Agent pour le traitement de gÉnodermatoses bulleuses

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

AGENT FOR TREATING HEREDITARY BULLOUS DERMATOSESDESCRIPTION OF THE INVENTIONField of the InventionThe present invention relates to the fields of pharmacology and biotechnology, and more particularly to a gene therapy agent for treating hereditary bullous dermatoses, which agent is a herpes simplex virus type 1 (HSV-1) comprising a recombinant HSV genome including one or two expression cassettes for expressing the human type VII collagen α1 chain; as well as to a pharmaceutical composition for use in gene therapy of hereditary bullous dermatoses, comprising a therapeutically effective amount of such a virus; to a vector comprising one or two polynucleotides independently encoding the human type VII collagen α1 chain; to a host cell comprising such a vector or modified by such a vector; to a method for the preventive, palliative or therapeutic treatment of skin and mucosal wounds in patients with epidermolysis bullosa, using such a virus, such a pharmaceutical composition or such a vector; and to the use of said virus, said pharmaceutical composition or said host cell both for the production of a medicinal product for the preventive, palliative or therapeutic treatment of skin and mucosal wounds in patients with epidermolysis bullosa, and for the preventive, palliative or therapeutic treatment of skin and mucosal wounds in patients with epidermolysis bullosa.The invention may be used for the preventive, palliative or therapeutic treatment, i.e., gene (gene replacement) therapy, of hereditary bullous dermatoses, such as epidermolysis bullosa and various clinical forms thereof, including, but not limited to, dominant dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa and other forms and variants of epidermolysis bullosa known to a person skilled in the art.Background of the InventionHereditary bullous dermatosis (epidermolysis bullosa) is a group of congenital (hereditary) skin diseases caused by mutations in the genes encoding structural proteins of the epidermis and the dermo-epidermal junction, which proteins are responsible for the adhesion strength between the epidermis and the dermis. Epidermolysis bullosa typically presents at birth or within the first days (sometimes months) of life. Clinical presentations of epidermolysis bullosa are diverse and depend on the clinical subtype. However, the main clinical sign of hereditary bullous dermatoses is the formation of blisters and / or erosions on the skin and mucous membranes in response to minor mechanical injury (Fine et al., 2014). These subtypes of epidermolysis bullosa having the most severe clinical course, as severe generalised recessive dystrophic epidermolysis bullosa (RDEB) or severe generalised junctional epidermolysis bullosa, are accompanied by widespread lesions of the skin, mucous membranes of internal organs and multiple erosions and ulcers.Hereditary bullous dermatosis is classified as an orphan disease. According to the National Institutes of Health (NIH, USA), a disease is considered an orphan disease if the number of cases is fewer than 200,000 in the USA (Cuatrecasas P., 2006; Wästfelt M. et al., 2006). In the Russian Federation, Federal Law No. 323-FZ On the Fundamentals of Health Protection of Citizens in the Russian Federation (as amended on 25 June 2012) legally introduces the concept of rare (orphan) diseases. According to said law, diseases with a prevalence of not more than 10 cases per 100,000 population are considered orphan diseases.To study the incidence and prevalence of hereditary bullous dermatosis and to simplify data collection, patient registries for individuals with this disease have been established in many countries (Pavičić et al., 1990; McKenna et al., 1992; Horn et al., 1997; Kho et al., 2010; Danescu et al., 2015; Fine, 2016).In the Russian Federation, the prevalence and incidence of hereditary bullous dermatosis have been studied since 2014 (Kubanov et al., 2015). As of the end of 2016, the number of registered cases of hereditary bullous dermatosis across 60 constituent entities of the Russian Federation was 417. The corresponding prevalence rate of this disease is 3.9 per 1 million population. The incidence rate of hereditary bullous dermatosis for 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 hereditary bullous dermatosis based on the layer of the epidermis in which the defective protein is predominantly localised and blisters are formed, 4 main types of the disease are recognised: epidermolysis bullosa simplex, junctional epidermolysis bullosa (JEB), dystrophic epidermolysis bullosa, and Kindler syndrome (Fine et al., 2014).Hereditary bullous dermatosis is a clinically and genetically heterogeneous group of diseases that develop as a result of mutations in the genes encoding structural proteins of the epidermis and the dermo-epidermal junction (Has et al., 2018). To date, more than 20 genes responsible for the development of hereditary bullous dermatosis are known to a person skilled in the art (Has C., Fischer J., 2018). For example, recessive and dominant dystrophic epidermolysis bullosa develop as a result of mutations in the COL7A1 gene encoding the type VII collagen α1 chain (Dang N., Murrell D.F., 2008), whereas a mutation in the KLHL24 gene (Kelch-like protein 24) is responsible for the development of a new clinical subtype of JEB (Lin Z., Li S., Feng C. et al., 2016; Lee J. Y. W., Liu L., Hsu C.-K. et al., 2017; He Y., Maier K., Leppert J. et al., 2016; Kim S., Coulombe P.A., 2007). Furthermore, new clinical phenotypes of hereditary bullous dermatosis 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; Gostyńska K. B., 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., Gostyński A. et al., 2017; He Y., Leppert J., Steinke H. et al., 2017; Cappuccio G., Pinelli M., Torella A. et al., 2017; McGrath J. A., Stone K. L., 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 L. et al., 2016; Has C., Sparta G., Kiritsi D. et al., 2012; Nicolaou N., Margadant C., Kevelam S. H. et al., 2012; Yalcin E. G., 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).The involvement of a large number of genes in the pathogenesis of hereditary bullous dermatosis underlies the diversity of the clinical manifestations of this disease. The extent of the lesions varies from localised eruptions limited to the skin of the palms and soles to generalised eruptions involving the mucous membranes of internal organs (Kubanov A.A. et al., 2020).In accordance with the current classification of the various types of epidermolysis bullosa, the types of this disease are classified based on skin morphology (specifically, the level of blister formation within the tissues). Kindler syndrome, associated with an autosomal recessive mutation in the kindlin-1 protein, is considered separately (Anwar M.I., Rashid A., Ghafoor R. et al., 2014; Gadzhimuradova K.M., Zhukova O.V. et al., 2022).According to said classification, the following types of the disease are recognised:epidermolysis bullosa simplex (EBS);junctional epidermolysis bullosa (JEB);dystrophic epidermolysis bullosa (DEB); andKindler syndrome.In turn, epidermolysis bullosa simplex comprises two subtypes, suprabasal epidermolysis bullosa simplex, in which, as a rule, a mutation in the placophilin-1 or desmoplakin genes occurs, and basal epidermolysis bullosa simplex, in which a mutation in the keratin-5, keratin-14 or α6β4 integrin genes occurs.Junctional epidermolysis bullosa comprises the Herlitz subtype, in which a mutation in laminin-332 (laminin-5) occurs, or by other subtypes in which a mutation in laminin-332, type XVII collagen or α6β4 integrin occurs.Dystrophic epidermolysis bullosa comprises a dominant subtype (dominant dystrophic epidermolysis bullosa), in which a dominant mutation occurs in type VII collagen, the main component of the anchoring fibrils of the dermo-epidermal zone of the cutaneous basement membrane, and a recessive subtype (recessive dystrophic epidermolysis bullosa), in which a recessive mutation occurs in the gene encoding said protein.Type VII collagen, previously also referred to as long-chain collagen, has an unusually long triple-helical domain spanning approximately 450 nm. This collagen comprises only one type of α-chains, α1(VII), and its triple-helical collagen domain is flanked by a large non-helical (non-collagenous) domain (NC-1) at the amino terminus and a shorter non-helical domain (NC-2) at the carboxyl terminus. Type VII collagen molecules extend from the lower part of the lamina densa to the papillary dermis, wherein they form antiparallel dimers linked by their carboxyl termini. The large amino-terminal non-collagenous domains of type VII collagen interact with type IV collagens and components of laminin-332 of the dermo-epidermal basement membrane; it is suggested that the majority of anchoring fibrils form U-shaped loops that enclose broad dermal collagen fibres consisting of type I and III collagens. Thus, alterations in the expression, structure or molecular interactions of type VII collagen with other components of the basement membrane may result in skin fragility. Such a condition is exemplified by dystrophic epidermolysis bullosa (DEB), a group of mechanobullous diseases characterised by blister formation on the skin as a result of minor injury. Characteristic mutations in the type VII collagen gene (COL7A1) have been detected in 500 families with various forms of DEB, and to date, none of these families have been found to have mutations in any genes other than those encoding the type VII collagen α1(VII) polypeptide. Furthermore, type VII collagen serves as an autoantigen in epidermolysis bullosa acquisita, an autoimmune disease characterised by the presence of circulating antibodies against the type VII collagen antigen.Severe generalised recessive dystrophic epidermolysis bullosa (RDEB) is classified as a clinical subtype of hereditary bullous dermatosis associated with the poorest prognosis (Fine, 2010).RDEB is characterised by multiple blisters, erosions and ulcers that recur spontaneously and / or as a result of injury and lead to progressive scarring and the development of widespread cicatricial atrophy areas, pseudosyndactyly, mitten-like deformities of the hands, and joint contractures (Fine et al., 2002), wherein such defects may persist on the skin from 1 month to several years even with rational topical therapy.The development of such erosions and ulcers in patients with severe generalised RDEB is associated with a number of pathogenetic factors that prevent normal healing: bacterial biofilm formation on the surface of erosions / ulcers; increased expression of pro-inflammatory cytokines (IL-6, CXCL13, MCP-1, HMGB-1, etc.); the absence of pilosebaceous follicles, which are a source of regional stem cells, in cicatricial atrophy areas; and a deficiency of type VII collagen, which leads to a reduced rate of keratinocyte migration from the wound edges and the maturation of granulation tissue.Severe generalised RDEB is accompanied by growth delay, anaemia, nutritional or protein-energy malnutrition, early development of complications, and early mortality (Fine et al., 2008; Zidorio et al., 2015).Excessive formation of scar tissue and the presence of long-term non-healing skin defects lead to the development of squamous cell carcinoma (SCC) of the skin (Fine et al., 2009; Mellerio et al., 2016). SCC in hereditary bullous dermatosis is characterised by a high risk of onset at an early age (20–25 years), rapid tumour growth, rapid metastasis, and multiple primary tumour foci (Mellerio et al., 2016). SCC is the leading cause of mortality in patients with recessive dystrophic epidermolysis bullosa.The cumulative risk of SCC in patients with severe generalised RDEB 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, and 90.1% at the age of 55 years (Mellerio et al., 2016). The 5-year survival rate of patients with RDEB-associated SCC is close to 0% (Fine et al., 2009; Mellerio et al., 2016).Other important and severe complications of epidermolysis bullosa that reduce patient quality of life, worsen disease prognosis and shorten life expectancy include cardiovascular and gastrointestinal tract lesions, as well as haematological disorders. Sepsis is a severe and life-threatening complication of epidermolysis bullosa.Management of hereditary bullous dermatoses currently comprises both symptomatic therapies for the disease, aimed at accelerating the healing of erosions and ulcers of the skin and mucous membranes of internal organs, preventing the development of secondary infection, and managing complications and comorbidities (Denyer et al., 2017), in particular, antibiotic therapy aimed at suppressing infections that affect open wounds of the skin and mucous membranes, as well as methods aimed at alleviating pain and pruritus experienced by patients, and surgical treatment of cicatricial atrophy areas, pseudosyndactyly, and joint contractures.The most promising emerging approaches to the treatment of hereditary bullous dermatoses include cellular therapy, protein replacement therapy, and gene therapy (Rashidghamat, McGrath, 2017). Cellular therapy typically comprises intradermal injections of allogeneic fibroblasts containing type VII collagen, whereas protein replacement therapy comprises injections of purified recombinant type VII collagen into the patient.It has been shown in the prior art that a single intradermal injection of allogeneic fibroblasts at a dose of 5 × 106 cells into an area of the superficial dermis of approximately 1 cm2 resulted in enhanced expression of the COL7A1 gene in the majority of patients, wherein low immunogenicity of said fibroblasts was noted in said study (Wong T., Gammon L. et al., 2008). Clinical trials have shown that a single intradermal injection of allogeneic fibroblasts increases the expression of the COL7A1 gene for 3–6 months and that of the type VII collagen protein, for 9–12 months (Nagy N., Almaani N. et al., 2011). In a Phase II, double-blind, placebo-controlled clinical trial, a significant reduction in the area and number of erosions was shown in RDEB patients who received a single intradermal injection of allogeneic fibroblasts, wherein said effect persisted for more than 28 days (Petrof G., Martinez-Queipo M., et al., 2013).However, effective cellular therapy, such as the aforementioned allogeneic fibroblast therapy, requires a readily available source of cells, to use a safe and cost-effective method for their isolation and processing, to eliminate or at least minimise the possibility of an immune incompatibility, to ensure complete biological safety for the patient, to adequately assess the technical complexity of the methods applied and the material costs, and to consider the legal and ethical restrictions of national laws in relation to the application of such methods.A solution to the aforementioned problems is provided by personalised cellular therapy (PCT) technologies, in which the patient’s own (autologous) cells, obtained and stored in advance or obtained immediately prior to the treatment, are used for treating the patient (Khabriev R.U., Kovalchuk L.V. et al., 2011; Patel S.A., King C.C. et al., 2010). However, each specific cellular technology is characterised by a unique algorithm for obtaining, storing, processing and using autologous cells or a cellular product, which significantly increases the labour intensity and cost of such therapy.Furthermore, patients with hereditary bullous dermatosis typically present with extensive wounds on skin areas subjected to constant mechanical impact; therefore, any intradermal injection would represent for such patients an extremely invasive and high-risk intervention, increasing the likelihood of severe, including life-threatening, complications.As for protein replacement therapy involving the administration of purified recombinant type VII collagen, the recombinant collagen cannot penetrate intact skin upon topical administration thereof, and therefore its effect is confined to lesion sites. Intradermal injections of purified recombinant type VII collagen represent another alternative for RDEB patients; however, limited diffusion and uncontrolled penetration depth during injection with a conventional needle necessitate the use of a sterile microneedle array, the widespread clinical use of which is not yet available (Nagarkar R. et al., 2020).On the other hand, it has been shown in the literature that systemic administration of type VII collagen may cause systemic toxicity (Hou Y., Guey L.T. et al., 2015), which significantly limits the applicability of protein replacement therapy in hereditary bullous dermatoses.The most advanced approaches to the treatment of hereditary bullous dermatoses currently under investigation include allogeneic bone marrow transplantation with or without mesenchymal stem cell infusion, the use of agents promoting translational read-through of premature termination codons or stop codons, systemic administration of recombinant type VII collagen, as well as gene therapy (Prodinger C., Reichelt J. et al., 2019).However, allogeneic bone marrow transplantation, the efficacy of which has been demonstrated by various authors in a small number of patients with recessive dystrophic epidermolysis bullosa (Boull C.L., Hylwa S.A., et al., 2016; Ebens C. L., McGrath J. A., Tamai K. et al., 2019; Geyer M. B., Radhakrishnan K. et al., 2015; Wagner J.E., Ishida-Yamamoto A. et al., 2010), has proved to be ineffective for the treatment of other clinical forms of epidermolysis bullosa, such as junctional epidermolysis bullosa (Hammersen J., Has C. et al., 2016).Particular mention should be made of methods utilising autologous transplantation of the patient’s keratinocytes. Such methods, which are currently being actively investigated, typically involve isolating the patient’s own keratinocytes from a small biopsy sample, with their subsequent expansion under ex vivo conditions and transplantation back to the patient. Successful application of these methods requires the presence of a sufficient number of healthy epidermal stem cells in the biopsy sample for their transfection and growth into epidermal equivalents that can be used for transplantation to the patient and that will be able to ensure permanent regeneration of both a healthy, functional, and renewing epidermis and the extracellular environment, which represents a significant problem in the case of patients with hereditary bullous dermatosis, since such patients have a very limited number of epidermal stem cells, and their number progressively decreases with age as a result of recurrent wounds and subsequent wound scarring.Herpesviruses, in particular herpes simplex virus (HSV), for example herpes simplex virus type 1 (HSV-1), represent one of the most versatile and flexible tools for gene replacement therapy. Herpes simplex virus is a complex, non-integrating DNA virus capable of infecting a very wide range of human and animal cells. The viral genome contains more than 80 genes and consists of two unique segments, UL and US, wherein each said segment is flanked by inverted repeats encoding critical duplicated genes. An important feature of viral replication is the expression of its genes in waves, referred to as cascade regulation (Rajcani, 2004). Deletion or inactivation of the essential immediate-early (IE) genes UL54 (ICP27) and RS1 (ICP4) renders the virus completely replication-defective and incapable of expressing early (E) genes involved in viral genome replication and late (L) genes involved in the assembly of progeny virions. These replication-defective viruses may be propagated in complementing cells that express the missing products of the RS1 (ICP4) and UL54 (ICP27) genes and may then be used to infect non-complementing cells, wherein the viral genome resides as a stable nuclear episome.The widespread use of HSV-based vectors is due to the natural properties of this virus, which make it attractive for vector development. Such properties include the latent nature of herpesvirus infection and the long-term expression of HSV genes, due to which HSV vectors may maintain a latent state in the patient, as well as the episomal (non-integrating) viral DNA genome, which avoids undesirable insertional mutagenesis in the host genome (Garber D.A. et al., 1993; Efstathiou S. et al., 1986; Jackson S.A. and DeLuca N.A., 2003), and the presence of non-essential genes for replication in HSV that may be deleted, thereby enabling cloning of large transgenes along with their regulatory sequences using HSV vectors (Roizman B., 1996). Hereinafter in the present description, the term ‘regulatory sequence’ or ‘regulatory element’ includes promoters, enhancers, insulators, polyadenylation signals, as well as other expression control elements well known to a person skilled in the art, including regulatory elements capable of increasing transgene expression (Powell et al., 2015; Maksimenko et al., 2015; Ermekova S.A., 2017).In addition, the features of HSV that make it particularly attractive for vector development include its wide cellular tropism, high transgene cargo capacity (approximately 30,000 base pairs), as well as its ability to transduce both dividing and quiescent cells (Spaete R.R. and Frenkel N., 1982).Attempts to develop various HSV-based vectors intended for the treatment of genetic diseases have been made in the prior art by various authors. For example, US Patent No. US 6319703 B1, published on 20 November 2001 (SPECK PETER G [US]), discloses a herpesvirus mutant comprising inactivating mutations in one or more essential viral genes required for its replication, in particular the gH gene, and comprising inactivating mutations in genes required for the lysis of virus-infected cells, in particular the VP16 gene. The herpesvirus mutant disclosed in US Patent No. US 6319703 B1 may be used to develop gene therapy vectors for severe diseases, including cystic fibrosis, Tay-Sachs disease (GM2 gangliosidosis), β-thalassaemia, and Hurler syndrome (mucopolysaccharidosis type IH). However, the patent does not address the use of this mutant for developing vectors for hereditary bullous dermatoses, specifically epidermolysis bullosa. Furthermore, no subsequent attempts to develop vectors for use in gene therapy of hereditary bullous dermatoses, such as epidermolysis bullosa, based on this herpesvirus mutant have been reported.Several attempts to develop a vector based on a replication-defective HSV for use in gene therapy of hereditary bullous dermatoses, such as epidermolysis bullosa, are also known in the prior art. For example, international patent application publication No. WO 2017 / 176336 A1, published on 12 October 2017, filed by KRYSTAL BIOTECH, INC. (US), discloses a replication-defective HSV-1 comprising a vector including one or more transgenes encoding a polypeptide selected from the type VII collagen α1 polypeptide chain, the lysyl hydroxylase 3 polypeptide chain, and a chimeric polypeptide of the type VII collagen α1 chain and lysyl hydroxylase 3, optionally comprising inactivating mutations in one or more immediate-early, early and late HSV-1 genes selected from RL2 (ICP0), RS1 (ICP4), US1 (ICP22), UL54 (ICP27), US12 (ICP47), UL23 (tk), UL41, and UL55. Hereinafter in the present description, the term ‘immediate-early genes’ (also referred to as immediate-early regulatory genes) of herpes simplex virus refers to genes encoding proteins that regulate the expression of early and late viral genes and are expressed first upon entry of the virus into the cell (Honess R.W. and Roizman B., 1974).Furthermore, WO 2017 / 176336 A1 discloses a pharmaceutical composition, optionally formulated in the form of an ointment, paste, cream, suspension, emulsion, lipophilic ointment, gel, powder, lotion, solution, spray, patch, a pre-filled microneedle array or an inhalation form, comprising such an HSV-1 virus together with a pharmaceutically acceptable carrier, intended for increasing, enhancing or maintaining the level of type VII collagen in cells, for example in skin cells, including epidermal or dermal cells, especially in patients with epidermolysis bullosa.The said virus or vector and the pharmaceutical composition comprising such a virus or vector according to WO 2017 / 176336 A1 may be used for the preventive, 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), pressure ulcers, erysipelas, ichthyosis vulgaris, dermatomyositis, acrodermatitis, stasis dermatitis, Netherton syndrome, epidermolysis bullosa simplex associated with a LAMB3 gene mutation, autosomal recessive congenital ichthyosis, xeroderma pigmentosum, and pemphigoid.WO 2017 / 176336 A1 discloses an illustrative example of such a replication-defective HSV-1 virus, KB103, which is proposed as the closest prior art of the virus according to the present invention and comprises an expression cassette including the human cytomegalovirus immediate-early promoter (HCMV IEp), a transgene encoding the human type VII collagen α1 chain (COL7A1), and the bovine growth hormone polyadenylation signal (BGHpA), wherein it is shown that infection of experimental animals (Balb / c and SKH1 mouse strains) with said virus provided a high number of type VII collagen transcripts in the mouse tissues and led to achieving a high level of type VII collagen in isolated mouse tissue samples and to the initiation of type VII collagen deposition in the region of the basement membrane.Nevertheless, there currently remains 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 human type VII collagen α1 chain, which, when incorporated into a recombinant HSV genome and into a virus (HSV-1) comprising such a genome, would ensure even more efficient expression of the transgene and, consequently, more effective gene therapy for hereditary bullous dermatosis, such as epidermolysis bullosa.Furthermore, the tasks of ensuring prolonged expression of the transgene (type VII collagen) and a higher viral yield during its production and developing an agent with higher therapeutic efficacy and fewer side effects, is also relevant.However, neither WO 2017 / 176336 A1 nor any other sources forming part of the prior art provide detailed information on the optimal combination of regulatory elements capable of ensuring maximum expression efficiency of the transgene (the COL7A1 gene), or on the efficiency of transgene expression and the level of type VII collagen production therefrom. At the same time, it is well known in the prior art that transgene expression depends on the type and combination of the regulatory sequences used (Powell et al., 2015; Maksimenko et al., 2015).Furthermore, according to published partially completed clinical trials of the agent developed by KRYSTAL BIOTECH, INC. (US) and disclosed in WO 2017 / 176336 A1 and WO 2019 / 210219 A1, which has received the international non-proprietary name beremagene geperpavec, the administration of said agent to a patient, such as a human, may be accompanied by a variety of side effects, including chills, cough, rhinorrhoea, pruritus, erythema, or skin rash. Moreover, in published clinical trials of said agent, three cases of squamous cell carcinoma of the skin were also reported in patients treated with this agent (Guide S.V., Gonzalez M.E. et al., 2022).In this regard, there currently remains a need for new, more advanced and safer viral vectors for the transduction of epidermal and dermal cells to express collagen therein and for the gene therapy of hereditary bullous dermatoses. Furthermore, as is well known to a person skilled in the art, universal regulatory elements with a clear mechanism of action have not yet been found (Powell et al., 2015; Maksimenko et al., 2015). Neither WO 2019 / 210219 A1 nor WO 2017 / 176336 A1 discloses in detail which specific regulatory sequences and what combinations thereof were used in the aforementioned viruses (HSV-1) and vectors based thereon for use in gene therapy of hereditary bullous dermatosis. However, it is well known that the choice of regulatory elements is critical for efficient transgene expression (Maksimenko O.G. et al., 2015).In this regard, a person skilled in the art is faced with the task of finding an optimal combination of regulatory sequences that would provide a higher expression of the transgene (the type VII collagen α1 chain gene), which would, in turn, allow to increase the efficiency of the gene therapy of hereditary bullous dermatosis. Furthermore, a person skilled in the art is faced with the tasks of providing prolonged expression of the transgene, achieving a higher viral yield during its production, and developing an agent with improved therapeutic efficacy and fewer side effects.Finally, despite the currently used therapies for hereditary bullous dermatosis approved for clinical use, there still remains a need to develop new, more effective agents for the treatment of this disease, including gene therapy agents, which would allow to improve the quality of life and increase the life expectancy of patients with hereditary bullous dermatosis, as well as to delay the onset of squamous cell carcinoma of the skin and other life-threatening complications of hereditary bullous dermatosis.Thus, the present invention aims to provide gene therapy agents based on herpes simplex virus that, in comparison with known analogues, would ensure the achievement of such technical results as more efficient and more stable expression of type VII collagen in the target tissues of a patient and, consequently, more effective therapy for hereditary bullous dermatosis, as well as ensuring prolonged expression of the transgene, achieving a higher viral yield during its production, and developing an agent with improved therapeutic efficacy and fewer side effects.Finally, the task of expanding the range of effective and safe gene therapy agents for the treatment (gene therapy) of hereditary bullous dermatosis, such as epidermolysis bullosa, including the medical, preventive or palliative treatment of this disease, is also relevant.Summary of the InventionIn one aspect, the present invention provides a herpes simplex virus type 1 (HSV-1) comprising a recombinant HSV genome, wherein said genome comprises one or two expression cassettes for the expression of the human type VII collagen α1 chain, wherein each of said expression cassettes independently includes a transgene encoding the human type VII collagen α1 chain and a combination of regulatory sequences selected from:- a human cytomegalovirus immediate-early promoter and enhancer (I);- a bovine growth hormone polyadenylation signal (II);- a Kozak sequence (III);- an SV40 intron (IV);- a UCOE intron (V),wherein said transgene together with said combination of regulatory sequences forms one of the following combinations:(A) human cytomegalovirus immediate-early promoter and enhancer–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(B) human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(C) human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(D) UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(E) UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(F) UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(G) UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(H) SV40 intron–human cytomegalovirus immediate-early promoter and enhancer–UCOE intron–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(I) SV40 intron–human cytomegalovirus immediate-early promoter and enhancer–UCOE intron–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal,wherein said recombinant HSV genome comprises inactivating mutations in one or more immediate-early, early or late HSV-1 genes.The inactivating mutations in one or more immediate-early, early or late genes of the said virus (HSV-1) are preferably selected from inactivating mutations in the RS1 (ICP4), UL54 (ICP27), UL41 and US1 (ICP22) genes, wherein the HSV-1 according to the present invention, preferably, does not comprise inactivating mutations in other genes.The aforementioned aim of the present invention is achieved and the technical results are attained by providing a herpes simplex virus type 1 (HSV-1) comprising a recombinant HSV genome, wherein said genome comprises one or two expression cassettes for the expression of the human type VII collagen α1 chain, wherein each of said expression cassettes independently includes a transgene encoding the human type VII collagen α1 chain and the following combination of regulatory sequences:(J) human cytomegalovirus immediate-early promoter and enhancer–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal,wherein said recombinant HSV genome comprises inactivating mutations in the RS1 (ICP4), UL54 (ICP27), US1 (ICP22), and UL41 genes and does not comprise inactivating mutations in the RL2 (ICP0) and / or US12 (ICP47) genes.In the proposed combinations (A)–(J), the human cytomegalovirus immediate-early promoter and enhancer is, preferably, represented by SEQ ID NO: 10 (Schmidt et al., 1990; Xia et al., 2006);the bovine growth hormone polyadenylation signal is, preferably, represented by SEQ ID NO: 11 (Iwakuma et al., 1999);the Kozak sequence is represented by SEQ ID NO: 12 (Kozak, 1984);the SV40 intron is, preferably, represented by SEQ ID NO: 13 (Xu et al., 2018);the UCOE intron is, preferably, represented by SEQ ID NO: 14 (Neville et al., 2017);and finally, the COL7A1 transgene encoding the type VII collagen α1 chain is, preferably, represented by SEQ ID NO: 15 (Christiano A.M. et al., 1994).In preferred, non-limiting embodiments of the invention, said recombinant HSV genome comprising one or two expression cassettes for the expression of the human type VII collagen α1 chain, wherein each of said expression cassettes independently includes a transgene encoding the human type VII collagen α1 chain and a combination of regulatory sequences, wherein said combination of regulatory sequences together with said transgene constitutes one of combinations (A)–(I), comprises inactivating mutations in one or more immediate-early, early or late HSV-1 genes selected from RS1 (ICP4), UL54 (ICP27), UL41, and US1 (ICP22) and does not comprise inactivating mutations in the RL2 (ICP0) and / or US12 (ICP47) genes.Moreover, in certain preferred, but also non-limiting embodiments of the invention, said recombinant HSV genome further comprises an inactivating mutation in the UL55 gene.Furthermore, in certain preferred, but also non-limiting embodiments of the invention, said recombinant HSV genome lacks the coding sequence of one or more HSV genes selected from RS1 (ICP4), UL54 (ICP27), US1 (ICP22), and UL41.Furthermore, in certain preferred, but non-limiting embodiments of the invention, the recombinant HSV genome according to the present invention lacks the regulatory sequences of the HSV US1 (ICP22) gene that drive its immediate-early expression.Furthermore, in certain preferred, but non-limiting embodiments of the invention, the recombinant HSV genome according to the present invention further comprises up to two single nucleotide substitutions in the UL27 gene.In more preferred, but also non-limiting embodiments of the invention, the said single nucleotide substitutions in the UL27 gene lead to an amino acid substitution at position 285 and an amino acid substitution at position 549 of the protein product of said gene, namely envelope glycoprotein B.In the most preferred, but also non-limiting embodiments of the invention, the said single nucleotide substitutions in the UL27 gene are selected from the list of possible substitutions: GAC→AAC, GAC→TAC, GAC→CAC, GAC→GTC, GAC→GGC, GAC→GCA, GAC→GAA, GAC→GAG for amino acid position 285, and GCC→ACC, GCC→TCC, GCC→GAC, GCC→GTC, GCC→GGC for amino acid position 549, wherein said single nucleotide substitutions in said HSV-1 gene lead to an amino acid substitution at position 285 and an amino acid substitution at position 549 of the protein product of said gene, namely envelope glycoprotein B (from aspartic acid to asparagine, tyrosine, histidine, valine, glycine, alanine, or glutamic acid, and from alanine to threonine, serine, aspartic acid, valine, or glycine, respectively).Furthermore, in preferred, but also non-limiting embodiments of the invention, the DNA sequence according to the present invention lacks the coding sequence of the UL41 gene.In alternative embodiments of the invention, the proposed mutant herpes simplex virus type 1 (HSV-1) viral DNA genome differs from wild-type HSV-1 in that it comprises inactivating mutations in one or more immediate-early, early and late HSV-1 genes selected from RS1 (ICP4), UL54 (ICP27), and UL41, and does not comprise inactivating mutations in the UL55 gene.In preferred, non-limiting embodiments of the invention, the DNA sequence according to the present invention further comprises up to two single nucleotide substitutions in the HSV-1 UL27 gene encoding the viral envelope glycoprotein B. In the most preferred, but also non-limiting embodiment of the invention, the said single nucleotide substitutions in the HSV-1 UL27 gene lead to an amino acid substitution at position 285 and an amino acid substitution at position 549 of the protein product of said gene, namely envelope glycoprotein B (from aspartic acid to asparagine, tyrosine, histidine, valine, glycine, alanine, or glutamic acid, and from alanine to threonine, serine, aspartic acid, valine, or glycine, respectively), wherein the combination of these substitutions is selected from the list of possible substitutions:GAC→AAC, GAC→TAC, GAC→CAC, GAC→GTC, GAC→GGC, GAC→GCA, GAC→GAA, GAC→GAG for amino acid position 285, and GCC→ACC, GCC→TCC, GCC→GAC, GCC→GTC, GCC→GGC for amino acid position 549.In another aspect, the present invention provides a pharmaceutical composition for use in gene therapy of hereditary bullous dermatoses, comprising a therapeutically effective amount of the virus (herpesvirus) as hereinbefore described and at least one pharmaceutically acceptable excipient, wherein, in the most preferred, but non-limiting embodiment of the invention, the said at least one pharmaceutically acceptable excipient is adapted for topical, transdermal, subcutaneous, and / or intradermal administration.In the most preferred, non-limiting embodiment of the invention, the herpes simplex virus is herpes simplex virus type 1 (HSV-1).In alternative, but also non-limiting embodiments of the invention, the herpes simplex virus may also be herpes simplex virus type 2 (HSV-2), or the herpesvirus may be varicella-zoster virus, human cytomegalovirus, human herpesvirus 6A, human herpesvirus 6B, human herpesvirus 7, or Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8).Most preferably, said pharmaceutical composition is intended for the treatment of hereditary bullous dermatoses associated with deficient production and / or structural and / or functional defects of the COL7A1 gene, as well as genes regulating its transcription.Unexpectedly, it has been found that a replication-defective herpes simplex virus type 1 (HSV-1) comprising a mutant HSV-1 DNA genome with inactivating mutations in one or more immediate-early, early and late viral genes selected from RS1 (ICP4), UL54 (ICP27), UL41, US1 (ICP22), and UL27, and one or two polynucleotides driving the expression of the COL7A1 gene, wherein said polynucleotide comprises or said two polynucleotides comprise the COL7A1 gene of the sequence of SEQ ID NO: 15 encoding the type VII collagen α1 chain, together with combinations of regulatory sequences including one or more elements selected from the human cytomegalovirus immediate-early promoter and enhancer (SEQ ID NO: 10), the bovine growth hormone polyadenylation signal (SEQ ID NO: 11), the Kozak sequence (SEQ ID NO: 12), the SV40 intron (SEQ ID NO: 13) and the UCOE intron (SEQ ID NO: 14), results, in comparison with known analogues, in the highest level of expression of the type VII collagen α1 chain. This effect has not been described in the prior art, could not have been predicted in light of the prior art, and suggests that such a virus (HSV-1), as well as a pharmaceutical composition comprising it, will be most effective as an agent for use in gene therapy of hereditary bullous dermatoses. Brief Description of the DrawingsFigure 1 schematically illustrates the structure of cassettes (A)–(J). Structural elements of the cassettes are designated as: I is human cytomegalovirus immediate-early promoter and enhancer; II is bovine growth hormone polyadenylation signal; III is Kozak sequence; IV is SV40 intron; V is UCOE intron; COL7A1 is transgene.Figure 2 shows the expression level of human type VII collagen protein (COL7) in HEK293T cells after transfection with a plasmid library expressing the COL7A1 gene, as assessed by Western blotting. Panel A is the expression level of human type VII collagen protein. Panel B is the expression level of pan-actin. Panel C is the expression level of green fluorescent protein (GFP). Detailed Description of the Preferred EmbodimentsIn a preferred, but non-limiting embodiment of the present invention, the proposed DNA sequence does not comprise the coding sequence of one or more HSV-1 immediate-early genes selected from RS1 (ICP4), UL54 (ICP27), and US1 (ICP22).Furthermore, in a preferred, but non-limiting embodiment of the present invention, the DNA sequence according to the present invention lacks the regulatory sequences of the US1 (ICP22) gene that drive its immediate-early expression.In certain embodiments of the invention, the virus (HSV-1) is attenuated. Furthermore, in certain embodiments of the invention, the virus exhibits reduced cytotoxicity compared to wild-type HSV-1. Furthermore, in certain embodiments of the invention, the HSV-1 virus is non-oncolytic.In a preferred, but non-limiting embodiment of the present invention, the DNA sequence proposed herein further comprises up to two single nucleotide substitutions in the UL27 gene. Most preferably, the DNA sequence according to the present invention further comprises up to two single nucleotide substitutions in the UL27 gene, wherein said single nucleotide substitutions in the UL27 gene lead to an amino acid substitution at position 285 and an amino acid substitution at position 549 of the protein product of said gene, namely envelope glycoprotein B (from aspartic acid to asparagine, tyrosine, histidine, valine, glycine, alanine, or glutamic acid, and from alanine to threonine, serine, aspartic acid, valine, or glycine, respectively), and, most preferably, the combination of these substitutions is selected from the list of possible substitutions:GAC→AAC, GAC→TAC, GAC→CAC, GAC→GTC, GAC→GGC, GAC→GCA, GAC→GAA, GAC→GAG for amino acid position 285, and GCC→ACC, GCC→TCC, GCC→GAC, GCC→GTC, GCC→GGC for amino acid position 549.Preferably, the DNA sequence according to the present invention also lacks the coding sequence of the UL41 gene.The aforementioned HSV-1 virus comprising the said DNA sequence in accordance with the present invention is propagated using host cell lines conventionally employed in the art, which are modified for this purpose to complement the deleted genes, as exemplified hereinafter in the present description.Non-limiting (illustrative) examples of such mammalian cell lines include, without limitation, the SV40-transformed monkey kidney CV1 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 (WI-38, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumour cells (MMT 060562, ATCC CCL51), TRI cells, MRC-5 cells, FS4 cells, a human hepatoma cell line (Hep G2), Chinese hamster ovary cells (CHO), including DHFR(-) cells, as well as myeloma cell lines, such as NS0 and Sp2 / 0. In certain embodiments of the invention, the host cell may be a human cell or a non-human primate cell. Examples of suitable host cells or cell lines include, without limitation, 293, HeLa, SH-SY5Y, Hep G2, CACO-2, A549, L929, 3T3, K562, CHO-K1, MDCK, HUVEC, Vero, N20, COS-7, PSN1, VCaP, CHO cells, and the like. It is understood by a person skilled in the art that other mammalian cell lines known in the art may also be used, in particular primate cell lines, including human cell lines, as well as non-human primate cell lines.In certain embodiments of the invention, the host cell is a target cell, such as a barrier tissue cell. Non-limiting examples of such target cells include, without limitation, epithelial cells, keratinocytes, and fibroblasts.In another aspect, the present invention provides a pharmaceutical composition comprising the said DNA sequence or the virus as hereinbefore described and at least one pharmaceutically acceptable excipient. Hereinafter in the present application, the term ‘pharmaceutically acceptable excipient’ refers to an inactive substance conventionally used in the art as a vehicle or carrier for medical products, which is non-toxic to the recipient at the doses and concentrations employed. Non-limiting (illustrative) examples of such pharmaceutically acceptable excipients include buffers (buffering 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 a person skilled in the art and conventionally employed in the art to formulate pharmaceutical compositions, including compositions for topical, and / or transdermal, and / or subcutaneous, and / or intradermal administration. A more detailed, but also non-limiting list of pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions according to the present invention is provided in ‘Remington: The Science and Practice of Pharmacy’, Academic Press, 23rd edition, edited by Adeboye Adejare, 13 November 2020, the contents of which are incorporated herein by reference.Said pharmaceutical composition may, without limitation, be formulated in a dosage form suitable for topical, transdermal, intradermal and / or subcutaneous 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. 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, intradermal, or topical administration. Non-limiting (illustrative) examples of such dosage forms include ointments, pastes, creams, suspensions, emulsions, lipophilic ointments, gels, powders, lotions, solutions, sprays, patches, pre-filled microneedle arrays, and injectable and inhalation forms. It is understood by a person skilled in the art 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.In alternative, but also illustrative and non-limiting embodiments of the invention, the pharmaceutical composition according to the present invention may comprise a pharmaceutically acceptable carrier, namely an inert carrier which is non-toxic to the recipient at the doses employed.In certain non-limiting embodiments of the invention, such a pharmaceutically acceptable carrier is a carrier suitable for formulating compositions for topical and / or transdermal administration. In certain other, also non-limiting embodiments of the invention, the pharmaceutically acceptable carrier is a carrier suitable for formulating compositions for topical, subcutaneous, transdermal, and / or intradermal administration.In the most preferred, but also non-limiting embodiments of the invention, the pharmaceutically acceptable carrier is a carrier conventionally employed in the art for providing ointments, pastes, creams, suspensions, emulsions, lipophilic ointments, gels, powders, lotions, solutions, sprays, patches, pre-filled microneedle arrays, and injectable and inhalation forms. In certain more preferred embodiments of the invention, such a pharmaceutically acceptable carrier is minimally invasive or non-invasive and comprises one or more excipients as hereinbefore described.In certain embodiments of the invention, the pharmaceutically acceptable carrier is suitable for providing dosage forms for topical, and / or transdermal, and / or intradermal administration. Non-limiting (illustrative) examples of such forms include ointments, pastes, creams, suspensions, emulsions, lipophilic ointments, gels, powders, lotions, solutions, sprays, patches, and inhalation forms.In certain non-limiting (illustrative) embodiments of the invention, such a pharmaceutically acceptable carrier is a carrier suitable for providing ointments, pastes, creams, suspensions, emulsions, gels, powders, lotions, solutions, sprays, patches, injectable forms, or inhalation forms.Non-limiting (illustrative) examples of such pharmaceutically acceptable carriers include binders, such as pregelatinised starch, including pregelatinised maize or potato starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, and the like; fillers, such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, and calcium hydrogen phosphate; lubricants, such as magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, maize starch, polyethylene glycols, sodium benzoate, and sodium acetate; disintegrants, such as starch and sodium starch glycolate; wetting agents, such as sodium lauryl sulfate; saline solutions; alcohols; polyethylene glycols; gelatin; lactose; amylose; magnesium stearate; talc; silicic acid; liquid paraffin; hydroxymethylcellulose; polyvinylpyrrolidone; sweeteners; flavourings; fragrances; colourants; moisturising agents; sunscreens; antibacterial agents; and agents stabilising polynucleotides and preventing their degradation.Other non-limiting (illustrative) examples of such pharmaceutically acceptable carriers can be found in the aforementioned monograph ‘Remington: The Science and Practice of Pharmacy’ (Academic Press, 23rd edition, edited by Adeboye Adejare, 13 November 2020), as well as in other similar publications well known to a person skilled in the art, for example, in the monograph ‘Handbook of Pharmaceutical Excipients’, 6th edition, 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.In an alternative embodiment of the invention, the carrier may be a microneedle array comprising the pharmaceutical composition according to the invention, wherein the methods for manufacturing and using such microneedles are generally known in the art, for example, from Kim Y.-C. et al., 2012, the contents of which are incorporated herein by reference.In certain embodiments of the invention, the pharmaceutical composition may comprise a combination of two, three, four or more pharmaceutically acceptable carriers suitable for providing dosage forms for topical, and / or transdermal, and / or intradermal administration.Furthermore, in certain non-limiting embodiments of the invention, the pharmaceutically acceptable carrier may optionally further include, without limitation, one or more of: binders, fillers, lubricants, disintegrants, wetting agents, electrolyte solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, liquid paraffin, hydroxypropyl cellulose, polyvinylpyrrolidone, colourants, sunscreens, antibacterial agents, agents capable of stabilising polynucleotides and preventing their degradation, and the like; wherein the binders, fillers, lubricants, disintegrants, wetting agents and other auxiliary components listed above may be used as conventionally employed in the art and well known to a person skilled in the art.Furthermore, it is understood by a person skilled in the art that the pharmaceutically acceptable carrier may comprise other excipients not explicitly mentioned in the present description, but conventionally employed in the art and well known to such a person, and that pharmaceutical compositions comprising such a pharmaceutically acceptable carrier or such pharmaceutically acceptable carriers are also encompassed by the present invention.The following examples, which demonstrate the embodiment of the present invention and the achievement of the stated technical results upon its implementation, are intended to illustrate the invention but not to limit the scope of the claims.The present invention is further illustrated, but not limited, by the following examples, which illustrate the present invention.Example 1. Production of the viral vector according to the present inventionTo construct the vector according to the present invention, a replication-defective viral vector based on the herpes simplex virus type 1 genome was employed.The herpes simplex virus genome was modified by introducing a bacterial artificial chromosome (BAC) into the UL37–UL38 intergenic region at Cre recombinase loxP sites according to the method described by Tanaka et al., 2003. The BAC cassette comprised gene sequences for selection, including a chloramphenicol resistance gene and an EGFP fluorescent marker protein gene, as well as loxP / Cre sequences, by means of which insertion into the viral genome was performed.To abrogate replicative capacity and reduce toxicity in human target cells, mutations in immediate-early genes, including RS1 / ICP4, US1 / ICP22, and UL54 / ICP27, including inactivating mutations, were sequentially introduced into the modified HSV-1 BAC genome. Inactivating mutations in the UL41 and / or UL27 genes were additionally introduced. For mutagenesis, plasmids expressing λ phage Red proteins and the I-SceI restriction enzyme were used.The cassette expressing the transgenes was integrated into the genomic locus vacated by the deletion of RS1 / ICP4.To obtain the cassette expressing the COL7A1 gene, a library of cassettes was constructed comprising a different set of regulatory elements selected from: UCOE (an insulator sequence consisting of CBX3 and HNRPA2B1 promoters with introns), a heterologous CMV promoter along with its enhancer, an SV40 chimeric intron, a Kozak sequence, a COL7A1 gene sequence, and a bovine growth hormone polyadenylation site (bGHpA).The library comprised:cassette (A) containing human cytomegalovirus immediate-early promoter and enhancer–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;cassette (B) containing human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;cassette (C) containing human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;cassette (D) containing UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;cassette (E) containing UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;cassette (F) containing UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;cassette (G) containing UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;andcassette (J) containing human cytomegalovirus immediate-early promoter and enhancer–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal.The most efficient regulatory element configuration of the expression cassette was determined experimentally by COL7A1 mRNA expression level and the production of the type VII collagen α1 chain upon transfection of the cassettes into HEK293 cells. The COL7A1 transcription level was assessed by reverse transcription quantitative real-time PCR (RT-qPCR) using COL7A1-specific primers and SYBR Green, with normalization to the B2M and TFRC housekeeping genes according to the ΔΔCt method. The expression level of type VII collagen was determined by Western blotting using anti-type VII collagen antibodies, adjusted for transfection efficiency based on green fluorescent protein, and with sample normalization to pan-actin (Figure 2). The protein expression level was determined by chemiluminescent band intensity on the membrane using a gel documentation system and was calculated using GelQuant software. Cassette (F) exhibited the highest efficiency of type VII collagen production based on the cell culture experiment results.Following the integration of the COL7A1 expression cassettes into the genomic loci vacated by the deletion of RS1 / ICP4, gene therapy replication-defective viral vectors based on the herpes simplex virus type 1 genome capable of efficient type VII collagen production were obtained. Example 2. Construction of a cell line for the production of replication-defective herpes simplex virus type 1 vectors according to the present inventionTo produce the replication-defective herpes simplex virus type 1 vectors, a modified Vero cell line complementing the expression of the proteins excluded from the viral vector was employed. To obtain the complementing Vero-based cell line, the cells were modified using lentiviral vectors carrying an expression cassette driving the expression of immediate-early herpes simplex virus type 1 genes, including RS1 / ICP4 and UL54 / ICP27.In the HEK293T cell line, lentiviral vectors were produced carrying an expression cassette of immediate-early herpes simplex virus type 1 genes, RS1 / ICP4 and UL54 / ICP27, as well as a gene for selection, namely the mCherry fluorescent protein. Alternatively, other fluorescent proteins may be used, for example, BFP (blue fluorescent protein), as well as antibiotic resistance genes. Subsequently, the Vero cell line was transduced with the obtained lentiviral vectors, and cell selection based on the selective marker was performed. The integration of the expression cassette and the expression of viral proteins therefrom were confirmed by PCR and Western blotting. Example 3. Production of replication-defective viruses according to the present inventionThe modified replication-defective viral vector DNA based on the herpes simplex virus type 1 genome according to the present invention, comprising the cassettes expressing type VII collagen, was transfected into cells of the complementing Vero line (ICP4+, ICP27+) capable of trans-complementing the expression of the products of the genes mutated in the vector and ensuring the replication and packaging of the replication-defective virus. To excise the BAC cassette from the DNA of the replication-defective viruses, the modified cells containing the replication-defective viral vector were additionally transfected with a plasmid expressing the Cre recombinase gene (Gierasch W.W. et al., 2006).The viruses were harvested 120–192 h post-transfection. For this purpose, the cells along with the medium were harvested and centrifuged at 300 g for 5 min, then the medium was separated and stored at -70 °C. Cell lysis in the cell pellet was performed using a lysis buffer (0.05 M Tris-HCl pH 8.0, 0.15 M NaCl), followed by three freeze-thaw cycles and centrifugation at 6,300 g for 10 min. The supernatant was collected and stored at -70 °C.The obtained replication-defective viruses were purified by standard techniques well known to a person skilled in the art, including gradient centrifugation and filtration. Example 4. Investigation of transgene expression in target cells in vitroTo evaluate transgene production in target cells in vitro, HaCaT keratinocytes and HDF dermal fibroblasts were transduced with the viruses according to the present invention obtained in Example 3 at a multiplicity of infection (MOI) ranging from 0.3 to 50. The HT-29 cell line, lacking the expression of type VII collagen, was also employed in the experiment, which was previously confirmed by a semi-quantitative assay of COL7A1 transcript levels. The COL7A1 transcription level was assessed by reverse transcription quantitative real-time PCR (RT-qPCR) using primers specific to COL7A1 and housekeeping genes according to the ΔΔCt method, with normalization to the mRNA level in untransduced cells. The expression of type VII collagen was determined by Western blotting using anti-type VII collagen antibodies and with normalization to pan-actin. In the transduced keratinocytes, fibroblasts and HT-29 cells, a significant MOI-dependent increase in the COL7A1 mRNA level and type VII collagen protein content was observed compared to the control.Thus, the presented examples confirm the industrial applicability of the present group of inventions.References1. Fine J.D., Bruckner-Tuderman L., Eady R.A. et al. Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification. J Am Acad Dermatol 2014; 70 (6): 1103–1126.2. Cuatrecasas P. Drug discovery in jeopardy. J Clin Invest. 2006 Nov 1; 116 (11): 2837–2842.3. Wästfelt M., Fadeel B., Henter J.I. A journey of hope: lessons learned from studies on rare diseases and orphan drugs. J Intern Med. 2006; 260: 1–10.4. Pavičić Z., Kmet-Vizintin P., Kansky A., Dobrić I. Occurrence of hereditary bullous epidermolyses in Croatia. Pediatr Dermatol 1990; 7 (2): 108–110.5. McKenna K.E., Walsh M.Y., Bingham E.A. Epidermolysis bullosa in Northern Ireland. Br J Dermatol 1992; 127 (4): 318–321.6. Horn H.M., Priestley G.C., Eady R.A., Tidman M.J. The prevalence of epidermolysis bullosa in Scotland. Br J Dermatol 1997; 136 (4): 560–564.7. Kho Y.C., Rhodes L.M., Robertson S.J. et al. Epidemiology of epidermolysis bullosa in the antipodes: the Australasian Epidermolysis Bullosa Registry with a focus on Herlitz junctional epidermolysis bullosa. Arch Dermatol 2010; 146: 635–640.8. Danescu S., Has C., Senila S. et al. Epidemiology of inherited epidermolysis bullosa in Romania and genotype-phenotype correlations in patients with dystrophic epidermolysis bullosa. J Eur Acad Dermatol Venereol 2015; 29 (5): 899–903.9. Fine J.-D. Epidemiology of Inherited Epidermolysis Bullosa Based on Incidence and Prevalence Estimates From the National Epidermolysis Bullosa Registry. JAMA Dermatol. 2016 Nov 1; 152(11): 1231–1238.10. Kubanov A.A., Albanova V.I., Karamova A.E., et al. [The prevalence of epidermolysis bullosa hereditaria in the population of the Russian Federation]. Vestnik Dermatologii i Venerologii. 2015; (3): 21–30. (in Russian).11. Kubanov A.A., Karamova A.E., Chikin V.V., Bogdanova E.V., Monchakovskaya E.S. [Epidemiology and the state of medical care for patients with congenital epidermolysis bullosa in the Russian Federation]. Vestnik Rossiiskoi Akademii Meditsinskikh Nauk. 2018; 73(6): 420–430. (in Russian).12. Has C., Nyström A., Saeidian A.H., Bruckner-Tuderman L., Uitto J. Epidermolysis bullosa: Molecular pathology of connective tissue components in the cutaneous basement membrane zone. Matrix Biol 2018; 7172: 31329.13. Has C., Fischer J. Inherited epidermolysis bullosa: new diagnostics and new clinical phenotypes. Exp Dermatol 2018; 28: 1146–1152.14. Dang N., Murrell D.F. Mutation analysis and characterization of COL7A1 mutations in dystrophic epidermolysis bullosa. Exp Dermatol 2008; 17: 553–68.15. Lin Z., Li S., Feng C. et al. Stabilizing mutations of KLHL24 ubiquitin ligase cause loss of keratin 14 and human skin fragility. Nat Genet 2016: 48: 1508–1516.16. Lee J. Y.W., Liu L., Hsu C.-K. et al. Mutations in KLHL24 Add to the Molecular Heterogeneity of Epidermolysis Bullosa Simplex. J Invest Dermatol 2017: 137: 1378–1380.17. He Y., Maier K., Leppert J. et al. Monoallelic Mutations in the Translation Initiation Codon of KLHL24 Cause Skin Fragility. Am J Hum Genet 2016: 99: 1395–1404.18. Kim S., Coulombe P. A. Intermediate filament scaffolds fulfil mechanical, organizational, and signalling functions in the cytoplasm. Genes Dev 2007; 21: 1581–97.19. Vahidnezhad H., Youssefian L., Saeidian A.H. et al. Recessive mutation in tetraspanin CD151 causes Kindler syndrome-like epidermolysis bullosa with multi-systemic manifestations including nephropathy. Matrix Biol 2018; 66: 22–33.20. Gostyńska K. B., Nijenhuis M., Lemmink H. et al. Mutation in exon 1a of PLEC, leading to disruption of plectin isoform 1a, causes autosomal-recessive skin-only epidermolysis bullosa simplex. Hum Mol Genet. 2015; 24 (11): 3155–62.21. Liu L., Dopping-Hepenstal P.J., Lovell P. A. et al. Autosomal recessive epidermolysis bullosa simplex due to loss of BPAG1-e expression. J Invest Dermatol 2012; 132: 742–744.22. Takeichi T., Nanda A., Liu L. et al. Founder mutation in dystonin-e underlying autosomal recessive epidermolysis bullosa simplex in Kuwait. Br J Dermatol 2015; 172 (2): 527–31.23. Turcan I., Pasmooij A.M.G., Gostynski A. et al. Epidermolysis Bullosa Simplex Caused by Distal Truncation of BPAG1-e: An Intermediate Generalized Phenotype with Prurigo Papules. J Invest Dermatol 2017; 137 (10): 2227–30.24. He Y., Leppert J., Steinke H. et al. Homozygous Nonsense Mutation and Additional Deletion of an Amino Acid in BPAG1e Causing Mild Localized Epidermolysis Bullosa Simplex. Acta Derm Venereol 2017; 97 (5): 657–9.25. Cappuccio G., Pinelli M., Torella A. et al. Expanding the phenotype of DST-related disorder: a case report suggesting a genotype / phenotype correlation. Am J Med Genet A 201; 173 (10): 2743–6.26. McGrath J. A., Stone K. L., Begum R. et al. Germline Mutation in EXPH5 Implicates the Rab27B Effector Protein Slac2-b in Inherited Skin Fragility. Am J Hum Genet 2012; 91: 1115–1121.27. Pigors M., Schwieger-Briel A., Leppert J. et al. Molecular heterogeneity of epidermolysis bullosa simplex: contribution of EXPH5 mutations. J Invest Dermatol 2014; 134: 842–845.28. Malchin N., Sarig O., Grafi-Cohen M. et al. A novel homozygous deletion in EXPH5 causes a skin fragility phenotype. Clin Exp Dermatol 2016; 41: 915–918.29. Rashidghamat E., Ozoemena L., Liu L., McGrath J. A., Martinez A. E., Mellerio J. E. Mutations in EXPH5 (exophilin-5) underlie a rare subtype of autosomal recessive epidermolysis bullosa simplex. Br J Dermatol 2016; 174 (2): 452–453.30. Has C., Sparta G., Kiritsi D. et al. Integrin alpha3 mutations with kidney, lung, and skin disease. N Engl J Med 2012; 366: 1508–14.31. Nicolaou N., Margadant C., Kevelam S.H. et al. Gain of glycosylation in integrin alpha3 causes lung disease and nephrotic syndrome. J Clin Invest 2012; 122: 4375–87.32. Yalcin E. G., He Y., Orhan D., Pazzagli C., Emiralioglu N., Has C. Crucial role of posttranslational modifications of integrin α3 in interstitial lung disease and nephrotic syndrome. Hum Mol Genet 2015; 24: 3679–88.33. Lovric S., Fang H., Vega-Warner V. et al. Rapid detection of monogenic causes of childhood-onset steroid-resistant nephrotic syndrome. Clin. J. Am. Soc. Nephrol. 2014; 9: 1109–1116.34. Shukrun R., Vivante A., Pleniceanu O. et al. A Human Integrin-α3 Mutation Confers Major Renal Developmental Defects. PLoS ONE 2014; 9 (3): e90879.35. Kubanov A.A., Karamova A.E., Monchakovskaya E.S. [Congenital epidermolysis bullosa: modern methods of diagnosis and therapy. Prospects for regenerative medicine]. Vestnik Dermatologii i Venerologii. 2020; 96(1): 10–17. (in Russian).36. Anwar M.I., Rashid A., Ghafoor R. et al. Kindler’s syndrome: a report of five cases in a family. J Coll Physicians Surg Pak. 2014; 24(10): 763–765.37. Gadzhimuradova K.M., Zhukova O.V., Gadzhimuradov M.N., Alieva S.N. [Kindler syndrome—a rare form of congenital epidermolysis bullosa]. Russkii Meditsinskii Zhurnal (RMJ). 2022; (3): 59–63. (in Russian).38. Fine J.-D. Inherited epidermolysis bullosa. Orphanet J Rare Dis. 2010 May 28; 5:12.39. Fine J.D., Johnson L. B., Weiner M. et al. Pseudosyndactyly and musculoskeletal deformities in inherited epidermolysis bullosa (EB): experience of the National EB Registry, 1986–2002. J Hand Surg (British and European Volume) 2002; 30B: 14–22.40. Fine J. D., Johnson L. B., Weiner M., Suchindran C. Cause-specific risks of childhood death in inherited epidermolysis bullosa. J Pediatr 2008; 152: 276–280.41. Zidorio A. P. C., Dutra E. S., Leão D. O. D., Costa I. M. C. Nutritional aspects of children and adolescents with epidermolysis bullosa: Literature review. An. Bras. Dermatol. 2015; 90: 217–223.42. Fine J. D., Johnson L. B., Weiner M., Li K. P., Suchindran C. Epidermolysis bullosa and the risk of life-threatening cancers: the national EB registry experience, 1986–2006. J Am Acad Dermatol 2009; 60: 203–211.43. Mellerio J. E., Robertson S. J., Bernardis C., Diem A., Fine J. D., George R., et al. Management of cutaneous squamous cell carcinoma in patients with epidermolysis bullosa: best clinical practice guidelines. Br J Dermatol 2016; 174: 56–67.44. Denyer J., Pillay E., Clapham J. Best practice guidelines for skin and wound care in epidermolysis bullosa. An International Consensus. Wounds International 2017.45. Rashidghamat E., McGrath J. A. Novel and emerging therapies in the treatment of recessive dystrophic epidermolysis bullosa. Intractable Rare Dis Res 2017; 6: 6–20.46. Wong T., Gammon L., Liu L., Mellerio J.E., Dopping-Hepenstal P.J., Pacy J., Elia G., Jeffery R., Leigh I.M., Navsaria H., McGrath J.A. Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa. J Invest Dermatol 2008; 128: 2179–2189.47. Nagy N., Almaani N., Tanaka A., Lai-Cheong J.E., Techanukul T., Mellerio J.E., McGrath J.A. HB-EGF induces COL7A1 expression in keratinocytes and fibroblasts: Possible mechanism underlying allogeneic fibroblast therapy in recessive dystrophic epidermolysis Bullosa. J Invest Dermatol. 2011; 131: 1771–1774.48. Petrof G., Martinez-Queipo M., Mellerio J.E., Kemp P., McGrath J.A. Fibroblast cell therapy enhances initial healing in recessive dystrophic epidermolysis bullosa wounds: Results of a randomized, vehicle-controlled trial. Br J Dermatol. 2013; 169: 1025–1033.49. Khabriev R.U., Kovalchuk L.V., Gankovskaya L.V., Dolgina E.N. [Method of personalised immunotherapy (Medical technology)]. Moscow: Federal Service for Surveillance in Healthcare (Roszdravnadzor), Certificate of Registration No. FS 2011 / 212 dated 28 July 2011. (in Russian).50. Patel S.A., King C.C., Lim P.K., Habiba U., Dave M., Porecha R., and Rameshwar P. Personalizing Stem Cell Research and Therapy: The Arduous Road Ahead or Missed Opportunity? Curr Pharmacogenomics Person Med. 2010 Mar 1; 8(1): 25–36.51. Nagarkar R., Singh M., Nguyen H.X., Jonnalagadda S. A Review of Recent Advances in Microneedle Technology for Transdermal Drug Delivery. J. Drug Deliv. Sci. Technol. 2020; 59:101923.52. Hou Y., Guey L.T., Wu T., Gao R., Cogan J., Wang X., Hong E., Ning W.V., Keene D., Liu N., Huang Y., Kaftan C., Tangarone B., Quinones-Garcia I., Uitto J., Francone O.L., Woodley D.T., Chen M. Intravenously Administered Recombinant Human Type VII Collagen Derived from Chinese Hamster Ovary Cells Reverses the Disease Phenotype in Recessive Dystrophic Epidermolysis Bullosa Mice. J Invest Dermatol 2015 Dec; 135(12): 3060–3067.53. Prodinger C., Reichelt J., Bauer J.W., and Laimer M. Epidermolysis bullosa: Advances in research and treatment. Exp Dermatol. 2019 Oct; 28(10):1176–1189.54. Boull C.L., Hylwa S.A., Sajic D., Wagner J.E., Tolar J., Hook K.P. Toxic Epidermal Necrolysis in Recessive Dystrophic Epidermolysis Bullosa following Bone Marrow Transplantation. The Journal of Pediatrics, vol. 173, June 2016, pp. 242–244.55. Ebens C. L., McGrath J. A., Tamai K., Hovnanian A., Wagner J. E., Riddle M. J., Keene D. R., DeFor T. E., Tryon R., Chen M., Woodley D. T., Hook K., Tolar J. Bone marrow transplant with post‐transplant cyclophosphamide for recessive dystrophic epidermolysis bullosa expands the related donor pool and permits tolerance of nonhaematopoietic cellular grafts. British Journal of Dermatology, Volume 181, Issue 6, 1 December 2019, pp. 1238–1246.56. Mark B Geyer, Kavita Radhakrishnan, Roger Giller, Noriko Umegaki, Sivan Harel, Maija Kiuru, Kimberly D Morel, Nicole LeBoeuf, Jessica Kandel, Anna Bruckner, Sandra Fabricatore, Mei Chen, David Woodley, John McGrath, LeeAnn Baxter-Lowe, Jouni Uitto, Angela M Christiano, Mitchell S Cairo. Reduced Toxicity Conditioning and Allogeneic Hematopoietic Progenitor Cell Transplantation for Recessive Dystrophic Epidermolysis Bullosa. Journal of Pediatrics 2015 Sep; 167(3): 765–9.e1.57. Wagner J. E., Ishida‐Yamamoto A., McGrath J. A., Hordinsky M., Keene D. R., Woodley D. T., Chen M., Riddle M. J., Osborn M. J., Lund T., Dolan M., Blazar B. R., Tolar J. Bone Marrow Transplantation for Recessive Dystrophic Epidermolysis Bullosa. N Engl J Med 2010; 363: 629–639.58. Johanna Hammersen, Cristina Has, Nora Naumann-Bartsch, Daniel Stachel, Dimitra Kiritsi, Stephan Söder, Mathilde Tardieu, Markus Metzler, Leena Bruckner-Tuderman, Holm Schneider. Genotype, Clinical Course, and Therapeutic Decision Making in 76 Infants with Severe Generalized Junctional Epidermolysis Bullosa. J Invest Dermatol 2016 Nov; 136(11): 2150–2157.59. Garber, D.A., Beverley, S.M., Coen, D.M. Demonstration of circularization of herpes simplex virus DNA following infection using pulsed field gel electrophoresis. Virology 1993 Nov, 197(1): 459–462.60. Efstathiou, S.; Minson, A.C.; Field, H.J.; Anderson, J.R.; Wildy, P. Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans. J. Virol. 1986 Feb; 57(2): 446–455.61. Jackson, S.A.; DeLuca, N.A. Relationship of herpes simplex virus genome configuration to productive and persistent infections. Proc. Natl. Acad. Sci. USA 2003, 100, 7871–7876.62. Roizman B. The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors. Proc Natl Acad Sci U S A 1996 Oct 15; 93(21): 11307–12.63. Powell S.K., Rivera-Soto R., Gray S.J. Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy. Discov Med 2015 Jan; 19(102): 49–57.64. Maksimenko O.G., Gasanov N.B., Georgiev P.G. [Regulatory elements in vectors for efficient generation of cell lines producing target proteins]. Acta Naturae. 2015; 7(3): 16–29. (in Russian).65. Guide S.V., Gonzalez M.E., Bağci I.S., Agostini B., Chen H., Feeney G., Steimer M., Kapadia B., Sridhar K., Sanchez L.Q., Gonzalez F., Van Ligten M. et al. Trial of Beremagene Geperpavec (B-VEC) for Dystrophic Epidermolysis Bullosa. N Engl J Med 2022; 387: 2211–2219.66. Ermekova S.A. [Molecular Biology]. Almaty: Republic of Kazakhstan; 2017. 539 p. – p. 167. (in Russian).67. Spaete R.R., Frenkel N. The herpes simplex virus amplicon: a new eukaryotic defective-virus cloning-amplifying vector. Cell, 1982 Aug, 30(1): 295–304.68. Krystal Biotech, Inc. Compositions and methods for the treatment of wounds, disorders, and diseases of the skin. WIPO PCT Patent Application WO 2017 / 176336 A1. Published 12 October 2017.69. Honess, R. W., and B. Roizman. Regulation of herpes virus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. Journal of Virology 1974 Jul; vol. 14, issue 1, pp. 8–19.70. Krystal Biotech, Inc. Recombinant nucleic acids encoding cosmetic protein(s) for aesthetic applications. WIPO PCT Patent Application WO 2019 / 210219 A1. Published 31 October 2019.71. Schmidt E.V., Christoph G., Zeller R., Leder P. The cytomegalovirus enhancer: a pan-active control element in transgenic mice. Mol Cell Biol. 1990 Aug; 10(8): 4406–4411.72. Xia W., Bringmann P., McClary J., Jones P.P., Manzana W., Zhu Y., Wang S., Liu Y., Harvey S., Madlansacay M.R., McLean K., Rosser M.P., MacRobbie J., Olsen C.L., Cobb R.R. High levels of protein expression using different mammalian CMV promoters in several cell lines. Protein Expression and Purification 2006 January; Volume 45, Issue 1, pp. 115–124.73. Iwakuma T., Cui Y., Chang L.J. Self-inactivating lentiviral vectors with U3 and U5 modifications. Virology 1999 Aug 15; 261(1): 120–132.74. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25; 12(2): 857–872.75. Xu D.-H., Wang X.-Y., Jia Y.-L., Wang T.-Y., Tian Z.-W., Feng X., Zhang Y.-N. SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells. J Cell Mol Med 2018 Apr; 22(4): 2231–2239.76. Neville J.J., Orlando J., Mann K., McCloskey B., Antoniou M.N. Ubiquitous Chromatin-opening Elements (UCOEs): Applications in biomanufacturing and gene therapy. Biotechnology Advances September 2017, Volume 35, Issue 5, pp. 557–564.77. Christiano A.M., Greenspan D.S., Lee S., and Uitto J. Cloning of human type VII collagen. Complete primary sequence of the alpha 1(VII) chain and identification of intragenic polymorphisms. J Biol Chem. 1994 Aug 12; 269(32): 20256-62.78. Remington: The Science and Practice of Pharmacy. Academic Press, 23rd edition, edited by Adeboye Adejare, 13 November 2020.79. Yeu-Chun Kim, Jung-Hwan Park, Mark R Prausnitz. Microneedles for drug and vaccine delivery. Adv Drug Deliv Rev 2012 Nov; 64(14): 1547–68.80. Tanaka M., Kagawa H., Yamanashi Y., Sata T., Kawaguchi Y. Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1: Viruses Reconstituted from the Clone Exhibit Wild-Type Properties In Vitro and In Vivo. Journal of Virology 2003 Jan; 77(2): 1382–1391.81. Gierasch W.W., Zimmerman D.L., Ward S.L., Vanheyningen T.K., Romine J.D., Leib D.A. Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS. J Virol Methods 2006 Aug; 135(2): 197–206.82. Pfaffl M.W., Horgan G.W., Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002 May 1; 30(9): e36.83. Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001 May 1; 29(9): e45.84. Woodley D.T., Keene D.R., Atha T., Huang Y., Lipman K., Li W., and Chen M. Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa. Nat Med. 2004 Jul; 10(7): 693–695.85. Masson-Meyers D.S., Andrade T.A.M., Caetano G.F., Guimaraes F.R., Leite M.N., Leite S.N., Frade M.A.C. Experimental models and methods for cutaneous wound healing assessment. Int. J. Exp. Pathol. 2020; 101: 21–37.86. Sluka K.A., Frey-Law L., Bement M.H. Exercise-induced pain and analgesia? Underlying mechanisms and clinical translation. Pain 2018 Sep; 159 Suppl 1 (Suppl 1): S91–S97.87. Bellare A., Epperly M.W., Greenberger J.S., Fisher R., Glowacki J. Development of tensile strength methodology for murine skin wound healing. MethodsX 2018 Apr 16; 5: 337–344.

Claims

1. A herpes simplex virus type 1 (HSV-1) comprising a recombinant HSV genome, wherein said genome comprises one or two expression cassettes for the expression of the human type VII collagen α1 chain, wherein each of said expression cassettes independently includes a transgene encoding the human type VII collagen α1 chain and a combination of regulatory sequences selected from:- a human cytomegalovirus immediate-early promoter and enhancer (I);- a bovine growth hormone polyadenylation signal (II);- a Kozak sequence (III);- an SV40 intron (IV);- a UCOE intron (V),wherein said transgene together with said combination of regulatory sequences forms one of the following combinations:(A) human cytomegalovirus immediate-early promoter and enhancer–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(B) human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(C) human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(D) UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(E) UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal; (F) UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(G) UCOE intron–human cytomegalovirus immediate-early promoter and enhancer–SV40 intron–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(H) SV40 intron–human cytomegalovirus immediate-early promoter and enhancer–UCOE intron–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal;(I) SV40 intron–human cytomegalovirus immediate-early promoter and enhancer–UCOE intron–Kozak sequence–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal,wherein said recombinant HSV genome comprises inactivating mutations in one or more immediate-early, early or late HSV-1 genes.

2. A herpes simplex virus type 1 (HSV-1) comprising a recombinant HSV genome, wherein said genome comprises one or two expression cassettes for the expression of the human type VII collagen α1 chain, each comprising the following combination of a transgene encoding the human type VII collagen α1 chain and regulatory sequences:(J) human cytomegalovirus immediate-early promoter and enhancer–transgene encoding the human type VII collagen α1 chain–bovine growth hormone polyadenylation signal,wherein said recombinant HSV genome comprises inactivating mutations in the RS1 (ICP4), UL54 (ICP27), US1 (ICP22), and UL41 genes and does not comprise inactivating mutations in the RL2 (ICP0) and / or US12 (ICP47) genes.

3. The virus according to any one of Claims 1 to 2, wherein said transgene encoding the human type VII collagen α1 chain has the sequence of SEQ ID NO:

15.

4. The virus according to any one of Claims 1 to 2, wherein said human cytomegalovirus immediate-early promoter and enhancer has the sequence of SEQ ID NO:

10.

5. The virus according to any one of Claims 1 to 2, wherein said bovine growth hormone polyadenylation signal has the sequence of SEQ ID NO:

11.

6. The virus according to any one of Claims 1 to 2, wherein said Kozak sequence has the sequence of SEQ ID NO:

12.

7. The virus according to any one of Claims 1 to 2, wherein said SV40 intron has the sequence of SEQ ID NO:

13.

8. The virus according to any one of Claims 1 to 2, wherein said UCOE intron has the sequence of SEQ ID NO:

14.

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

10. The virus according to any one of Claims 1 to 9, wherein said recombinant HSV genome further comprises an inactivating mutation in the UL55 gene.

11. The virus according to Claim 1, wherein said recombinant HSV genome comprises inactivating mutations in the RS1 (ICP4), UL54 (ICP27), US1 (ICP22), and UL41 genes and does not comprise inactivating mutations in the RL2 (ICP0) and / or US12 (ICP47) genes.

12. The virus according to any one of Claims 1 to 11, wherein said recombinant HSV genome lacks inactivating mutations in other genes.

13. The virus according to any one of Claims 1 to 12, wherein said recombinant HSV genome lacks the coding sequence of one or more HSV-1 genes selected from RS1 (ICP4), UL54 (ICP27), and US1 (ICP22).

14. The virus according to any one of Claims 1 to 13, wherein said recombinant HSV genome lacks the regulatory sequences of the US1 (ICP22) gene that drive its immediate-early expression.

15. The virus according to any one of Claims 1 to 13, wherein said recombinant HSV genome further comprises up to two single nucleotide substitutions in the UL27 gene.

16. The virus according to Claim 15, wherein said single nucleotide substitutions in the UL27 gene lead to an amino acid substitution at position 285 and an amino acid substitution at position 549 of the protein product of said gene, wherein the combination of these substitutions is selected from the list of possible substitutions: GAC→AAC, GAC→TAC, GAC→CAC, GAC→GTC, GAC→GGC, GAC→GCA, GAC→GAA, GAC→GAG for amino acid position 285, and GCC→ACC, GCC→TCC, GCC→GAC, GCC→GTC, GCC→GGC for amino acid position 549.

17. The virus according to any one of Claims 1 to 16, wherein said recombinant HSV genome lacks the coding sequence of the UL41 gene.

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

19. The pharmaceutical composition according to Claim 18, wherein said composition is formulated in a dosage form for topical administration.

20. The pharmaceutical composition according to Claim 18, wherein said composition is formulated in a dosage form for transdermal administration.

21. The pharmaceutical composition according to Claim 18, wherein said composition is formulated in a dosage form for intradermal administration.

22. A vector comprising one or two polynucleotides encoding a type VII collagen α1 chain polypeptide, wherein said polynucleotide comprises, or said two polynucleotides each independently comprise, an expression cassette for the expression of the human type VII collagen α1 chain comprising a transgene encoding the human type VII collagen α1 chain and a combination of regulatory sequences selected from a human cytomegalovirus immediate-early promoter and enhancer, a bovine growth hormone polyadenylation signal, a Kozak sequence, an SV40 intron, and a UCOE intron, wherein said transgene together with said regulatory sequences forms one of combinations (A) to (I) as specified in Claim 1, or combination (J) as specified in Claim 2, and wherein said vector is a recombinant HSV genome, and wherein said recombinant HSV genome comprises inactivating mutations in one or more immediate-early, early, or late HSV-1 genes.

23. The vector according to Claim 22, wherein said recombinant HSV genome further comprises an inactivating mutation in the UL55 gene.

24. The vector according to Claim 22, wherein said recombinant HSV genome comprises inactivating mutations in the RS1 (ICP4), UL54 (ICP27), US1 (ICP22), and UL41 genes and does not comprise inactivating mutations in the RL2 (ICP0) and / or US12 (ICP47) genes.

25. The vector according to Claim 22, wherein said recombinant HSV genome does not comprise inactivating mutations in other genes.

26. The vector according to Claim 22, wherein said recombinant HSV genome lacks the coding sequence of one or more HSV-1 genes selected from RS1 (ICP4), UL54 (ICP27), and US1 (ICP22).

27. The vector according to Claim 22, wherein said recombinant HSV genome lacks the regulatory sequences of the US1 (ICP22) gene that drive its immediate-early expression.

28. The vector according to Claim 22, wherein said recombinant HSV genome further comprises up to two single nucleotide substitutions in the UL27 gene.

29. The vector according to Claim 28, wherein said single nucleotide substitutions in the UL27 gene lead to an amino acid substitution at position 285 and an amino acid substitution at position 549 of the protein product of said gene, wherein the combination of these substitutions is selected from the list of possible substitutions: GAC→AAC, GAC→TAC, GAC→CAC, GAC→GTC, GAC→GGC, GAC→GCA, GAC→GAA, GAC→GAG for amino acid position 285, and GCC→ACC, GCC→TCC, GCC→GAC, GCC→GTC, GCC→GGC for amino acid position 549.

30. The vector according to Claim 22, wherein said recombinant HSV genome lacks the coding sequence of the UL41 gene.

31. The vector according to Claim 22, wherein said transgene encoding the human type VII collagen α1 chain has the sequence of SEQ ID NO:

15.

32. The vector according to Claim 22, wherein said human cytomegalovirus immediate-early promoter and enhancer has the sequence of SEQ ID NO:

10.

33. The vector according to Claim 22, wherein said bovine growth hormone polyadenylation signal has the sequence of SEQ ID NO:

11.

34. The vector according to Claim 22, wherein said Kozak sequence has the sequence of SEQ ID NO:

12.

35. The vector according to Claim 22, wherein said SV40 intron has the sequence of SEQ ID NO:

13.

36. The vector according to Claim 22, wherein said UCOE intron has the sequence of SEQ ID NO:

14.

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

38. A host cell comprising the vector according to any one of Claims 22 to 37, or modified with the vector according to any one of Claims 22 to 37.

39. The cell according to Claim 38, wherein said cell is a mammalian cell.

40. The cell according to Claim 39, wherein said host cell is selected from an SV40-transformed monkey kidney cell, 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 canine kidney cell, a Buffalo rat liver cell, a human lung cell, a human liver cell, a mouse mammary tumour cell (MMT 060562, ATCC CCL51), a TRI cell, an MRC-5 cell, an FS4 cell, a human hepatoma cell (Hep G2), a Chinese hamster ovary (CHO) cell, a DHFR(-) cell, an NS0 myeloma cell, and a Sp2 / 0 myeloma cell.

41. The cell according to Claim 38, wherein said cell is a target cell.

42. The cell according to Claim 41, wherein said target cell is selected from barrier tissue cells, including, without limitation, epithelial cells, keratinocytes, and fibroblasts.

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

44. The use of the virus according to any one of Claims 1 to 17, the pharmaceutical composition according to any one of Claims 18 to 21, the vector according to any one of Claims 22 to 37, or the cell according to any one of Claims 38 to 42, for the manufacture of a medical product for the preventive, palliative, or therapeutic treatment (improvement) of skin and mucosal wounds in a patient with epidermolysis bullosa, and / or for the preventive, palliative, or therapeutic treatment of impaired integrity or condition of skin and mucous membranes caused by a mutation in the COL7A1 gene in a patient with epidermolysis bullosa.

45. The use of the virus according to any one of Claims 1 to 17, the pharmaceutical composition according to any one of Claims 18 to 21, the vector according to any one of Claims 22 to 37, or the cell according to any one of Claims 38 to 42, for the preventive, palliative, or therapeutic treatment (improvement) of skin and mucosal wounds in a patient with epidermolysis bullosa, and / or for the preventive, palliative, or therapeutic treatment of impaired integrity or condition of skin and mucous membranes caused by a mutation in the COL7A1 gene in a patient with epidermolysis bullosa.

Images