human cervical carcinoma cell, a dog kidney cell, a Buffalo rat liver cell, a human lung cell, a human liver cell, a mouse breast tumor cell (MMT 060562, ATCC CCL51), a TRI cell, a MRC 5 cell, a FS4 cell, a human hepatoma cell (Hep G2), a Chinese hamster ovary cell (CHO), a DHFR(-) cell, an NSO myeloma cell, and an Sp2 / 0 myeloma cell.

41. The cell according to paragraph 38, characterized in that said cell is a target cell.

42. The cell according to paragraph 41, characterized in that said target cell is selected from barrier tissue cells, including epithelial cells, keratinocytes and fibroblasts.

43. A method for the prophylactic, palliative or therapeutic treatment (improvement of the condition) of wounds of the skin and mucous membranes in a patient suffering from epidermolysis bullosa, as well as the prophylactic, palliative or therapeutic treatment of a disruption of the integrity or condition of the skin and mucous membranes caused by a mutation of the COL7A1 gene in a patient suffering from epidermolysis bullosa, wherein said method comprises administering to said patient a therapeutically effective amount of a virus according to any of paragraphs 1 to 17, a pharmaceutical composition according to any of paragraphs 18 to 21 or a vector according to any of paragraphs 22 to 37.

44. The use of a virus according to any of paragraphs 1 to 17, a pharmaceutical composition according to any of paragraphs 18 to 21, a vector according to any of paragraphs 22 to 37, or a cell according to any of paragraphs 38 to 42 for the manufacture of a medicinal product for the prophylactic, palliative, or therapeutic treatment (improvement of the condition) of wounds of the skin and mucous membranes in a patient suffering from epidermolysis bullosa, as well as for the prophylactic, palliative, or therapeutic treatment of a disruption of the integrity or condition of the skin and mucous membranes caused by a mutation of the COL7A1 gene in a patient suffering from epidermolysis bullosa.

45. The use of a virus according to any of paragraphs 1 to 17, a pharmaceutical composition according to any of paragraphs 18 to 21, a vector according to any of paragraphs 22 to 37, or a cell according to any of paragraphs 38 to 42 for the prophylactic, palliative, or therapeutic treatment (improvement of the condition) of wounds of the skin and mucous membranes in a patient suffering from epidermolysis bullosa, as well as for the prophylactic, palliative, or therapeutic treatment of a violation of the integrity or condition of the skin and mucous membranes caused by a mutation of the COL7A1 gene in a patient suffering from epidermolysis bullosa.

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