Compositions and methods for treating wounds, conditions and diseases of the skin

Abstract

The present invention relates to compositions and methods for treating wounds, conditions, and diseases of the skin. In particular, the present invention relates, in part, to pharmaceutical compositions comprising one or more polynucleotides suitable for enhancing, increasing, augmenting, and / or potentiating levels of collagen alpha-1 (VII) chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeletal 17 polypeptide in a subject. The present invention also relates, in part, to pharmaceutical compositions and methods for providing prophylactic, palliative, or therapeutic relief of a wound, condition, or disease of the skin in a subject, including a subject having or at risk of developing one or more symptoms of epidermolysis bullosa.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680084419.7, filed on December 28, 2016, entitled "Compositions and methods for treating wounds, ailments and diseases of the skin".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 320,316, filed April 8, 2016, which is incorporated herein by reference in its entirety.

[0004] Sequence list submitted on an ASCII text file

[0005] The following content, submitted on an ASCII text file, is incorporated herein by reference in its entirety: Sequence List in Computer-Readable Form (CRF) (filename: 761342000140SEQLIST.txt, record date: December 28, 2016, size: 394 KB). Technical Field

[0006] This disclosure relates in part to compositions and methods for providing preventive, alleviating, or therapeutic relief of wounds, conditions, or diseases of the skin in subjects, including subjects having one or more symptoms of epidermolysis bullosa or at risk of developing one or more symptoms of epidermolysis bullosa. Background Technology

[0007] Many serious disease-related skin conditions are associated with one or more inherited conditions in patients with these diseases. One such condition, epidermolysis bullosa (EB), is a group of inherited conditions that cause the skin and mucous membranes of affected individuals to blister and erode when subjected to minor injuries or friction, such as scrapes, rubs, or abrasions. Dystrophic epidermolysis bullosa (DEB) is one of the major forms of EB. The signs and symptoms of this disease vary greatly among affected individuals, ranging from mild (blistering may only affect the hands, feet, knees, and elbows) to severe (extensive blistering and scarring, which can lead to vision loss, disfigurement, and other serious and sometimes fatal medical conditions).

[0008] Dystrophic epidermolysis bullosa is classified into three main types. Autosomal dominant dystrophic epidermolysis bullosa (DDEB), typically the mildest form, usually involves blistering confined to the hands, feet, knees, and elbows. The other two types, Halllopeau-Siemens recessive dystrophic epidermolysis bullosa and non-Hallopeau-Siemens recessive dystrophic epidermolysis bullosa (collectively referred to as recessive dystrophic epidermolysis bullosa or RDEB), are more severe. RDEB is typically characterized by extensive blistering and scarring of the skin and mucous membranes. Blisters are often found throughout the body, including mucous membranes such as the lining of the mouth and digestive tract, and the healing of these blisters leads to extensive scarring. Damage to the mouth and esophagus makes food difficult to chew and swallow, resulting in chronic malnutrition and slow growth. Complications of extensive scarring can include finger and toe fusion, joint deformities, and eye inflammation leading to vision loss. Furthermore, patients with RDEB have a high risk of developing squamous cell carcinoma, which can be unusually aggressive in this patient population and often becomes life-threatening. Although these three types of dystrophic epidermolysis bullosa differ in severity, they share many common characteristics and are caused by the same gene mutation.

[0009] Dystrophic epidermolysis bullosa is caused by mutations in the Col7a1 gene, which encodes collagen alpha-1 (VII) chain protein (Collagen 7). More than 240 different mutations in this gene have been identified in DEB patients. Furthermore, a significant decrease in the expression of the PLOD3 gene, which encodes collagen-modifying lysine hydroxylase 3 (LH3), has also been observed in DEB patients. Collagen alpha-1 (VII) chain protein plays a role in strengthening and stabilizing the skin, while lysine hydroxylase 3 plays a crucial role in the synthesis and secretion of functional collagen alpha-1 (VII) chain protein. In short, the Col7a1 transcript is translated, and the resulting peptide undergoes post-translational modification by hydroxylating proline residues (via prolyl hydroxylase) and lysine residues (via lysine hydroxylase, such as LH3). Then, hydroxylysine residues can be glycosylated, and subsequently, three glycosylated peptides form a triple helix (called procollagen) and are secreted from the cell. The secreted procollagen can then associate with higher-order structures to form anchoring fibrils. These anchoring fibrils can then be used to aid tissue, stabilize, and facilitate adhesion of the epithelial basement membrane. The epithelial basement membrane is responsible for anchoring the epithelium to the underlying loose connective tissue and is essential for dermal-epidermal stability (dermal-epidermal junction integrity). Mutations in the Col7a1 gene and decreased PLOD3 expression levels impair the ability of collagen α-1 (VII) chain proteins to properly connect the epidermis to the dermis in patients with dystrophic epidermolysis bullosa, resulting in skin fragility.

[0010] Treatment options for patients with epidermolysis bullosa are limited. Current care focuses on managing the symptoms, including providing medications to control pain and itching, administering oral antibiotics to prevent infection from open wounds on the skin and mucous membranes, and surgical strategies to address scarring and deformities. Research approaches to treating the underlying cause of epidermolysis bullosa include intradermal injections of purified collagen 7, collagen 7-containing fibroblasts, or viral vectors encoding collagen 7. Because many DEB patients have multiple wounds in large, vulnerable areas such as the sacrum, buttocks, feet, lower back, and hands, any treatment involving intradermal injections would be highly invasive, as these large wound areas would require injections, potentially repeatedly, even at unclear intervals.

[0011] Therefore, there is a clear need for minimally invasive / minimally invasive / non-invasive treatment options for patients with epidermolysis bullosa that address the defects in collagen α-1 (VII) chain protein and lysyl hydroxylase 3 protein observed in this patient population.

[0012] All references cited in this article, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot accessions, are incorporated herein by reference in their entirety, as if each individual reference were specifically and individually indicated as incorporated by reference. Summary of the Invention

[0013] To meet these needs, this disclosure relates in part to compositions and methods for providing preventative, alleviating, or therapeutic relief of wounds, conditions, or diseases of the skin in subjects, particularly those with one or more symptoms of epidermolysis bullosa or at risk of developing one or more symptoms of epidermolysis bullosa. In particular, this disclosure relates in part to methods of treating an individual by administering (e.g., topically or transdermally) a pharmaceutical composition comprising one or more polynucleotides encoding a collagen α-1 (VII) chain polypeptide, a lysine hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, and / or chimeric polypeptides thereof.

[0014] Therefore, certain aspects of this disclosure relate to a pharmaceutical composition comprising: a virus comprising a vector, wherein the vector comprises one or more transgenic molecules encoding a collagen α-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, or a chimeric polypeptide thereof; and a pharmaceutically acceptable carrier. In some embodiments, the virus is an adenovirus, adeno-associated virus, a retrovirus, a lentivirus, Sendai virus, herpes simplex virus, vaccinia virus, or any hybrid virus thereof. In some embodiments, the virus is replication-defective. In some embodiments, the virus is herpes simplex virus (HSV). In some embodiments, the herpes simplex virus is herpes simplex virus type 1, herpes simplex virus type 2, or any derivative thereof. In some embodiments, the herpes simplex virus comprises a modified envelope. In some embodiments, the modified envelope alters the tissue tropism of the herpes simplex virus relative to wild-type herpes simplex virus. In some embodiments, the modified envelope comprises a mutant herpes simplex virus glycoprotein. In some embodiments, the vector is an HSV-1 amplicon or an HSV-1 heterozygous amplicon. In some embodiments, the HSV-1 heterozygous amplicon is an HSV / AAV heterozygous amplicon, an HSV / EBV heterozygous amplicon, an HSV / EBV / RV heterozygous amplicon, or an HSV / Sleeping Beauty heterozygous amplicon. In some embodiments, the vector is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the immediate early herpes simplex virus genes. In some embodiments, the herpes simplex virus genes are ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41, or UL55. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4, ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutations in the ICP4, ICP27, and UL55 genes are deletions of the coding sequences of the ICP4, ICP27, and UL55 genes. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions in the promoter regions of the ICP22 and ICP47 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0, ICP4, and ICP22 genes.In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0, ICP4, ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the gene. In some embodiments, the recombinant herpes simplex virus genome also includes an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in both the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome includes one or more transgenes within one or more viral loci. In some embodiments, the recombinant herpes simplex virus genome includes one or more transgenes within one or more ICP4 viral loci. In some embodiments, the recombinant herpes simplex virus genome includes one or more transgenes within the UL41 viral locus. In some embodiments, the vector is capable of replicating within the target cells upon delivery. In some embodiments, the pharmaceutically acceptable carrier is suitable for topical or transdermal administration. In some embodiments, the one or more transgenes contain a miRNA binding site. In some embodiments, the one or more transgenes are operatively linked to one or more heterologous promoters. In some embodiments, the one or more heterologous promoters are one or more of the human cytomegalovirus (HCMV) immediate early promoter, elongation factor-1 (EF1) promoter, and / or any combination thereof. In some embodiments, the vector contains a transgene encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the vector contains two transgenes, each encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the collagen α-1 (VII) chain polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 2. In some embodiments, the collagen α-1 (VII) chain polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 2. In some embodiments, when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces anchoring fibrillary formation in the subject. In some embodiments, when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces the subject's epithelial basement membrane tissue and / or epithelial basement adhesion. In some embodiments, the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 4. In some embodiments, the lysyl hydroxylase 3 polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 4.In some embodiments, when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of a subject, the lysyl hydroxylase 3 polypeptide enhances, increases, strengthens, and / or reinforces the formation of hydroxylysine residues on one or more collagen polypeptides in the subject. In some embodiments, when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces anchoring fibrillation, epithelial basement membrane tissue, and / or epithelial basement membrane adhesion in the subject. In some embodiments, the vector contains at least a first transgene and a second transgene. In some embodiments, the first transgene encodes a collagen α-1 (VII) chain polypeptide, and the second transgene encodes the lysyl hydroxylase 3 polypeptide. In some embodiments, the vector contains a polycistronic transgene. In some embodiments, the polycistronic transgene encodes a collagen α-1 (VII) chain polypeptide in a first open reading frame (ORF) and encodes the lysyl hydroxylase 3 polypeptide in a second open reading frame (ORF). In some embodiments, the first and second ORFs are separated by an internal ribosome entry site (IRES). In some embodiments, when the collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide are expressed in one or more target cells of a subject, these polypeptides are in approximately an equimolar ratio. In some embodiments, when the collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide are expressed in one or more target cells of a subject, these polypeptides enhance, increase, strengthen, and / or reinforce anchoring fibrillation, epithelial basement membrane tissue, and / or epithelial basement membrane adhesion of the subject. In some embodiments, the chimeric polypeptide comprises a linker polypeptide between the collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide. In some embodiments, the linker polypeptide is a T2A, P2A, E2A, or F2A linker polypeptide. In some embodiments, the linker polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12. In some embodiments, the chimeric peptide has at least 80% sequence identity with the sequences of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28. In some embodiments, when the chimeric peptide is expressed in one or more target cells of a subject, the peptide enhances, increases, strengthens, and / or reinforces anchoring fibrillation, epithelial basement membrane tissue, and / or epithelial basement membrane adhesion in the subject.

[0015] Other aspects of this disclosure relate to a method of providing preventative, palliative, or therapeutic relief of skin wounds, conditions, or diseases in a subject, the method comprising administering, topically or transdermally, a pharmaceutical composition capable of enhancing, increasing, strengthening, and / or reinforcing levels of collagen α-1 (VII) chain polypeptides and / or lysyl hydroxylase 3 polypeptides in one or more cells of the subject. In some embodiments, the pharmaceutical composition comprises: a virus comprising a vector, wherein the vector comprises one or more transgenes encoding collagen α-1 (VII) chain polypeptides, lysyl hydroxylase 3 polypeptides, or chimeric polypeptides thereof; and a pharmaceutically acceptable carrier. In some embodiments, the virus is an adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, herpes simplex virus, vaccinia virus, or any hybrid virus thereof. In some embodiments, the virus is replication-defective. In some embodiments, the virus is herpes simplex virus (HSV). In some embodiments, the herpes simplex virus is herpes simplex virus type 1, herpes simplex virus type 2, or any derivative thereof. In some embodiments, the herpes simplex virus comprises a modified envelope. In some embodiments, the modified envelope alters the tissue tropism of the herpes simplex virus relative to wild-type herpes simplex virus. In some embodiments, the modified envelope contains a mutated herpes simplex virus glycoprotein. In some embodiments, the vector is an HSV-1 amplicon or an HSV-1 heterozygous amplicon. In some embodiments, the HSV-1 heterozygous amplicon is an HSV / AAV heterozygous amplicon, an HSV / EBV heterozygous amplicon, and an HSV / EBV / RV heterozygous amplicon, or an HSV / Sleeping Beauty heterozygous amplicon. In some embodiments, the vector is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome contains an inactivating mutation in the immediate early herpes simplex virus gene. In some embodiments, the herpes simplex virus gene is ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41, or UL55. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP4, ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutations in the ICP4, ICP27, and UL55 genes are deletions of the coding sequences of the ICP4, ICP27, and UL55 genes. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions in the promoter regions of the ICP22 and ICP47 genes.In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0, ICP4, and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0, ICP4, ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutation is a deletion of the coding sequence of a gene. In some embodiments, the recombinant herpes simplex virus genome also includes an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in both the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome includes one or more of the aforementioned transgenes within one or more viral loci. In some embodiments, the recombinant herpes simplex virus genome contains one or more transgenes within one or more ICP4 viral loci. In some embodiments, the recombinant herpes simplex virus genome contains one or more transgenes within the UL41 viral locus. In some embodiments, the vector is capable of replicating within the target cells upon delivery. In some embodiments, the pharmaceutically acceptable carrier is suitable for topical or transdermal administration. In some embodiments, the one or more transgenes contain a miRNA binding site. In some embodiments, the one or more transgenes are operatively linked to one or more heterologous promoters. In some embodiments, the one or more heterologous promoters are one or more of the human cytomegalovirus (HCMV) immediate early promoter, elongation factor-1 (EF1) promoter, and / or any combination thereof. In some embodiments, the vector contains a transgene encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the vector contains two transgenes, each encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the collagen α-1 (VII) chain polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 2. In some embodiments, the collagen α-1 (VII) chain polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 2. In some embodiments, when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces anchoring fibrillation in the subject.In some embodiments, when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces the subject's epithelial basement membrane tissue and / or epithelial basement adhesion. In some embodiments, the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 4. In some embodiments, the lysyl hydroxylase 3 polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 4. In some embodiments, when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of a subject, the lysyl hydroxylase 3 polypeptide enhances, increases, strengthens, and / or reinforces the formation of hydroxylysine residues on one or more collagen polypeptides of the subject. In some embodiments, when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces the subject's anchoring fibrillation, epithelial basement membrane tissue, and / or epithelial basement adhesion. In some embodiments, the vector contains at least a first transgene and a second transgene. In some embodiments, the first transgene encodes a collagen α-1 (VII) chain polypeptide, and the second transgene encodes a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector contains a polycistronic transgene. In some embodiments, the polycistronic transgene encodes a collagen α-1 (VII) chain polypeptide in a first open reading frame (ORF) and a lysyl hydroxylase 3 polypeptide in a second open reading frame (ORF). In some embodiments, the first and second ORFs are separated by an internal ribosome entry site (IRES). In some embodiments, when the collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide are expressed in one or more target cells of a subject, these polypeptides are in approximately an equimolar ratio. In some embodiments, when the collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide are expressed in one or more target cells of a subject, these polypeptides enhance, increase, strengthen, and / or reinforce anchoring fibrillation, epithelial basement membrane tissue, and / or epithelial basement membrane adhesion in the subject. In some embodiments, the chimeric polypeptide comprises a linker polypeptide between the collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide. In some embodiments, the linker polypeptide is a T2A, P2A, E2A, or F2A linker polypeptide. In some embodiments, the linker polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.In some embodiments, the chimeric peptide has at least 80% sequence identity with the sequences of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28. In some embodiments, when the chimeric peptide is expressed in one or more target cells of a subject, the peptide enhances, increases, strengthens, and / or reinforces anchoring fibrillation, epithelial basement membrane tissue, and / or epithelial basement membrane adhesion. In some embodiments, the pharmaceutical composition is administered once, twice, three times, four times, five times, or more daily. In some embodiments, the pharmaceutical composition is applied to one or more affected and / or unaffected areas of a subject. In some implementations, the skin disease or condition is one or more of the following: epidermolysis bullosa, skin cancer, psoriasis, lichen planus, lupus, rosacea, eczema, cutaneous candidiasis, cellulitis, impetigo, bedsores, erysipelas, ichthyosis vulgaris, dermatomyositis, acrodermatitis, stasis dermatitis, Netherton's syndrome, epidermolysis bullosa simplex (LAMB3 gene), autosomal recessive congenital ichthyosis, xeroderma pigmentosum, and pemphigoid.

[0016] Other aspects of this disclosure relate to isolated chimeric polypeptides comprising a collagen α-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and a linker polypeptide, wherein the collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide are separated by the linker polypeptide; to polynucleotides encoding said isolated chimeric polypeptides; to vectors comprising said polynucleotides; and to host cells comprising said vectors. In some embodiments, the vector is an HSV-1 amplicon or an HSV-1 heterozygous amplicon. In some embodiments, the HSV-1 heterozygous amplicon is an HSV / AAV heterozygous amplicon, an HSV / EBV heterozygous amplicon, and an HSV / EBV / RV heterozygous amplicon, or an HSV / Sleeping Beauty heterozygous amplicon. In some embodiments, the vector is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the immediate early herpes simplex virus genome. In some embodiments, the herpes simplex virus gene is ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41, or UL55. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4, ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutations in the ICP4, ICP27, and UL55 genes are deletions of the coding sequences of the ICP4, ICP27, and UL55 genes. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions in the promoter regions of the ICP22 and ICP47 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0, ICP4, and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0, ICP4, ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutation is a deletion of the coding sequence of a gene. In some embodiments, the recombinant herpes simplex virus genome also includes an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in both the ICP47 and UL41 genes.In some embodiments, the recombinant herpes simplex virus genome contains polynucleotides within one or more viral loci. In some embodiments, the recombinant herpes simplex virus genome contains polynucleotides within one or more ICP4 viral loci. In some embodiments, the recombinant herpes simplex virus genome contains polynucleotides within the UL41 viral locus.

[0017] Other aspects of this disclosure relate to a vector comprising one or more polynucleotides encoding a collagen α-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, or any combination thereof, wherein the vector is a recombinant herpes simplex virus genome; and relates to a host cell containing the vector. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in immediate early herpes simplex virus genes. In some embodiments, the herpes simplex virus genes are ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41, or UL55. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4, ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutations in the ICP4, ICP27, and UL55 genes are deletions of the coding sequences of the ICP4, ICP27, and UL55 genes. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions in the promoter regions of the ICP22 and ICP47 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0, ICP4, and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0, ICP4, ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutation is a deletion of the coding sequence of a gene. In some embodiments, the recombinant herpes simplex virus genome further includes an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in both the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome includes the one or more polynucleotides within one or more viral loci. In some embodiments, the recombinant herpes simplex virus genome includes the one or more polynucleotides within one or more ICP4 viral loci. In some embodiments, the recombinant herpes simplex virus genome includes the one or more polynucleotides within the UL41 viral locus.In some embodiments, the vector comprises a polynucleotide encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the vector comprises two polynucleotides encoding a collagen α-1 (VII) chain polypeptide.

[0018] Other aspects of this disclosure relate to methods for collecting herpes simplex virus, wherein a target vector is encapsulated within the herpes simplex virus. In some embodiments, the method includes the steps of: contacting a host cell with a vector encoding a herpes virus, contacting the host cell with an HSV-1 amplicon or an HSV-1 heterozygous amplicon containing one or more polynucleotides described herein, and collecting the herpes simplex virus produced by the host cell. In some embodiments, the method includes the steps of: contacting a complementary host cell with a recombinant herpes simplex virus genome vector containing one or more polynucleotides described herein, and collecting the herpes simplex virus produced by the complementary host cell. In some embodiments, the collected herpes simplex virus is herpes simplex virus type 1, herpes simplex virus type 2, or any derivative thereof.

[0019] Other aspects of this disclosure relate to a medicine box containing the pharmaceutical composition described herein and instructions for administering the pharmaceutical composition.

[0020] Other aspects of this disclosure relate to a pharmaceutical composition comprising: a virus comprising a vector, wherein the vector comprises one or more transgenic polypeptides encoding collagen α-1 (VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, keratin type I cytoskeleton 17 polypeptide, or chimeric polypeptides thereof; and a pharmaceutically acceptable carrier. In some embodiments, the virus is an adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, herpes simplex virus, vaccinia virus, or any hybrid virus thereof. In some embodiments, the virus is replication-defective. In some embodiments, the virus is herpes simplex virus (HSV). In some embodiments, the herpes simplex virus is herpes simplex virus type 1, herpes simplex virus type 2, or any derivative thereof. In some embodiments, the vector is an HSV-1 amplicon or an HSV-1 heterozygous amplicon. In some embodiments, the HSV-1 heterozygous amplicon is an HSV / AAV heterozygous amplicon, an HSV / EBV heterozygous amplicon, and an HSV / EBV / RV heterozygous amplicon, or an HSV / Sleeping Beauty heterozygous amplicon. In some embodiments, the vector is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the immediate early herpes simplex virus genes. In some embodiments, the herpes simplex virus genes are ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41, or UL55. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4, ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutations in the ICP4, ICP27, and UL55 genes are deletions of the coding sequences of the ICP4, ICP27, and UL55 genes. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions in the promoter regions of the ICP22 and ICP47 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0, ICP4, and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes.In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0, ICP4, ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the gene. In some embodiments, the recombinant herpes simplex virus genome also includes an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in both the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome includes one or more transgenes within one or more viral loci. In some embodiments, the recombinant herpes simplex virus genome includes one or more transgenes within one or more ICP4 viral loci. In some embodiments, the recombinant herpes simplex virus genome includes one or more transgenes within the UL41 viral locus. In some embodiments, the vector is capable of replicating within the target cells upon delivery. In some embodiments, the pharmaceutically acceptable carrier is suitable for topical or transdermal administration. In some embodiments, the pharmaceutically acceptable carrier is suitable for subcutaneous or intradermal administration. In some embodiments, the one or more transgenes include miRNA binding sites. In some embodiments, the vector contains a transgene encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the vector contains a transgene encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector contains a transgene encoding a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the vector contains two transgenes, each encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the collagen α-1 (VII) chain polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 2. In some embodiments, the collagen α-1 (VII) chain polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 2. In some embodiments, when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces anchoring fibrillation in the subject. In some embodiments, when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces epithelial basement membrane tissue and / or epithelial basement membrane adhesion in the subject. In some embodiments, the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 4. In some embodiments, the lysyl hydroxylase 3 polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 4.In some embodiments, when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of a subject, the lysyl hydroxylase 3 polypeptide enhances, increases, strengthens, and / or reinforces the formation of hydroxylysine residues on one or more collagen polypeptides of the subject. In some embodiments, when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces anchoring fibrillation, epithelial basement membrane tissue, and / or epithelial basement membrane adhesion of the subject. In some embodiments, the keratin type I cytoskeleton 17 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 30. In some embodiments, the keratin type I cytoskeleton 17 polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 30. In some embodiments, the keratin type I cytoskeleton 17 polypeptide enhances, increases, strengthens, and / or reinforces wound healing of the subject. In some embodiments, the vector contains at least a first transgene and a second transgene. In some embodiments, the first transgene and the second transgene each encode a collagen α-1 (VII) chain polypeptide. In some embodiments, the first transgene encodes a collagen α-1 (VII) chain polypeptide, and the second transgene encodes a lysyl hydroxylase 3 polypeptide. In some embodiments, the first transgene encodes a collagen α-1 (VII) chain polypeptide, and the second transgene encodes a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the first transgene encodes a lysyl hydroxylase 3 polypeptide, and the second transgene encodes a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the vector contains at least the first transgene, the second transgene, and the third transgene. In some embodiments, the first transgene encodes a collagen α-1 (VII) chain polypeptide, the second transgene encodes a lysyl hydroxylase 3 polypeptide, and the third transgene encodes a keratin type I cytoskeleton 17 polypeptide.

[0021] Other aspects of this disclosure relate to a method of providing preventative, palliative, or therapeutic relief of a wound, symptom, or disease of the skin in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a vector, wherein the vector is a recombinant herpes simplex virus genome, and wherein the pharmaceutical composition is capable of enhancing, increasing, strengthening, and / or reinforcing the levels of collagen α-1 (VII) chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the subject. In some embodiments, the pharmaceutical composition comprises: a virus comprising a vector, wherein the vector comprises one or more transgenic molecules encoding collagen α-1 (VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, keratin type I cytoskeleton 17 polypeptide, or chimeric polypeptides thereof; and a pharmaceutically acceptable carrier. In some embodiments, the virus is an adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, herpes simplex virus, vaccinia virus, or any hybrid virus thereof. In some embodiments, the virus is replication-deficient. In some embodiments, the virus is herpes simplex virus (HSV). In some embodiments, the herpes simplex virus is herpes simplex virus type 1, herpes simplex virus type 2, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the immediate early herpes simplex virus genes. In some embodiments, the herpes simplex virus genes are ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41, or UL55. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4, ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutations in the ICP4, ICP27, and UL55 genes are deletions of the coding sequences of the ICP4, ICP27, and UL55 genes. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions in the promoter regions of the ICP22 and ICP47 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0, ICP4, and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes.In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0, ICP4, ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the gene. In some embodiments, the recombinant herpes simplex virus genome also includes an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in both the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome includes one or more transgenes within one or more viral loci. In some embodiments, the recombinant herpes simplex virus genome includes one or more transgenes within one or more ICP4 viral loci. In some embodiments, the recombinant herpes simplex virus genome includes one or more transgenes within the UL41 viral locus. In some embodiments, the vector is capable of replicating within the target cells upon delivery. In some embodiments, the pharmaceutically acceptable carrier is suitable for topical or transdermal administration. In some embodiments, the pharmaceutically acceptable carrier is suitable for subcutaneous or intradermal administration. In some embodiments, the one or more transgenes include miRNA binding sites. In some embodiments, the vector contains a transgene encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the vector contains a transgene encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector contains a transgene encoding a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the collagen α-1 (VII) chain polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 2. In some embodiments, the collagen α-1 (VII) chain polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 2. In some embodiments, when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces anchoring fibrillation in the subject. In some embodiments, when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces epithelial basement membrane tissue and / or epithelial basement membrane adhesion in the subject. In some embodiments, the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 4. In some embodiments, the lysyl hydroxylase 3 polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 4. In some embodiments, when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of a subject, the lysyl hydroxylase 3 polypeptide enhances, increases, strengthens, and / or reinforces the formation of hydroxylysine residues on one or more collagen polypeptides of the subject.In some embodiments, when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens, and / or reinforces anchoring fibrillation, epithelial basement membrane tissue, and / or epithelial basement membrane adhesion in the subject. In some embodiments, the keratin type I cytoskeleton 17 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 30. In some embodiments, the keratin type I cytoskeleton 17 polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 30. In some embodiments, the keratin type I cytoskeleton 17 polypeptide enhances, increases, strengthens, and / or reinforces wound healing in the subject. In some embodiments, the vector contains at least a first transgene and a second transgene. In some embodiments, the first transgene and the second transgene each encode a collagen α-1 (VII) chain polypeptide. In some embodiments, the first transgene encodes a collagen α-1 (VII) chain polypeptide, and the second transgene encodes a lysyl hydroxylase 3 polypeptide. In some embodiments, the first transgene encodes a collagen α-1 (VII) chain polypeptide, and the second transgene encodes a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the first transgene encodes a lysyl hydroxylase 3 polypeptide, and the second transgene encodes a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the vector package contains at least the first, second, and third transgenes. In some embodiments, the first transgene encodes a collagen α-1 (VII) chain polypeptide, the second transgene encodes a lysyl hydroxylase 3 polypeptide, and the third transgene encodes a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the pharmaceutical composition is administered to the subject topically or transdermally. In some embodiments, the pharmaceutical composition is administered to the subject subcutaneously or intradermally. In some embodiments, the pharmaceutical composition is administered once, twice, three times, four times, five times, or more daily. In some embodiments, the pharmaceutical composition is administered to one or more affected and / or unaffected areas of the subject. In some implementations, the skin disease or condition is one or more of the following: epidermolysis bullosa, skin cancer, psoriasis, lichen planus, lupus, rosacea, eczema, cutaneous candidiasis, cellulitis, impetigo, bedsores, erysipelas, ichthyosis vulgaris, dermatomyositis, acrodermatitis, stasis dermatitis, Natherton's syndrome, epidermolysis bullosa simplex (LAMB3 gene), autosomal recessive congenital ichthyosis, xeroderma pigmentosum, and bullous pemphigoid. Attached Figure Description

[0022] Figures 1A to 1F A schematic diagram showing the wild-type herpes simplex virus genome and the modified herpes simplex virus genome. Figure 1A The genome of wild-type herpes simplex virus is shown. Figure 1BThe modified herpes simplex virus genome is shown, the genome containing a transgene encoding a collagen α-1 (VII) chain polypeptide. Figure 1C The modified herpes simplex virus genome is shown, which contains two transgenes, one encoding a collagen α-1 (VII) chain polypeptide and the other encoding a lysine hydroxylase 3 polypeptide, wherein these transgenes are encoded on the same DNA strand. Figure 1D The modified herpes simplex virus genome is shown, which contains two transgenes, one encoding a collagen α-1 (VII) chain polypeptide and the other encoding a lysine hydroxylase 3 polypeptide, wherein these transgenes are encoded in an antisense orientation on opposite DNA strands. Figure 1E The modified herpes simplex virus genome is shown, which contains a polycistronic transgene encoding a collagen α-1 (VII) chain polypeptide and a lysine hydroxylase 3 polypeptide separated by the internal ribosome entry site (IRES). Figure 1F The modified herpes simplex virus genome is shown, the genome containing a transgene encoding a chimeric polypeptide comprising a collagen α-1 (VII) chain polypeptide, a linker polypeptide, and a lysine hydroxylase 3 polypeptide.

[0023] Figures 2A to 2G Further schematic diagrams showing the wild-type herpes simplex virus genome and the modified herpes simplex virus genome are shown. Figure 2A The genome of wild-type herpes simplex virus is shown. Figure 2B The modified herpes simplex virus genome is shown, which contains deletions of the coding sequences for ICP4 (two copies), ICP27 and UL55, as well as deletions of the promoter sequences for ICP22 and ICP47, wherein two transgenes encoding collagen α-1 (VII) chain polypeptides are integrated at the ICP4 locus. Figure 2C The modified herpes simplex virus genome is shown, which contains deletions of the coding sequences for ICP4 (two copies) and ICP22, wherein two transgenes encoding collagen α-1 (VII) chain polypeptides are integrated at the ICP4 locus. Figure 2D The modified herpes simplex virus genome is shown, which contains deletions of the coding sequences for ICP0 and ICP4 (two copies), wherein two transgenes encoding collagen α-1 (VII) chain polypeptides are integrated at the ICP4 locus. Figure 2E The modified herpes simplex virus genome is shown, which contains deletions of the coding sequences for ICP0, ICP4 (two copies) and ICP22, wherein two transgenes encoding collagen α-1 (VII) chain polypeptides are integrated at the ICP4 locus. Figure 2FThe modified herpes simplex virus genome is shown, which contains deletions of coding sequences for ICP0, ICP4 (two copies), ICP22 and ICP27, wherein two transgenes encoding collagen α-1 (VII) chain polypeptides are integrated at the ICP4 locus. Figure 2G The modified herpes simplex virus genome is shown, which contains deletions of coding sequences for ICP0, ICP4 (two copies), ICP22, ICP27 and UL55, wherein two transgenes encoding collagen α-1 (VII) chain polypeptides are integrated at the ICP4 locus.

[0024] Figure 3 The diagram shows “KB103”, which is a replication-defective type 1 herpes simplex virus (HSV-1) carrying a human collagen 7 (COL7A1) expression cassette.

[0025] Figures 4A to 4B This shows KB103-infected RDEB human skin keratinocytes ( Figure 4A ) and RDEB human skin fibroblasts ( Figure 4B The levels of COL7 transcripts in the cells showed a dose-dependent increase. The transcripts were quantified relative to β-actin levels and normalized to expression in uninfected cells.

[0026] Figures 5A to 5B The expression of human Col7 protein detected in KB103-infected cells is shown. Figure 5A Human Col7 protein expression is shown in uninfected normal and RDEB fibroblasts, as well as in fibroblasts infected with KB103 at a specified multiplicity of infection (MOI). Figure 5B Human Col7 protein expression is shown in uninfected normal and RDEB keratinocytes, as well as in keratinocytes infected with KB103 at a specified multiplicity of infection (MOI). Human GAPDH protein expression is shown as a loading control.

[0027] Figure 6 Human COL7A1 protein expression is shown in uninfected (control) or KB103-infected (C7, MOI 3) RDEB human skin fibroblasts (EB HDF), normal human skin keratinocytes (normal HDK), and RDEB human skin keratinocytes (RDEB HDK), as assessed by immunofluorescence.

[0028] Figure 7 Human Col7 and LH3 protein expression is shown in uninfected normal and RDEB human skin keratinocytes, as well as in keratinocytes infected with KB103 at the specified MOI. Human GAPDH protein expression is shown as a loading control.

[0029] Figure 8 Human TSP-1 protein expression is shown in uninfected normal and RDEB human skin fibroblasts, as well as in fibroblasts infected with KB103 at a specified MOI. Human GAPDH protein expression is shown as a loading control.

[0030] Figures 9A to 9B The image shows uninfected (control) RDEB human skin keratinocytes and keratinocytes infected with KB103 at the specified MOI, compared with those infected with increased concentrations of rat tail collagen 1 (…). Figure 9A ) and human fibronectin ( Figure 9B Cell adhesion in the treated pores.

[0031] Figure 10 This shows Col7 deposition in the basement membrane zone (BMZ) of a skin-equivalent organoid culture infected with KB103, as determined by immunofluorescence.

[0032] Figure 11 This study shows the quantification of viral genome copy number and human Col7 transcript levels in tissues isolated from KB103-infected mice.

[0033] Figure 12 The expression of human Col7 protein in skin tissue from KB103-infected mice, as determined by immunofluorescence, is shown, including the initiation of human Col7 deposition in the basement membrane zone (BMZ). Detailed Implementation

[0034] This disclosure relates in part to pharmaceutical compositions comprising one or more polynucleotides encoding collagen α-1 (VII) chain polypeptides, lysyl hydroxylase 3 polypeptides, keratin type I cytoskeleton 17 polypeptides, and / or chimeric polypeptides thereof. In some embodiments, the pharmaceutical composition comprises a carrier, wherein the carrier comprises one or more transgenic molecules encoding collagen α-1 (VII) chain polypeptides, lysyl hydroxylase 3 polypeptides, keratin type I cytoskeleton 17 polypeptides, and / or chimeric polypeptides thereof. In some embodiments, the carrier comprises one or more transgenic molecules adapted to enhance, increase, strengthen, and / or reinforce the levels of collagen α-1 (VII) chain polypeptides and / or lysyl hydroxylase 3 polypeptides and / or keratin type I cytoskeleton 17 polypeptides in one or more cells of a subject. This disclosure also relates in part to methods of providing prophylactic, palliative, or therapeutic relief of wounds, conditions, or diseases of the skin (e.g., dystrophic epidermolysis bullosa) in a subject by administering (e.g., topically or transdermally) the pharmaceutical compositions described herein.

[0035] The following description illustrates exemplary methods, parameters, etc. However, it should be understood that such description is not intended to limit the scope of this disclosure, but is provided as a description of exemplary embodiments.

[0036] General technology

[0037] The techniques and procedures described or cited herein are commonly known to those skilled in the art and frequently used using conventional methods, such as those widely used as described in the following literature: Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMAusubel et al., eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BDHames and G. G. Taylor, eds. (1995)), Harlow and Lane, eds. (1988); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998), Academic Press; Animal Cell Culture (RIFreshney). eds., 1987); Introduction to Cell and Tissue Culture (JP Mather and PERoberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths and DG Newell, eds., 1993-8) J.Wiley and Sons; Gene Transfers for Mammalian Cells (JMMiller and MPCalos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0038] definition

[0039] Before describing the invention in detail, it should be understood that the invention is not limited to specific compositions or biological systems, which can of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0040] As used herein, unless the context clearly states otherwise, the singular forms “a” and “the” include a plural referent. Thus, for example, reference to “a molecule” may optionally include a combination of two or more such molecules, etc.

[0041] As used herein, the term "about" refers to a common range of error for a corresponding value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) implementations for that value or parameter itself.

[0042] As used herein, the terms “polynucleotide,” “nucleic acid sequence,” “nucleic acid,” and their variants shall generally refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, as well as other polymers containing a non-nucleotide backbone, provided that these polymers contain nucleobases whose conformation allows for base pairing and base stacking, as seen in DNA and RNA. Therefore, these terms encompass known types of nucleic acid sequence modifications, such as substitutions and internucleotide modifications of one or more naturally occurring nucleotides in analogue form.

[0043] As used herein, when a nucleic acid is functionally related to another nucleic acid sequence, that nucleic acid is "operationally ligated" or "operationally ligated." For example, if a promoter or enhancer affects the transcription of a sequence, then the promoter or enhancer is operably ligated to a coding sequence; or if a ribosome binding site is positioned to facilitate translation, then it is operably ligated to a coding sequence. Generally, "operationally ligated" means that the ligated DNA sequences are contiguous.

[0044] As used herein, the term "vector" refers to a discrete element used to introduce heterologous nucleic acids into a cell to facilitate the expression or replication of those nucleic acids. Expression vectors include vectors capable of expressing nucleic acids operatively linked to regulatory sequences, such as promoter regions, which can influence the expression of such nucleic acids. Thus, an expression vector can refer to DNA or RNA constructs, such as plasmids, bacteriophages, recombinant viruses, or other vectors that induce nucleic acid expression upon introduction into a suitable host cell. Suitable expression vectors are well known to those skilled in the art and include vectors that can replicate in eukaryotic cells and vectors that remain as appendages or are integrated into the host cell genome.

[0045] As used in this article, an "open reading frame" or "ORF" refers to a continuous nucleic acid sequence, DNA or RNA, that encodes a protein or polypeptide. Typically, nucleic acids contain a translation initiation signal or start codon (such as ATG or AUG) and a stop codon.

[0046] As used in this article, "internal ribosome entry site" or "IRES" refers to a nucleotide sequence that allows translation to begin in the middle of an mRNA sequence, such as after the first start codon.

[0047] As used in this article, "untranslated region" or "UTR" refers to the untranslated nucleic acid at the 5' and / or 3' end of an open reading frame. The presence of one or more UTRs in a polynucleotide can affect the post-transcriptional regulation of the polynucleotide, mRNA stability, and / or translation.

[0048] As used herein, the term "transgenic" refers to a polynucleotide that, upon introduction into a cell, is transcribed into RNA and translated and / or expressed under appropriate conditions. In some respects, it confers desired properties upon the cell into which it is introduced, or otherwise produces desired therapeutic or diagnostic outcomes.

[0049] As used herein, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably and can refer to a polymer of two or more amino acids.

[0050] As used herein, “subject,” “host,” or “individual” means any animal classified as a mammal, including humans, livestock, and farm animals; as well as zoo animals, sporting animals, or pet animals such as dogs, horses, cats, and cattle; and animals used in studies such as mice and rats. In some implementations, the mammal is a human.

[0051] As used herein, “topical application” or “application in a local manner” means the delivery of the composition to a subject by bringing the formulation containing the composition into direct or otherwise contact with all or part of the subject’s skin. This term covers several routes of administration, including but not limited to local and transdermal. Local application is used as a means of delivering the composition to the epidermis or dermis or a specific layer thereof of a subject.

[0052] As used herein, an "effective amount" is at least the minimum amount required to achieve measurable improvement or prevention of one or more symptoms of a particular disease. An effective amount is also the amount at which the beneficial therapeutic effect outweighs any toxic or harmful effects of the treatment. For prophylactic use, beneficial or desired outcomes include results such as eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the occurrence of disease, disease complications, and intermediate pathological phenotypes that occur during disease development. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing one or more symptoms caused by the disease, improving the quality of life of individuals with the disease, delaying disease progression, and / or prolonging survival. An effective amount may be administered in one or more doses.

[0053] Pharmaceutical Composition

[0054] Nucleotides

[0055] In one aspect, this document provides a pharmaceutical composition comprising one or more polynucleotides encoding a collagen α-1 (VII) chain (Col7) polypeptide, a lysyl hydroxylase 3 (LH3) polypeptide, a keratin type I cytoskeleton 17 (KRT17) polypeptide, and / or chimeric polypeptides thereof. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a chimeric polypeptide. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a collagen α-1 (VII) chain polypeptide and a lysyl hydroxylase 3 polypeptide. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a collagen α-1 (VII) chain polypeptide and a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a lysyl hydroxylase 3 polypeptide and a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a collagen α-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and a keratin type I cytoskeleton 17 polypeptide.

[0056] In some embodiments, the pharmaceutical composition comprises a vector encoding one or more transgenic molecules comprising the polynucleotides described herein. In some embodiments, the pharmaceutical composition comprises a vector encoding one or more transgenic molecules encoding a collagen α-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, and / or chimeric polypeptides thereof. In some embodiments, the vector comprises one or more transgenic molecules encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the vector comprises one or more transgenic molecules encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector comprises one or more transgenic molecules encoding a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the vector comprises one or more transgenic molecules encoding a chimeric polypeptide. In some embodiments, the vector comprises one or more transgenic molecules encoding a collagen α-1 (VII) chain polypeptide and one or more transgenic molecules encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector comprises one or more transgenic molecules encoding a collagen α-1 (VII) chain polypeptide and one or more transgenic molecules encoding a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the vector comprises one or more transgenes encoding lysyl hydroxylase 3 polypeptide and one or more transgenes encoding keratin type I cytoskeleton 17 polypeptide. In some embodiments, the vector comprises one or more transgenes encoding collagen α-1 (VII) chain polypeptide, one or more transgenes encoding lysyl hydroxylase 3 polypeptide, and one or more transgenes encoding keratin type I cytoskeleton 17 polypeptide.

[0057] In some embodiments, the pharmaceutical composition comprises synthetic RNA, wherein the synthetic RNA encodes one or more transgenic molecules comprising the polynucleotides described herein. In some embodiments, the pharmaceutical composition comprises synthetic RNA, wherein the synthetic RNA encodes one or more transgenic molecules encoding collagen α-1 (VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, keratin type I cytoskeleton 17 polypeptide, and / or chimeric polypeptides thereof. In some embodiments, the synthetic RNA encodes one or more transgenic molecules encoding collagen α-1 (VII) chain polypeptide. In some embodiments, the synthetic RNA encodes one or more transgenic molecules encoding lysyl hydroxylase 3 polypeptide. In some embodiments, the synthetic RNA encodes one or more transgenic molecules encoding keratin type I cytoskeleton 17 polypeptide. In some embodiments, the synthetic RNA encodes one or more transgenic molecules encoding chimeric polypeptides. In some embodiments, the synthetic RNA encodes one or more transgenic molecules encoding collagen α-1 (VII) chain polypeptide and one or more transgenic molecules encoding lysyl hydroxylase 3 polypeptide. In some embodiments, the synthetic RNA encodes one or more transgenic molecules encoding collagen α-1 (VII) chain polypeptide and one or more transgenic molecules encoding keratin type I cytoskeleton 17 polypeptide. In some embodiments, the synthetic RNA comprises one or more transgenes encoding a lysyl hydroxylase 3 polypeptide and one or more transgenes encoding a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the synthetic RNA comprises one or more transgenes encoding a collagen α-1 (VII) chain polypeptide, one or more transgenes encoding a lysyl hydroxylase 3 polypeptide, and one or more transgenes encoding a keratin type I cytoskeleton 17 polypeptide.

[0058] Collagen α-1 (VII) chain

[0059] In some aspects, the polynucleotides of this disclosure encode collagen α-1 (VII) chain polypeptides. An example of a polynucleotide encoding a collagen α-1 (VII) chain polypeptide is SEQ ID NO: 1. The polynucleotides of this disclosure also include polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1.

[0060] In some embodiments, the polynucleotide encoding the collagen α-1 (VII) chain polypeptide is a polynucleotide encoding an N-terminal truncated, C-terminal truncated, or fragmented portion of the collagen α-1 (VII) chain polypeptide. The polynucleotide encoding an N-terminal truncated, C-terminal truncated, or fragmented portion of the collagen α-1 (VII) chain polypeptide includes polynucleotides having at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, or at least 350, at least 500, at least 1000, at least 2500, at least 5000, or at least 7500 (but less than 8835) consecutive nucleotides as specified in SEQ ID NO: 1.

[0061] In some embodiments, the polynucleotide encoding the collagen α-1 (VII) chain polypeptide is a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the polynucleotide encoding the collagen α-1 (VII) chain polypeptide is a polynucleotide encoding a polypeptide whose amino acid sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2. In some embodiments, this disclosure relates to a polynucleotide encoding a polypeptide that is a homolog of the Homo sapiens collagen α-1 (VII) chain polypeptide. Methods for identifying polypeptides as homologs of the target polypeptide are well known to those skilled in the art.

[0062] In some embodiments, the polynucleotide encoding the collagen α-1 (VII) chain polypeptide is a polynucleotide encoding an N-terminal truncated, C-terminal truncated, or fragmented amino acid sequence of SEQ ID NO: 2. The N-terminal truncated, C-terminal truncated, or fragmented polynucleotide may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, or at least 2500 (but less than 2944) consecutive amino acids of SEQ ID NO: 2.

[0063] In some embodiments, when a polynucleotide encoding a collagen α-1 (VII) chain polypeptide is delivered to one or more target cells of a subject, the polynucleotide expresses the collagen α-1 (VII) chain polypeptide. In some embodiments, the expression of the collagen α-1 (VII) chain polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased levels of the collagen α-1 chain polypeptide in one or more target cells. In some embodiments, the expression of the collagen α-1 (VII) chain polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased function of the collagen α-1 chain polypeptide in one or more target cells. In some embodiments, the expression of the collagen α-1 (VII) chain polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased activity of the collagen α-1 chain polypeptide in one or more target cells. In some embodiments, the expression of the collagen α-1 (VII) chain polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased anchoring fibrillation in the subject. In some embodiments, the expression of the collagen α-1 (VII) chain polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased epithelial basement membrane tissue and / or epithelial basement membrane adhesion in the subject. In some implementations, the expression of collagen α-1 (VII) chain peptides is enhanced, increased, strengthened, and / or reinforced the dermal-epidermal junction integrity of the subject.

[0064] Lysyl hydroxylase 3

[0065] In some aspects, the polynucleotides of this disclosure encode a lysyl hydroxylase 3 polypeptide. An example of a polynucleotide encoding a lysyl hydroxylase 3 polypeptide is SEQ ID NO: 3. The polynucleotides of this disclosure also include polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 3.

[0066] In some embodiments, the polynucleotide encoding the lysyl hydroxylase 3 polypeptide is a polynucleotide encoding an N-terminal truncated, C-terminal truncated, or fragmented lysyl hydroxylase 3 polypeptide. The N-terminal truncated, C-terminal truncated, or fragmented polynucleotide encoding the lysyl hydroxylase 3 polypeptide includes polynucleotides having at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 500, at least 750, at least 1000, at least 1500, or at least 2000 (but less than 2217) consecutive nucleotides as specified in SEQ ID NO: 3.

[0067] In some embodiments, the polynucleotide encoding the lysyl hydroxylase 3 polypeptide is a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO:4. In some embodiments, the polynucleotide encoding the lysyl hydroxylase 3 polypeptide is a polynucleotide encoding a polypeptide whose amino acid sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO:4. In some embodiments, this disclosure relates to a polynucleotide encoding a polypeptide that is a homolog of the Homo sapiens lysyl hydroxylase 3 polypeptide. Methods for identifying polypeptides as homologs of the target polypeptide are well known to those skilled in the art.

[0068] In some embodiments, the polynucleotide encoding the lysyl hydroxylase 3 polypeptide is a polynucleotide encoding an N-terminal truncated, C-terminal truncated, or fragmented amino acid sequence of SEQ ID NO: 4. The N-terminal truncated, C-terminal truncated, or fragmented may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, or at least 700 (but less than 738) consecutive amino acids of SEQ ID NO: 4.

[0069] In some embodiments, when a polynucleotide encoding a lysyl hydroxylase 3 polypeptide is delivered to one or more target cells of a subject, the polynucleotide expresses the lysyl hydroxylase 3 polypeptide. In some embodiments, the expression of the lysyl hydroxylase 3 polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased levels of the lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, the expression of the lysyl hydroxylase 3 polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased function of the lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, the expression of the lysyl hydroxylase 3 polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased activity of the lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, the expression of the lysyl hydroxylase 3 polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased formation of hydroxylysine residues on one or more collagen polypeptides in the subject. In some embodiments, the expression of the lysyl hydroxylase 3 polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased anchoring fibrils formation in the subject. In some embodiments, the expression of lysyl hydroxylase 3 peptide is enhanced, increased, strengthened, and / or reinforced in the subject's epithelial basement membrane tissue and / or epithelial basement adhesion. In some embodiments, the expression of lysyl hydroxylase 3 peptide is enhanced, increased, strengthened, and / or reinforced in the subject's dermal-epidermal junction integrity.

[0070] In some embodiments, a polynucleotide encoding a collagen α-1 (VII) chain polypeptide and a polynucleotide encoding a lysyl hydroxylase 3 polypeptide are delivered to the same cells of the subject. In some embodiments, when the polynucleotides encoding the collagen α-1 (VII) chain polypeptide and the polynucleotide encoding the lysyl hydroxylase 3 polypeptide are delivered to the same cells of the subject, these polynucleotides express both the collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide. In some embodiments, the polynucleotides encoding the collagen α-1 (VII) chain polypeptide and the polynucleotide encoding the lysyl hydroxylase 3 polypeptide express the collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide in an equimolar ratio.

[0071] Keratin type I cytoskeleton 17

[0072] In some aspects, the polynucleotides of this disclosure encode a keratin type I cytoskeleton 17 polypeptide. An example of a polynucleotide encoding a keratin type I cytoskeleton 17 polypeptide is SEQ ID NO: 29. The polynucleotides of this disclosure also include polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 29.

[0073] In some embodiments, the polynucleotide encoding the keratin type I cytoskeleton 17 polypeptide is a polynucleotide encoding an N-terminal truncated, C-terminal truncated, or fragmented portion of the keratin type I cytoskeleton 17 polypeptide. The N-terminal truncated, C-terminal truncated, or fragmented polynucleotide encoding the collagen α-1 (VII) chain polypeptide includes polynucleotides having at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, or at least 350, at least 500, at least 1000, or at least 1250 (but less than 1299) consecutive nucleotides as specified in SEQ ID NO: 29.

[0074] In some embodiments, the polynucleotide encoding the keratin type I cytoskeleton 17 polypeptide is a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide encoding the keratin type I cytoskeleton 17 polypeptide is a polynucleotide encoding a polypeptide whose amino acid sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 30. In some embodiments, this disclosure relates to a polynucleotide encoding a polypeptide that is a homolog of the Homo sapiens keratin type I cytoskeleton 17 polypeptide. Methods for identifying polypeptides as homologs of the target polypeptide are well known to those skilled in the art.

[0075] In some embodiments, the polynucleotide encoding the keratin type I cytoskeleton 17 polypeptide is an N-terminal truncated, C-terminal truncated, or fragmented polynucleotide encoding the amino acid sequence of SEQ ID NO: 30. The N-terminal truncated, C-terminal truncated, or fragmented polynucleotide may contain at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 425 (but less than 432) consecutive amino acids of SEQ ID NO: 30.

[0076] In some embodiments, when a polynucleotide encoding a keratin type I cytoskeleton 17 polypeptide is delivered to one or more target cells of a subject, the polynucleotide expresses the keratin type I cytoskeleton 17 polypeptide. In some embodiments, the expression of the keratin type I cytoskeleton 17 polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased levels of the keratin type I cytoskeleton 17 polypeptide in one or more target cells. In some embodiments, the expression of the keratin type I cytoskeleton 17 polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased function of the keratin type I cytoskeleton 17 polypeptide in one or more target cells. In some embodiments, the expression of the keratin type I cytoskeleton 17 polypeptide is enhanced, increased, strengthened, and / or amplified, resulting in increased wound healing in the subject.

[0077] Chimeric peptides containing linkers

[0078] In some embodiments, the polynucleotide of this disclosure encodes a chimeric polypeptide comprising a collagen α-1 (VII) chain polypeptide and a lysine hydroxylase 3 polypeptide. In some embodiments, the polynucleotide encoding the chimeric polypeptide further comprises a polynucleotide encoding a connector polypeptide. In some embodiments, the polynucleotide encoding the connector polypeptide is a polynucleotide encoding a cleavable connector polypeptide. Examples of polynucleotides encoding cleavable connector polypeptides may include, but are not limited to, polynucleotides encoding T2A, P2A, E2A, or F2A connector polypeptides. In some embodiments, the polynucleotide encoding the connector polypeptide is a polynucleotide encoding a T2A connector polypeptide. In some embodiments, the polynucleotide encoding the connector polypeptide is a polynucleotide encoding a P2A connector polypeptide. In some embodiments, the polynucleotide encoding the connector polypeptide is a polynucleotide encoding an E2A connector polypeptide. In some embodiments, the polynucleotide encoding the connector polypeptide is a polynucleotide encoding an F2A connector polypeptide.

[0079] In some aspects, the polynucleotides of this disclosure encode adapter polypeptides. Examples of polynucleotides encoding adapter polypeptides are SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11. The polynucleotides of this disclosure also include polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

[0080] In some embodiments, the polynucleotide encoding the adapter polypeptide is a polynucleotide encoding an N-terminal truncated, C-terminal truncated, or fragmented portion of the adapter polypeptide. The polynucleotide encoding an N-terminal truncated, C-terminal truncated, or fragmented portion of the adapter polypeptide includes polynucleotides having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 (but less than 66) consecutive nucleotides of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

[0081] In some embodiments, the polynucleotide encoding the adapter polypeptide is a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. In some embodiments, the polynucleotide encoding the adapter polypeptide is a polynucleotide encoding a polypeptide whose amino acid sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.

[0082] In some embodiments, the polynucleotide encoding the adapter polypeptide is an N-terminal truncated, C-terminal truncated, or fragmented polynucleotide encoding the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. The N-terminal truncated, C-terminal truncated, or fragmented polynucleotide may comprise at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 (but less than 22) consecutive amino acids of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.

[0083] In some embodiments, the polynucleotide encoding the adaptor polypeptide further comprises a polynucleotide encoding one or more furin cleavage sites. In some embodiments, the polynucleotide encoding one or more furin cleavage sites encodes an amino acid sequence that is identical or substantially similar to the sequence of a classic furin cleavage site (Arg-X-(Arg / Lys)-Arg). In some embodiments, the one or more furin cleavage sites are encoded upstream of the adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the T2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the T2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the P2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the P2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the E2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the E2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the F2A adapter peptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the F2A adapter peptide.

[0084] In some embodiments, the polynucleotide encoding the chimeric polypeptide encodes a chimeric polypeptide comprising a collagen α-1 (VII) chain polypeptide, a linker polypeptide, and a lysine hydroxylase 3 polypeptide. In some embodiments, the polynucleotide encoding the chimeric polypeptide comprises, from 5' to 3', a polynucleotide encoding a collagen α-1 (VII) chain polypeptide, a polynucleotide encoding a linker polypeptide, and a polynucleotide encoding a lysine hydroxylase 3 polypeptide. In some embodiments, the polynucleotide encoding the chimeric polypeptide comprises, from 5' to 3', a polynucleotide encoding a lysine hydroxylase 3 polypeptide, a polynucleotide encoding a linker polypeptide, and a polynucleotide encoding a collagen α-1 (VII) chain polypeptide.

[0085] Examples of polynucleotides encoding chimeric polypeptides comprising collagen α-1 (VII) chain polypeptide, adaptor polypeptide, and lysyl hydroxylase 3 polypeptide are SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27. The polynucleotides disclosed herein also include polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequences of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27.

[0086] In some embodiments, the polynucleotide encoding the chimeric polypeptide is a polynucleotide encoding an N-terminal truncated, C-terminal truncated, or fragmented portion of the chimeric polypeptide. Polynucleotides encoding N-terminal truncated, C-terminal truncated, or fragmented portions of the chimeric polypeptide include those having SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 16. 27. At least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, or at least 10000 (but less than 11121) consecutive nucleotide polynucleotides.

[0087] In some embodiments, the polynucleotide encoding a chimeric polypeptide comprising a collagen α-1 (VII) chain polypeptide, a linker polypeptide, and a lysine hydroxylase 3 polypeptide is a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28. In some embodiments, the polynucleotide encoding a chimeric polypeptide comprising a collagen α-1 (VII) chain polypeptide, a linker polypeptide, and a lysine hydroxylase 3 polypeptide is a polynucleotide encoding a polypeptide whose amino acid sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28.

[0088] In some embodiments, the polynucleotide encoding the chimeric polypeptide comprising the collagen α-1 (VII) chain polypeptide, the linker polypeptide, and the lysyl hydroxylase 3 polypeptide is a polynucleotide encoding an N-terminal truncated, C-terminal truncated, or fragmented amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28. The N-terminal truncated, C-terminal truncated, or fragmented polynucleotide may comprise SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28. At least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3250, or at least 3500 (but less than 3706) consecutive amino acids.

[0089] In some embodiments, the polynucleotide encoding the chimeric polypeptide expresses the chimeric polypeptide when it is delivered to one or more target cells of a subject. In some embodiments, the chimeric polypeptide is cleaved after expression in one or more target cells. In some embodiments, the chimeric polypeptide is cleaved within a linker polypeptide when expressed in one or more target cells. In some embodiments, the chimeric polypeptide is cleaved into two polypeptides, one containing a collagen α-1 (VII) chain polypeptide and the other containing a lysyl hydroxylase 3 polypeptide. In some embodiments, the expression of the chimeric polypeptide enhances, increases, strengthens, and / or amplifies the levels of the collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, the expression of the chimeric polypeptide enhances, increases, strengthens, and / or amplifies the function of the collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, the expression of the chimeric polypeptide enhances, increases, strengthens, and / or amplifies the activity of the collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, the expression of the chimeric peptide enhances, increases, strengthens, and / or reinforces the formation of hydroxylysine residues on one or more collagen peptides in the subject. In some embodiments, the expression of the chimeric peptide enhances, increases, strengthens, and / or reinforces anchoring fibrils formation in the subject. In some embodiments, the expression of the chimeric peptide enhances, increases, strengthens, and / or reinforces epithelial basement membrane tissue and / or epithelial basement membrane adhesion in the subject. In some embodiments, the expression of the chimeric peptide enhances, increases, strengthens, and / or reinforces the dermal-epidermal junction integrity in the subject.

[0090] The polynucleotides disclosed herein may be codon-optimized. In some embodiments, the polynucleotides of this disclosure have been codon-optimized for human cells. In some embodiments, the polynucleotides of this disclosure have been codon-optimized for mouse cells. In some embodiments, the polynucleotides of this disclosure have been codon-optimized for rat cells. In some embodiments, the polynucleotides of this disclosure have been codon-optimized for hamster cells. In some embodiments, the polynucleotides of this disclosure have been codon-optimized for canine cells. In some embodiments, the polynucleotides of this disclosure have been codon-optimized for yeast cells. In some embodiments, the polynucleotides of this disclosure have been codon-optimized for bacterial cells. The polynucleotides of this disclosure may be DNA polynucleotides, RNA polynucleotides, or a combination of one or more DNA polynucleotides and one or more RNA polynucleotides.

[0091] carrier

[0092] In some aspects, this disclosure relates to vectors containing one or more polynucleotides described herein, preferably expression vectors. In some embodiments, the vector is a DNA vector. Generally, vectors suitable for maintaining, propagating, or expressing polynucleotides to produce one or more polypeptides in a subject can be used. Examples of suitable vectors include, but are not limited to, plasmids, granules, episomes, transposons, and viral vectors (e.g., adenovirus, vaccinia virus, Sindbisvirus, measles, herpesvirus, lentivirus, retrovirus, adeno-associated virus vector, etc.). In some embodiments, the vector is capable of autonomous replication in host cells. In some embodiments, the vector cannot autonomously replicate in host cells. In some embodiments, the vector is capable of integrating into host DNA. Methods for preparing vectors containing one or more target polynucleotides are well known to those skilled in the art.

[0093] In some embodiments, the vector is a herpes simplex virus vector. In some embodiments, the herpes simplex virus vector is a herpes virus amplicon vector. Herpes virus amplicon vectors (including structural features) and methods for preparing vectors are well known in the art (de Silva S. and Bowers W. “Herpes Virus Amplicon Vectors”. Viruses 2009, 1, 594-629). In some embodiments, the vector is an HSV-1 amplicon. In some embodiments, the vector is an HSV-1 heterozygous amplicon. Examples of HSV-1 heterozygous amplicon include, but are not limited to, HSV / AAV heterozygous amplicon, HSV / EBV heterozygous amplicon, HSV / EBV / RV heterozygous amplicon, and HSV / Sleeping Beauty heterozygous amplicon. In some embodiments, the vector is an HSV / AAV heterozygous amplicon. In some embodiments, the vector is an HSV / EBV heterozygous amplicon. In some embodiments, the vector is an HSV / EBV / RV heterozygous amplicon. In some implementations, the vector is an HSV / Sleeping Beauty hybrid amplicon.

[0094] In some embodiments, the herpes simplex virus vector is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome has been engineered to reduce or eliminate the expression of one or more virulent herpes simplex virus genes. Methods for engineering recombinant herpes simplex virus genomes are broadly described in WO2015 / 009952. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations. Examples of inactivating mutations may include, but are not limited to, deletions, insertions, point mutations, and rearrangements. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41, and UL55 herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome contains an inactivating mutation in the ICP0 gene. In some embodiments, the recombinant herpes simplex virus genome contains an inactivating mutation in the ICP4 gene (one or two copies). In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in the ICP27 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in the ICP47 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-2 genome.

[0095] In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 (one or two copies), ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 (one or two copies), ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutations in the ICP4 (one or two copies), ICP27, and / or UL55 genes are deletions of the coding sequences of the ICP4 (one or two copies), ICP27, and / or UL55 genes. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions in the promoter regions of the ICP22 and ICP47 genes (e.g., the ICP22 and ICP47 coding sequences are intact but lack transcriptional activity). In some embodiments, the recombinant herpes simplex virus genome contains deletions in the coding sequences of the ICP4 (one or two copies), ICP27, and UL55 genes, and deletions in the promoter regions of the ICP22 and ICP47 genes. In some embodiments, the recombinant herpes simplex virus genome further includes an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-2 genome.

[0096] In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 (one or two copies) and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0 and ICP4 (one or two copies) genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0, ICP4 (one or two copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0, ICP4 (one or two copies), ICP22, ICP27, and UL55 genes. In some embodiments, the inactivation mutation in the ICP0, ICP4 (one or two copies), ICP22, ICP27, and / or UL55 genes comprises a deletion of the coding sequences of the ICP0, ICP4 (one or two copies), ICP22, ICP27, and / or UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises deletions in the coding sequences of the ICP0, ICP4 (one or two copies), ICP22, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome also comprises an inactivation mutation in the ICP47 gene. In some embodiments, the recombinant herpes simplex virus genome also comprises an inactivation mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome also comprises inactivation mutations in the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. In some implementations, the recombinant herpes simplex virus genome is the recombinant HSV-2 genome.

[0097] In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 (one or two copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 (one or two copies), ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP0, ICP4 (one or two copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-2 genome.

[0098] In some embodiments, the recombinant herpes simplex virus genome is contained in one or more polynucleotides of the present disclosure within one, two, three, four, five, six, seven or more viral loci. Examples of suitable viral loci may include, but are not limited to, the herpes simplex virus loci ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41 and UL55. In some embodiments, the recombinant herpes simplex virus genome contains one or more polynucleotides of this disclosure within one or more viral ICP4 loci (e.g., the recombinant virus carries a polynucleotide encoding Col7 in one or two ICP4 loci; the recombinant virus carries a polynucleotide encoding LH3 in one or two ICP4 loci; the recombinant virus carries a polynucleotide encoding KRT17 in one or two ICP4 loci; the recombinant virus carries a polynucleotide encoding Col7 in one ICP4 locus and a polynucleotide encoding KRT17 in another ICP4 locus; the recombinant virus carries a polynucleotide encoding Col7 in one ICP4 locus and a polynucleotide encoding LH3 in another ICP4 locus; the recombinant virus carries a polynucleotide encoding LH3 in one ICP4 locus and a polynucleotide encoding KRT17 in another ICP4 locus, etc.). In some embodiments, the recombinant herpes simplex virus genome contains one or more polynucleotides of this disclosure within the viral UL41 locus. In some embodiments, the recombinant herpes simplex virus genome contains one or more polynucleotides of this disclosure within the viral ICP47 locus.In some embodiments, the recombinant herpes simplex virus genome contains one or more polynucleotides of the present disclosure within one or more viral ICP4 loci, and within the viral UL41 locus (e.g., the recombinant virus carries a polynucleotide encoding Col7 in one or two ICP4 loci and a polynucleotide encoding LH3 in the UL41 locus; the recombinant virus carries a polynucleotide encoding Col7 in one or two ICP4 loci and a polynucleotide encoding Col7 in the UL41 locus; the recombinant virus carries a polynucleotide encoding Col7 in one or two ICP4 loci and a polynucleotide encoding KRT17 in the UL41 locus; the recombinant virus carries a polynucleotide encoding LH3 in one or two ICP4 loci and a polynucleotide encoding KRT17 in the UL41 locus). Polynucleotides encoding LH3; recombinant viruses carrying polynucleotides encoding LH3 at one or two ICP4 loci and polynucleotides encoding Col7 at the UL41 locus; recombinant viruses carrying polynucleotides encoding LH3 at one or two ICP4 loci and polynucleotides encoding KRT17 at the UL41 locus; recombinant viruses carrying polynucleotides encoding KRT17 at one or two ICP4 loci and polynucleotides encoding LH3 at the UL41 locus; recombinant viruses carrying polynucleotides encoding KRT17 at one or two ICP4 loci and polynucleotides encoding Col7 at the UL41 locus; recombinant viruses carrying polynucleotides encoding KRT17 at one or two ICP4 loci and polynucleotides encoding KRT17 at the UL41 locus, etc.

[0099] The vector may include the polynucleotide of this disclosure in a form suitable for expression of the polynucleotide in a host cell. The expression vector may include one or more regulatory sequences operatively linked to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (such as globulins, elastases, albumins, alpha-fetoprotein, insulin, etc.) and enhancer sequences from eukaryotic viruses (such as the SV40 enhancer (bp 100-270) post-OMI, the cytomegalovirus early promoter enhancer, the polyoma enhancer post-OMI, the adenovirus enhancer, etc.). Examples of promoters suitable for transcription in mammalian host cells include, but are not limited to, promoters derived from the genomes of viruses (such as polyomavirus, fowlpox virus, adenoviruses (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), etc.) or promoters from heterologous mammals (such as actin promoters, immunoglobulin promoters, promoters from heat shock, etc.), provided that these promoters are compatible with the host cell. In some embodiments, the polynucleotides of this disclosure are operatively linked to one or more heterologous promoters. In some embodiments, the one or more heterologous promoters are one or more of the human cytomegalovirus (HCMV) immediate early promoter, elongation factor-1 (EF1) promoter, and / or any combination thereof. In some embodiments, the one or more heterologous promoters are one or more of constitutive promoters, tissue-specific promoters, time promoters, spatial promoters, inducible promoters, and repressive promoters. The regulatory sequence may include a constitutive regulatory sequence that directs the expression of the nucleotide sequence, as well as tissue-specific regulatory sequences and / or inducible sequences. The design of the expression vector may depend on factors such as the host cell in contact with the polynucleotide of this disclosure, the desired protein expression level, etc. The expression vector of this disclosure may be introduced into a host cell to produce a protein or polypeptide encoded by the polynucleotide described herein (e.g., collagen α-1 (VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, keratin type I cytoskeleton 17 polypeptide, chimeric polypeptide, etc.).

[0100] In some embodiments, the vector of this disclosure comprises one or more transgenes, which comprise one or more polynucleotides described herein. The one or more transgenes may be inserted into the vector in any orientation. If the vector comprises two or more transgenes (e.g., two or more, three or more, etc.), these transgenes may be inserted in the same orientation or in opposite orientations. Not wishing to be bound by theory, incorporating two transgenes in an antisense orientation into the vector can help avoid readthrough and ensure correct expression of each transgene. In some embodiments, the vector comprises one or more transgenes encoding a polypeptide selected from the group consisting of: collagen α-1 (VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, keratin type I cytoskeleton 17 polypeptide, and / or chimeric polypeptides thereof. In some embodiments, the vector comprises a single transgene encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the vector comprises two transgenes, each encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the vector comprises three transgenes, each encoding a collagen α-1 (VII) chain polypeptide. In some embodiments, the vector comprises a single transgene encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector contains two transgenes, each encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector contains three transgenes, each encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector contains a single transgene encoding a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the vector contains two transgenes, each encoding a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the vector contains three transgenes, each encoding a keratin type I cytoskeleton 17 polypeptide. In some embodiments, the vector contains a single transgene encoding a chimeric polypeptide comprising a collagen α-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeleton 17 polypeptide.

[0101] In some embodiments, the vector contains at least two transgenes (e.g., two, three, four, five, six, seven, or more transgenes). In some embodiments, at least a first transgene encodes a collagen α-1 (VII) chain polypeptide, and at least a second transgene encodes a lysyl hydroxylase 3 polypeptide. In some embodiments, at least a first transgene encodes a lysyl hydroxylase 3 polypeptide, and at least a second transgene encodes a collagen α-1 (VII) chain polypeptide. In some embodiments, at least a first transgene encodes a collagen α-1 (VII) chain polypeptide, and at least a second transgene encodes a keratin type I cytoskeleton 17 polypeptide. In some embodiments, at least a first transgene encodes a keratin type I cytoskeleton 17 polypeptide, and at least a second transgene encodes a collagen α-1 (VII) chain polypeptide. In some embodiments, at least a first transgene encodes a lysyl hydroxylase 3 polypeptide, and at least a second transgene encodes a keratin type I cytoskeleton 17 polypeptide. In some embodiments, at least a first transgene encodes a keratin type I cytoskeleton 17 polypeptide, and at least a second transgene encodes a lysyl hydroxylase 3 polypeptide. In some embodiments, at least a first transgene encodes a collagen α-1 (VII) chain polypeptide, and at least a second transgene encodes a chimeric polypeptide comprising a collagen α-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeleton 17 polypeptide. In some embodiments, at least a first transgene encodes a lysyl hydroxylase 3 polypeptide, and at least a second transgene encodes a chimeric polypeptide comprising a collagen α-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeleton 17 polypeptide. In some embodiments, at least a first transgene encodes a keratin type I cytoskeleton 17 polypeptide, and at least a second transgene encodes a chimeric polypeptide comprising a collagen α-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeleton 17 polypeptide.

[0102] In some embodiments, the vector contains at least three transgenes (e.g., three, four, five, six, seven or more transgenes). In some embodiments, at least the first transgene encodes a collagen α-1 (VII) chain polypeptide, at least the second transgene encodes a lysine hydroxylase 3 polypeptide, and at least the third transgene encodes a keratin type I cytoskeleton 17 polypeptide.

[0103] In some embodiments, the vector contains a polycistronic transgene. In some embodiments, the polycistronic transgene encodes a collagen α-1 (VII) chain polypeptide in the first open reading frame (ORF) and a lysine hydroxylase 3 polypeptide in the second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a lysine hydroxylase 3 polypeptide in the first open reading frame (ORF) and a collagen α-1 (VII) chain polypeptide in the second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a collagen α-1 (VII) chain polypeptide in the first open reading frame (ORF) and a keratin type I cytoskeleton 17 polypeptide in the second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a keratin type I cytoskeleton polypeptide in the first open reading frame (ORF) and a collagen α-1 (VII) chain polypeptide in the second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a lysine hydroxylase 3 polypeptide in the first open reading frame (ORF) and a keratin type I cytoskeleton polypeptide in the second open reading frame (ORF).

[0104] In some embodiments, the polycistronic transgene encodes a collagen α-1(VII) chain polypeptide in the first open reading frame (ORF) and a chimeric polypeptide comprising a collagen α-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeleton 17 polypeptide in the second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a lysyl hydroxylase 3 polypeptide in the first open reading frame (ORF) and a chimeric polypeptide comprising a collagen α-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeleton 17 polypeptide in the second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a keratin type I cytoskeleton 17 polypeptide in the first open reading frame (ORF) and a chimeric polypeptide comprising a collagen α-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeleton 17 polypeptide in the second open reading frame (ORF). In some implementations, the first and second ORFs are separated by the internal ribosome entry site (IRES).

[0105] In some embodiments, the polycistronic transgene encodes a collagen α-1 (VII) chain polypeptide in the first open reading frame (ORF), a keratin type I cytoskeleton polypeptide in the second open reading frame (ORF), and a lysine hydroxylase 3 polypeptide in the third open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a lysine hydroxylase 3 polypeptide in the first open reading frame (ORF), a keratin type I cytoskeleton 17 polypeptide in the second open reading frame (ORF), and a collagen α-1 (VII) chain polypeptide in the third open reading frame (ORF). In some embodiments, the first, second, and third ORFs are separated by an internal ribosome entry site (IRES).

[0106] Suitable examples of IRES may include, but are not limited to, virus-derived IRES (e.g., IRES derived from poliovirus, rhinovirus, encephalomyocytovirus, foot-and-mouth disease virus, hepatitis C virus, classical swine fever virus, Raul's sarcoma virus, human immunodeficiency virus, cricket paralysis virus, Kaposi's sarcoma-associated herpesvirus, etc.) and cell mRNA-derived IRES (e.g., IRES derived from growth factor mRNA, such as fibroblast growth factor 2, platelet-derived growth factor B, and vascular endothelial growth factor; IRES derived from transcription factor mRNA, such as antennal factor, ultrapithoraxm, and NF-κB inhibitor; and IRES derived from oncogene mRNA, such as c-myc, pim-1, and protein kinase p58). PITSLRE (IRES, etc.).

[0107] The vector disclosed herein can further encode additional coding and non-coding sequences. Examples of additional coding and non-coding sequences may include, but are not limited to, sequences encoding additional peptide tags, introns, 5' and 3' UTRs, etc. Examples of suitable peptide tags may include, but are not limited to, any combination of the following tags: purification tags, such as his tags, flag tags, maltose-binding proteins, and glutathione S-transferase tags; detection tags, such as photometrically detectable tags (e.g., red fluorescent protein) and tags with detectable enzyme activity (e.g., alkaline phosphatase, etc.); tags containing secretory sequences, leader sequences, and / or stable sequences; protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites), etc. In some embodiments, the 5' and / or 3' UTR increases the stability, localization, and / or translation efficiency of polynucleotides. In some embodiments, the 5' and / or 3' UTR is modified to increase the stability, localization, and / or translation efficiency of one or more polynucleotides. In some embodiments, the 5' and / or 3' UTR improves the level and / or duration of protein expression. In some embodiments, the 5' and / or 3' UTR includes elements (e.g., one or more miRNA binding sites, etc.) that can block or reduce off-target transgene expression (e.g., inhibit expression in a specific cell type (e.g., neurons) at a specific time during a specific developmental stage). In some embodiments, the 5' and / or 3' UTR includes elements (e.g., one or more miRNA binding sites, etc.) that can enhance transgene expression in a specific cell type.

[0108] Synthetic RNA polynucleotides

[0109] In some aspects, this disclosure relates to synthetic RNA, particularly synthetic mRNA, containing one or more polynucleotides described herein. In some embodiments, the synthetic mRNA polynucleotide comprises a 5'-cap structure. Examples of 5'-cap structures may include, but are not limited to, cap-0, cap-1, cap-2, and cap-3 structures and derivatives thereof. In some embodiments, the synthetic mRNA polynucleotide comprises a 3'-poly(A) tail. In some embodiments, the synthetic mRNA polynucleotide comprises one or more 5' and / or 3' UTRs flanking one or more coding sequences contained within the synthetic mRNA polynucleotide. In some embodiments, the 5' and / or 3' UTRs increase the stability, localization, and / or translation efficiency of the synthetic mRNA polynucleotide. In some embodiments, the 5' and / or 3' UTRs are modified to increase the stability, localization, and / or translation efficiency of the synthetic mRNA polynucleotide. In some embodiments, the 5' and / or 3' UTRs improve the level and / or duration of protein expression. In some embodiments, the 5' and / or 3' UTRs are modified to improve the level and / or duration of protein expression. In some embodiments, the 5' and / or 3' UTR includes elements (e.g., miRNA binding sites, etc.) that can restrict off-target expression (e.g., inhibit expression in a specific cell type (e.g., neurons) at a specific time during a specific developmental stage). In some embodiments, the 5' UTR contains a Kozak sequence. In some embodiments, the Kozak sequence is identical or substantially similar to a Kozak consortium sequence. Methods for preparing synthetic mRNA polynucleotides containing one or more target polynucleotides are well known to those skilled in the art.

[0110] In some aspects, the synthetic mRNA polynucleotides disclosed herein comprise one or more modified ribonucleotides. Examples of modified ribonucleotides may include, but are not limited to, 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-aminouridine, 5-hydroxyuridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseuuridine, 5-aminopseuuridine, 5-hydroxypseuuridine, 4-thio-5-azauridine, 4-thiopseuuridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-aza ... 4-Thio-5-aminopseudouridine, 4-Thio-5-hydroxypseudouridine, 2-Thiocytidine, 5-azacytidine, pseudocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudocytidine, 5-aminopseudocytidine, 5-hydroxypseudocytidine, N4-methyl-5-azacytidine, N4-methylpseudocytidine, 2-Thio-5-azacytidine, 2-Thiopseudocytidine, 2-Thio-N4-methylcytidine, 2-Thio-N4-aminocytidine, 2-Thio-N4-hydroxycytidine, 2-Thio- 5-Methylcytidine, 2-Thio-5-aminocytidine, 2-Thio-5-hydroxycytidine, 2-Thio-5-methyl-5-azacytidine, 2-Thio-5-amino-5-azacytidine, 2-Thio-5-hydroxy-5-azacytidine, 2-Thio-5-methylpseucytidine, 2-Thio-5-aminopseucytidine, 2-Thio-5-hydroxypseucytidine, 2-Thio-N4-methyl-5-azacytidine, 2-Thio-N4-methylpseucytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-methylcytidine -Azacytidine, N4-methyl-5-methylpseudocytidine, N4-methyl-5-aminopseudocytidine, N4-methyl-5-hydroxypseudocytidine, N4-amino-5-azacytidine, N4-aminopseudocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminopseudocytidine, N4-amino-5-aminopseudocytidine, N4-amino-5-hydroxypseudocytidine, N4-amino-5-methylpseudocytidine, N4-amino-5-aminopseudocytidine, N4-amino-5-hydroxypseudocytidine, N4-hydroxypseudocytidine, N4-hydroxypseudocytidine, N4-hydroxypseudocytidine, N4-hydroxypseudocytidine, N4-hydroxypseudocytidine, N4-hydroxy-5-methylcytidineN4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudomytidine, N4-hydroxy-5-aminopseudomytidine, N4-hydroxy-5-hydroxypseudomytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2 -Thio-N4-methyl-5-methylpseudocytoplasmic acid, 2-Thio-N4-methyl-5-aminopseudocytoplasmic acid, 2-Thio-N4-methyl-5-hydroxypseudocytoplasmic acid, 2-Thio-N4-amino-5-azacytoplasmic acid, 2-Thio-N4-aminopseudocytoplasmic acid, 2-Thio-N4-amino-5-methylpseudocytoplasmic acid, 2-Thio-N4-amino-5-aminopseudocytoplasmic acid, 2-Thio-N4-amino-5-hydroxypseudocytoplasmic acid, 2-Thio-N4-amino-5-methyl-5-azacytoplasmic acid, 2-Thio-N4-amino-5-amino-5-azacytoplasmic acid, 2-Thio-N4-amino-5-hydroxypseudocytoplasmic acid, 2-Thio-N4-amino-5-aminopseudocytoplasmic acid Cytidine, 2-thio-N4-amino-5-hydroxypsytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypsytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylcytidine, 2-thio-N4-hydroxy-5-methylcytidine, 2-thio-N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxypsytidine, N6-methyladenosine, N6-aminoadenosine, N 6-Hydroxyadenosine, 7-Deazoadenosine, 8-azaadenosine, N6-methyl-7-deazoadenosine, N6-methyl-8-azaadenosine, 7-deazo-8-azaadenosine, N6-methyl-7-deazo-8-azaadenosine, N6-amino-7-deazoadenosine, N6-amino-8-azaadenosine, N6-amino-7-deazo-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazoadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deazoadenosine, N6-hydroxy-7-deazo-8-azaadenosine, 6-thioguanosine, 7-deazoguanosine, 8-azaguanosine, 6-thio-7-deazoguanosine, 6-thio-8-azaguanosine, 7-deazo-8-azaguanosine, and 6-thio-7-deazo-8-azaguanosine.

[0111] In some embodiments, the polynucleotide encoding the collagen α-1 (VII) chain polypeptide and the polynucleotide encoding the lysyl hydroxylase 3 polypeptide are contained within two separate synthetic mRNA polynucleotides. In some embodiments, the polynucleotide encoding the collagen α-1 (VII) chain polypeptide and the polynucleotide encoding the keratin type I cytoskeleton 17 polypeptide are contained within two separate synthetic mRNA polynucleotides. In some embodiments, the polynucleotide encoding the lysyl hydroxylase 3 polypeptide and the polynucleotide encoding the keratin type I cytoskeleton 17 polypeptide are contained within two separate synthetic mRNA polynucleotides. In some embodiments, the polynucleotide encoding the collagen α-1 (VII) chain polypeptide, the polynucleotide encoding the lysyl hydroxylase 3 polypeptide, and the polynucleotide encoding the keratin type I cytoskeleton 17 polypeptide are contained within three separate synthetic mRNA polynucleotides.

[0112] In some embodiments, the polynucleotide encoding the collagen α-1 (VII) chain polypeptide, the polynucleotide encoding the lysyl hydroxylase 3 polypeptide, and / or the polynucleotide encoding the keratin type I cytoskeleton 17 polypeptide are single continuous polynucleotides contained within a single synthetic mRNA polynucleotide. In some embodiments, in the single synthetic mRNA, the single continuous polynucleotide encodes the collagen α-1 (VII) chain polypeptide in a first open reading frame (ORF) and the lysyl hydroxylase 3 polypeptide in a second open reading frame (ORF). In some embodiments, in the single synthetic mRNA, the single continuous polynucleotide encodes the lysyl hydroxylase 3 polypeptide in a first open reading frame (ORF) and the collagen α-1 (VII) chain polypeptide in a second open reading frame (ORF). In some embodiments, in the single synthetic mRNA, the single continuous polynucleotide encodes the collagen α-1 (VII) chain polypeptide in a first open reading frame (ORF) and the keratin type I cytoskeleton 17 polypeptide in a second open reading frame (ORF). In some embodiments, a single continuous polynucleotide in a single synthetic mRNA encodes a keratin type I cytoskeleton polypeptide in a first open reading frame (ORF) and a collagen α-1(VII) chain polypeptide in a second open reading frame (ORF). In some embodiments, a single continuous polynucleotide in a single synthetic mRNA encodes a lysine hydroxylase 3 polypeptide in a first open reading frame (ORF) and a keratin type I cytoskeleton 17 polypeptide in a second open reading frame (ORF). In some embodiments, a single continuous polynucleotide in a single synthetic mRNA encodes a keratin type I cytoskeleton polypeptide in a first open reading frame (ORF) and a lysine hydroxylase 3 polypeptide in a second open reading frame (ORF). In some embodiments, in a single synthetic mRNA, a single continuous polynucleotide encodes a collagen α-1 (VII) chain polypeptide in a first open reading frame (ORF) and encodes a chimeric polypeptide comprising a collagen α-1 (VII) chain polypeptide, a lysine hydroxylase 3 polypeptide, and / or a keratin type I cytoskeleton 17 polypeptide in a second open reading frame (ORF). In some embodiments, in a single synthetic mRNA, a single continuous polynucleotide encodes a keratin type I cytoskeleton 17 polypeptide on a first open reading frame (ORF) and encodes a chimeric polypeptide comprising a collagen α-1 (VII) chain polypeptide, a lysine hydroxylase 3 polypeptide, and / or a keratin type I cytoskeleton 17 polypeptide on a second open reading frame (ORF).In some implementations, the two ORFs are separated by IRES.

[0113] In some embodiments, in a single synthetic mRNA, a single continuous polynucleotide encodes a collagen α-1 (VII) chain polypeptide in a first open reading frame (ORF), a keratin type I cytoskeleton polypeptide in a second open reading frame (ORF), and a lysine hydroxylase 3 polypeptide in a third open reading frame (ORF). In some embodiments, in a single synthetic mRNA, a single continuous polynucleotide encodes a lysine hydroxylase 3 polypeptide in a first open reading frame (ORF), a keratin type I cytoskeleton 17 polypeptide in a second open reading frame (ORF), and a collagen α-1 (VII) chain polypeptide in a third open reading frame (ORF). In some implementations, the first, second, and third ORFs are separated by the internal ribosome entry site (IRES).

[0114] Suitable examples of IRES may include, but are not limited to, virus-derived IRES (e.g., IRES derived from poliovirus, rhinovirus, encephalomyocytovirus, foot-and-mouth disease virus, hepatitis C virus, classical swine fever virus, Raul's sarcoma virus, human immunodeficiency virus, cricket paralysis virus, Kaposi's sarcoma-associated herpesvirus, etc.) and cell mRNA-derived IRES (e.g., IRES derived from growth factor mRNA, such as fibroblast growth factor 2, platelet-derived growth factor B, and vascular endothelial growth factor; IRES derived from transcription factor mRNA, such as antennal factor, ultrapithoraxm, and NF-κB inhibitor; and IRES derived from oncogene mRNA, such as c-myc, pim-1, and protein kinase p58). PITSLRE (IRES, etc.).

[0115] In some embodiments, the polynucleotide encoding any chimeric polypeptide comprising the collagen α-1 (VII) chain polypeptide, lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide described herein is encoded within a single ORF of the synthetic mRNA polynucleotide.

[0116] The synthetic mRNA polynucleotides disclosed herein can further encode additional coding sequences. Examples of additional coding sequences may include, but are not limited to, sequences encoding additional polypeptide tags. Examples of suitable polypeptide tags may include, but are not limited to, any combination of the following tags: purification tags, such as his tags, flag tags, maltose-binding proteins, and glutathione S-transferase tags; detection tags, such as photometrically detectable tags (e.g., red fluorescent protein) and tags with detectable enzyme activity (e.g., alkaline phosphatase); tags containing secretory sequences, leader sequences, and / or stable sequences; protease cleavage sites (such as furin cleavage sites), etc.

[0117] Delivery medium

[0118] Some aspects of this disclosure relate to a pharmaceutical composition comprising a delivery medium that includes one or more polynucleotides described herein. In some embodiments, the delivery medium is adapted to deliver one or more polynucleotides to one or more target cells.

[0119] In some embodiments, the delivery medium is a virus. Examples of viral delivery media include, but are not limited to, adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, herpes simplex virus, vaccinia virus, or any hybrid virus thereof. In some embodiments, the virus is replication-defective. In some embodiments, the virus is capable of replication. In some embodiments, the virus has been modified to alter its tissue tropism relative to that of an unmodified wild-type virus. Methods for preparing viruses comprising one or more polynucleotides are well known to those skilled in the art.

[0120] In some embodiments, the viral delivery medium is herpes simplex virus. The herpes simplex virus delivery medium can be prepared by methods disclosed, for example, in WO2015 / 009952. In some embodiments, the herpes simplex virus comprises a modified envelope. In some embodiments, the modified envelope comprises one or more (e.g., one, two, three, four, or more) mutated herpes simplex virus glycoproteins. Examples of herpes simplex virus glycoproteins may include, but are not limited to, glycoproteins gB, gD, gH, and gL. In some embodiments, the modified envelope alters the tissue tropism of the herpes simplex virus relative to wild-type herpes simplex virus. In some embodiments, the herpes simplex virus is herpes simplex virus type 1, herpes simplex virus type 2, or any derivative thereof. In some embodiments, the virus is herpes simplex virus type 1. In some embodiments, the virus is herpes simplex virus type 2.

[0121] In some embodiments, the delivery medium is a non-viral delivery medium. In some embodiments, the non-viral delivery medium is a chemical-based delivery medium (a chemical-based delivery reagent). Examples of chemical-based delivery media include, but are not limited to, calcium phosphate, dendrites, liposomes (cationic liposomes, non-cationic liposomes, and mixtures), exogenous bodies, charged lipids, and cationic polymers (such as DEAE-dextran, polyethyleneimine, etc.). In some embodiments, the non-viral delivery medium is a non-chemical delivery medium. Examples of non-chemical delivery media include, but are not limited to, electroporation, nuclear transfection, sonoporosis, optical transfection, and particle-based media (such as gene guns, magnetically assisted transfection, impale infection, particle bombardment, etc.). In some embodiments, the non-viral delivery medium is a dendrite, liposome, exogenous body, charged lipid, or cationic polymer. In some embodiments, the non-viral delivery medium is a dendrite. In some embodiments, the non-viral delivery medium is a liposome. In some embodiments, the non-viral delivery medium is an exogenous body. In some embodiments, the non-viral delivery medium is a charged lipid. In some embodiments, the non-viral delivery medium is a cationic polymer. Methods for generating one or more target polynucleotides in a complex containing a non-viral delivery medium are well known to those skilled in the art.

[0122] Pharmaceutically acceptable carrier

[0123] Certain aspects of this disclosure relate to a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is one sufficient for topical and / or transdermal administration / application. In some embodiments, the pharmaceutically acceptable carrier is one sufficient for subcutaneous and / or intradermal administration / application. In some embodiments, the pharmaceutically acceptable carrier is minimally invasive or non-invasive. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, etc.). Chloride, benzalkonium chloride, benzyl chloride; phenol, butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gel or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; polyols, such as glycerol (e.g., formulations containing 10% glycerol); salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). A more detailed discussion of pharmaceutically acceptable carriers can be found in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub.Co., NJ 1991).

[0124] In some embodiments, pharmaceutically acceptable carriers are suitable for topical or transdermal application / administration. Examples of carriers suitable for topical or transdermal application / administration may include, but are not limited to, ointments, pastes, creams, suspensions, emulsions, fat-based ointments, gels, powders, lotions, solutions, sprays, patches, microneedle arrays, and inhalants. In some embodiments, pharmaceutically acceptable carriers include one or more of ointments, pastes, creams, suspensions, emulsions, fat-based ointments, gels, powders, lotions, solutions, sprays, and inhalants. In some embodiments, pharmaceutically acceptable carriers comprise ointments. In some embodiments, pharmaceutically acceptable carriers comprise pastes. In some embodiments, pharmaceutically acceptable carriers comprise creams. In some embodiments, pharmaceutically acceptable carriers comprise suspensions. In some embodiments, pharmaceutically acceptable carriers comprise emulsions. In some embodiments, pharmaceutically acceptable carriers comprise gels. In some embodiments, pharmaceutically acceptable carriers comprise powders. In some embodiments, pharmaceutically acceptable carriers comprise lotions. In some embodiments, the pharmaceutically acceptable carrier comprises a solution. In some embodiments, the pharmaceutically acceptable carrier comprises a spray. In some embodiments, the pharmaceutically acceptable carrier comprises an inhaler. In some embodiments, the pharmaceutically acceptable carrier comprises a patch (e.g., a patch applied to the skin). In some embodiments, the pharmaceutically acceptable carrier comprises a microneedle array. Methods for preparing and using microneedle arrays suitable for the delivery of pharmaceutical compositions are well known in the art (Kim Y. et al., “Microneedles for drug and vaccine delivery”. Advanced Drug Delivery Reviews 2012, 64 (14): 1547-68).

[0125] In some implementations, a pharmaceutically acceptable carrier comprises a combination of two, three, four, five or more different pharmaceutically acceptable carriers suitable for topical or transdermal application / administration.

[0126] In some embodiments, pharmaceutically acceptable carriers may further comprise one or more additional components. Examples of additional components may include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, or dicalcium phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium glycolate); wetting agents (e.g., sodium dodecyl sulfate); salt solutions; alcohols; polyethylene glycol; gelatin; lactose; amylase; magnesium stearate; talc; silicic acid; viscous paraffin; hydroxymethylcellulose; polyvinylpyrrolidone; sweeteners; flavoring agents; fragrances; coloring agents; humectants; sunscreens; antibacterial agents; agents capable of stabilizing polynucleotides or preventing their degradation, etc.

[0127] The pharmaceutical compositions and formulations described herein can be prepared by mixing a delivery medium comprising one or more polynucleotides described herein with one or more pharmaceutically acceptable carriers. Formulations intended for in vivo administration are typically sterile. Sterility can be readily achieved, for example, through filtration using a sterile filter membrane.

[0128] Treatment

[0129] This disclosure relates in part to pharmaceutical compositions and methods for providing preventive, alleviating, or therapeutic relief of wounds, conditions, or diseases of the skin in a subject. Examples of skin diseases or conditions may include, but are not limited to, one or more of epidermolysis bullosa, skin cancer, psoriasis, lichen planus, lupus, rosacea, eczema, cutaneous candidiasis, cellulitis, impetigo, bedsores, erysipelas, ichthyosis vulgaris, dermatomyositis, acrodermatitis, stasis dermatitis, Natherton's syndrome, epidermolysis bullosa simplex (LAMB3 gene), autosomal recessive congenital ichthyosis, xeroderma pigmentosum, and bullous pemphigoid. In some embodiments, the skin disease or condition is epidermolysis bullosa. In some embodiments, the subject has one or more symptoms of epidermolysis bullosa or is at risk of developing one or more symptoms of epidermolysis bullosa.

[0130] The polynucleotides and pharmaceutical compositions described herein may be used to provide preventative, palliative, or therapeutic relief of wounds, conditions, or diseases of the skin in subjects, including treatment of one or more symptoms of epidermolysis bullosa (e.g., subclinical epidermolysis bullosa, overt epidermolysis bullosa, etc.). The pharmaceutical compositions disclosed herein may be administered by any suitable method known in the art, including but not limited to oral administration, sublingual administration, buccal administration, topical administration, rectal administration, inhalation, transdermal administration, subcutaneous injection, intradermal injection, intravenous (IV) injection, intra-arterial injection, intramuscular injection, intracardiac injection, intraosseous injection, intraperitoneal injection, mucosal administration, vaginal administration, intravitreal administration, intra-articular administration, periarticular administration, topical administration, epidermal administration, or any combination thereof. The pharmaceutical compositions may be delivered to an individual via various routes, including but not limited to subcutaneous, intradermal, topical, transdermal, and mucosal administration. Therefore, this disclosure also covers methods of delivering any of the polynucleotides or pharmaceutical compositions described herein to an individual, such as an individual with epidermolysis bullosa or at risk of developing epidermolysis bullosa.

[0131] In some embodiments, preventative, palliative, or therapeutic relief of a subject's skin wounds, conditions, or diseases is provided, including the administration of an effective amount of a pharmaceutical composition capable of enhancing, increasing, strengthening, and / or reinforcing the levels of collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the subject. In some embodiments, the pharmaceutical composition is administered intradermally or subcutaneously. In some embodiments, the pharmaceutical composition is administered transdermally or topically. In some embodiments, preventative, palliative, or therapeutic relief of a subject's skin wounds, conditions, or diseases is provided, including the topical administration of an effective amount of a pharmaceutical composition capable of enhancing, increasing, strengthening, and / or reinforcing the levels of collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the subject. The pharmaceutical composition may be any pharmaceutical composition described herein. In some embodiments, the individual suffers from epidermolysis bullosa. In some embodiments, the individual suffers from dystrophic epidermolysis bullosa. In some embodiments, the individual has overt dystrophic epidermolysis bullosa. In some embodiments, the individual has latent dystrophic epidermolysis bullosa. In some embodiments, the pharmaceutical composition is applied once, twice, three times, four times, five times, or more daily. In some embodiments, the pharmaceutical composition is applied to one or more affected areas of the individual. In some embodiments, the pharmaceutical composition is applied to one or more unaffected areas of the individual.

[0132] In some embodiments, the pharmaceutical compositions described herein may be used to treat or alleviate one or more symptoms of epidermolysis bullosa. Symptoms of epidermolysis bullosa (e.g., latent dystrophic epidermolysis bullosa, overt dystrophic epidermolysis bullosa, etc.) may include, but are not limited to, blisters on the skin (especially blisters on the hands, feet, knees, and elbows), blisters on the mucous membranes, skin scarring, mucosal scarring, skin erosion, nail and / or toenail deformities, nail and / or toenail loss, internal blistering (including blistering on the vocal cords, esophagus, and upper respiratory tract), thickening of the skin (especially thickening of the skin on the palms and soles), scalp blistering, scalp scarring, hair loss (scarring alopecia), thinning of the skin, atrophic scarring, milia, dental conditions (such as tooth decay and enamel dysplasia), joint deformities, fusion of fingers and toes, and dysphagia.

[0133] In some embodiments, a method of treating an individual suffering from epidermolysis bullosa is provided, the method comprising administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, strengthening, and / or reinforcing the levels of collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the individual. In some embodiments, the pharmaceutical composition is administered intradermally or subcutaneously; in some embodiments, the pharmaceutical composition is administered transdermally or locally. In some embodiments, a method of therapeutically treating an individual suffering from epidermolysis bullosa is provided, the method comprising locally administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, strengthening, and / or reinforcing the levels of collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the individual. The pharmaceutical composition may be any pharmaceutical composition described herein. In some embodiments, the individual suffers from dystrophic epidermolysis bullosa. In some embodiments, the individual suffers from overt dystrophic epidermolysis bullosa. In some embodiments, the individual suffers from subclinical dystrophic epidermolysis bullosa. In some embodiments, the pharmaceutical composition is applied once, twice, three times, four times, five times, or more daily. In some embodiments, the pharmaceutical composition is applied to one or more affected areas of the individual. In some embodiments, the pharmaceutical composition is applied to one or more unaffected areas of the individual.

[0134] In some embodiments, a method of prophylactic treatment for an individual suffering from epidermolysis bullosa is provided, the method comprising administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, strengthening, and / or reinforcing the levels of collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the individual. In some embodiments, the pharmaceutical composition is administered intradermally or subcutaneously; in some embodiments, the pharmaceutical composition is administered transdermally or locally. In some embodiments, a method of prophylactic treatment for an individual suffering from epidermolysis bullosa is provided, the method comprising locally administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, strengthening, and / or reinforcing the levels of collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the individual. The pharmaceutical composition may be any pharmaceutical composition described herein. In some embodiments, the individual suffers from dystrophic epidermolysis bullosa. In some embodiments, the individual suffers from overt dystrophic epidermolysis bullosa. In some embodiments, the individual suffers from subclinical dystrophic epidermolysis bullosa. In some embodiments, the pharmaceutical composition is applied once, twice, three times, four times, five times, or more daily. In some embodiments, the pharmaceutical composition is applied to one or more affected areas of the individual. In some embodiments, the pharmaceutical composition is applied to one or more unaffected areas of the individual.

[0135] In some embodiments, a method is provided for the prophylactic treatment of an individual at risk of developing epidermolysis bullosa, the method comprising administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, strengthening, and / or reinforcing the levels of collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the individual. In some embodiments, the pharmaceutical composition is administered intradermally or subcutaneously; in some embodiments, the pharmaceutical composition is administered transdermally or locally. In some embodiments, a method is provided for the prophylactic treatment of an individual at risk of developing epidermolysis bullosa, the method comprising administering locally an effective amount of a pharmaceutical composition capable of enhancing, increasing, strengthening, and / or reinforcing the levels of collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the individual. The pharmaceutical composition may be any pharmaceutical composition described herein. In some embodiments, the individual is at risk of developing dystrophic epidermolysis bullosa. In some embodiments, the individual is at risk of developing overt dystrophic epidermolysis bullosa. In some embodiments, an individual is at risk of developing subclinical dystrophic epidermolysis bullosa. In some embodiments, the pharmaceutical composition is applied once, twice, three times, four times, five times, or more daily. In some embodiments, the pharmaceutical composition is applied to one or more affected areas of an individual. In some embodiments, the pharmaceutical composition is applied to one or more unaffected areas of an individual.

[0136] In some embodiments, administration of an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, strengthens, and / or reinforces the levels of collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the individual. In some embodiments, administration of an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, strengthens, and / or reinforces the function of collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the individual. In some embodiments, administration of an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, strengthens, and / or reinforces the activity of collagen α-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the individual.

[0137] In some embodiments, application of an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, strengthens, and / or reinforces the formation of anchoring fibrils. In some embodiments, application of an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, strengthens, and / or reinforces the epithelial basement membrane tissue. In some embodiments, application of an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, strengthens, and / or reinforces the epithelial basement membrane adhesion. In some embodiments, application of an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, strengthens, and / or reinforces the dermal-epidermal junction integrity. In some embodiments, application of an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, strengthens, and / or reinforces wound healing. Without wishing to be bound by theory, it is believed that increasing, strengthening, and / or reinforcing the level of collagen α-1 (VII) chain polypeptides in one or more cells of an individual by applying one or more of the pharmaceutical compositions described herein will allow for increased production and secretion of functional collagen α-1 (VII) chain proteins in the individual. Without being bound by theory, it is believed that increasing, enhancing, and / or strengthening the level of lysyl hydroxylase 3 peptide in one or more cells of an individual by administering one or more of the pharmaceutical compositions described herein will increase post-translational modifications of collagen α-1(VII) chain peptides in the individual, enhancing the production and / or secretion of functional collagen α-1(VII) chain protein. Without being bound by theory, it is further believed that increasing, enhancing, and / or strengthening the levels of collagen α-1(VII) chain peptide and lysyl hydroxylase 3 peptide in the same cells of an individual by administering one or more of the pharmaceutical compositions described herein (by contacting cells with two separate polynucleotides expressing peptides, by contacting cells with a single consecutive polynucleotide expressing two peptides alone, or by contacting cells with a single consecutive polynucleotide expressing a chimeric peptide) will have an additive effect on enhancing the production and secretion of functional collagen α-1(VII) chain protein. Without being bound by theory, it is believed that increased production and secretion of functional collagen α-1(VII) chain protein will allow for improved anchoring fibrillation in the individual, aiding in tissue, stabilization, and assisting in the adhesion of the epithelial basement membrane. Without being bound by theory, it is believed that this will ultimately increase dermal-epidermal stability in individuals with epidermolysis bullosa, treat existing wounds, and prevent or delay the recurrence of wounds in the treated area.

[0138] Isolated polynucleotides and polypeptides

[0139] Some aspects of this disclosure relate to isolated polynucleotides comprising a polynucleotide encoding a collagen α-1(VII) chain polypeptide. Other aspects of this disclosure relate to isolated polynucleotides comprising a polynucleotide encoding a lysine hydroxylase 3 polypeptide. Still other aspects of this disclosure relate to isolated polynucleotides comprising a polynucleotide encoding a keratin type I cytoskeleton 17 polypeptide.

[0140] Other aspects of this disclosure relate to isolated polynucleotides comprising a polynucleotide encoding a collagen α-1 (VII) chain polypeptide and a polynucleotide encoding a lysine hydroxylase 3 polypeptide, separated by a polynucleotide encoding a linker polypeptide. In some embodiments, the isolated polynucleotide encodes a chimeric polypeptide comprising a collagen α-1 (VII) chain polypeptide, a linker polypeptide, and a lysine hydroxylase 3 polypeptide.

[0141] In some embodiments, the polynucleotide encoding the adaptor polypeptide further comprises a polynucleotide encoding one or more furin cleavage sites. In some embodiments, the one or more furin cleavage sites are encoded upstream of the adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the T2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the T2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the P2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the P2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the E2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the E2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the F2A adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the F2A adaptor polypeptide.

[0142] An example of a polynucleotide encoding a collagen α-1 (VII) chain polypeptide is SEQ ID NO: 1. Polynucleotides encoding collagen α-1 (VII) chain polypeptides also include polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1.

[0143] An example of a polynucleotide encoding a lysyl hydroxylase 3 polypeptide is SEQ ID NO: 3. Polynucleotides encoding a lysyl hydroxylase 3 polypeptide also include polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 3.

[0144] An example of a polynucleotide encoding a keratin type I cytoskeleton 17 polypeptide is SEQ ID NO: 29. Polynucleotides encoding a keratin type I cytoskeleton 17 polypeptide also include polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 29.

[0145] Examples of polynucleotides encoding adapter polypeptides are SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11. Polynucleotides encoding adapter polypeptides also include polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

[0146] Examples of polynucleotides encoding chimeric polypeptides comprising collagen α-1 (VII) chain polypeptide, adaptor polypeptide, and lysyl hydroxylase 3 polypeptide are SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27. The polynucleotide encoding the chimeric polypeptide also includes polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27.

[0147] Other aspects of this disclosure relate to one or more (e.g., one or more, two or more, three or more, etc.) isolated polynucleotides described herein contained within a vector. In some embodiments, the vector is an adenovirus vector, an adeno-associated virus vector, a retroviral vector, a lentiviral vector, a herpes simplex virus vector, a vaccinia virus vector, or any hybrid viral vector thereof. In some embodiments, the vector is a herpes simplex virus vector. In some embodiments, the vector contains one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) transgenes.

[0148] In some embodiments, the herpes simplex virus vector is a herpesvirus amplicon vector. In some embodiments, the vector is an HSV-1 amplicon. In some embodiments, the vector is an HSV-1 heterozygous amplicon. Examples of HSV-1 heterozygous amplicon may include, but are not limited to, HSV / AAV heterozygous amplicon, HSV / EBV heterozygous amplicon, HSV / EBV / RV heterozygous amplicon, and HSV / Sleeping Beauty heterozygous amplicon. In some embodiments, the vector is an HSV / AAV heterozygous amplicon. In some embodiments, the vector is an HSV / EBV heterozygous amplicon. In some embodiments, the vector is an HSV / EBV / RV heterozygous amplicon. In some embodiments, the vector is an HSV / Sleeping Beauty heterozygous amplicon. Other aspects of this disclosure relate to a method for preparing a viral delivery medium containing one or more polynucleotides described herein. In some embodiments, the method includes contacting a host cell with one or more viral vectors containing one or more isolated polynucleotides described herein and collecting the viral delivery medium generated from the host cell. Methods for culturing cells and exposing cells to one or more target viral vectors (e.g., through transduction or transfection) are well known to those skilled in the art.

[0149] In some embodiments, the herpes simplex virus vector is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome has been engineered to reduce or eliminate the expression of one or more virulent herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations. Examples of inactivating mutations may include, but are not limited to, deletions (e.g., deletions of gene coding sequences or deletions of one or more gene transcriptional regulatory elements), insertions, point mutations, and rearrangements. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in one or more immediate early genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41, and UL55 herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome contains an inactivating mutation in the ICP0 gene. In some embodiments, the recombinant herpes simplex virus genome contains an inactivating mutation in the ICP4 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in the ICP27 gene. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP4, ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome includes inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-2 genome.

[0150] In some embodiments, the isolated recombinant herpes simplex virus genome contains one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) polynucleotides (e.g., transgenes) of this disclosure within one, two, three, four, seven or more viral loci. Examples of suitable viral loci may include, but are not limited to, the herpes simplex virus loci ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41, and UL55. In some embodiments, the isolated recombinant herpes simplex virus genome contains one or more polynucleotides of this disclosure within one or more viral ICP4 loci (e.g., the recombinant virus carries a polynucleotide encoding Col7 in one or two ICP4 loci; the recombinant virus carries a polynucleotide encoding LH3 in one or two ICP4 loci; the recombinant virus carries a polynucleotide encoding KRT17 in one or two ICP4 loci; the recombinant virus carries a polynucleotide encoding Col7 in one ICP4 locus and a polynucleotide encoding KRT17 in another ICP4 locus; the recombinant virus carries a polynucleotide encoding Col7 in one ICP4 locus and a polynucleotide encoding LH3 in another ICP4 locus; the recombinant virus carries a polynucleotide encoding LH3 in one ICP4 locus and a polynucleotide encoding KRT17 in another ICP4 locus, etc.). In some embodiments, the isolated recombinant herpes simplex virus genome contains one or more polynucleotides of this disclosure within the viral UL41 locus. In some embodiments, the isolated recombinant herpes simplex virus genome contains one or more polynucleotides of this disclosure within the viral ICP47 locus.In some embodiments, the isolated recombinant herpes simplex virus genome contains one or more polynucleotides of this disclosure within one or more viral ICP4 loci and within the viral UL41 locus (e.g., the recombinant virus carries a polynucleotide encoding Col7 in one or two ICP4 loci and a polynucleotide encoding LH3 in the UL41 locus; the recombinant virus carries a polynucleotide encoding Col7 in one or two ICP4 loci and a polynucleotide encoding Col7 in the UL41 locus; the recombinant virus carries a polynucleotide encoding Col7 in one or two ICP4 loci and a polynucleotide encoding KRT17 in the UL41 locus; the recombinant virus carries a polynucleotide encoding LH3 in one or two ICP4 loci and a polynucleotide encoding KRT17 in the UL41 locus). The recombinant virus carries a polynucleotide encoding LH3; a recombinant virus carries a polynucleotide encoding LH3 in one or two ICP4 loci and a polynucleotide encoding Col7 in the UL41 locus; a recombinant virus carries a polynucleotide encoding LH3 in one or two ICP4 loci and a polynucleotide encoding KRT17 in the UL41 locus; a recombinant virus carries a polynucleotide encoding KRT17 in one or two ICP4 loci and a polynucleotide encoding LH3 in the UL41 locus; a recombinant virus carries a polynucleotide encoding KRT17 in one or two ICP4 loci and a polynucleotide encoding Col7 in the UL41 locus; a recombinant virus carries a polynucleotide encoding KRT17 in one or two ICP4 loci and a polynucleotide encoding KRT17 in the UL41 locus, etc.

[0151] In some respects, the isolated polynucleotides described herein are contained within synthetic mRNA. In some embodiments, the synthetic mRNA comprises one or more modified ribonucleotides.

[0152] Some aspects of this disclosure relate to isolated polypeptides comprising collagen α-1 (VII) chain polypeptides. Other aspects of this disclosure relate to isolated polypeptides comprising lysyl hydroxylase 3 polypeptides. Still other aspects of this disclosure relate to isolated polypeptides comprising keratin type I cytoskeleton 17 polypeptides.

[0153] Other aspects of this disclosure relate to isolated chimeric polypeptides comprising a collagen α-1 (VII) chain polypeptide and a lysine hydroxylase 3 polypeptide separated by a linker polypeptide.

[0154] In some embodiments, the adaptor polypeptide further comprises one or more furin cleavage sites. In some embodiments, the amino acid sequence of the furin cleavage site is identical or substantially similar to that of the classic furin cleavage site (Arg-X-(Arg / Lys)-Arg). In some embodiments, the one or more furin cleavage sites are located at the N-terminus of the adaptor polypeptide. In some embodiments, the one or more furin cleavage sites are located at the C-terminus of the adaptor polypeptide. In some embodiments, the adaptor polypeptide comprises one or more furin cleavage sites and a T2A adaptor polypeptide from the N-terminus to the C-terminus. In some embodiments, the adaptor polypeptide comprises a T2A adaptor polypeptide and one or more furin cleavage sites from the N-terminus to the C-terminus. In some embodiments, the adaptor polypeptide comprises one or more furin cleavage sites and a P2A adaptor polypeptide from the N-terminus to the C-terminus. In some embodiments, the adaptor polypeptide comprises a P2A adaptor polypeptide and one or more furin cleavage sites from the N-terminus to the C-terminus. In some embodiments, the adaptor polypeptide comprises one or more furin cleavage sites and an E2A adaptor polypeptide from the N-terminus to the C-terminus. In some embodiments, the adaptor polypeptide comprises an E2A adaptor polypeptide and one or more furin cleavage sites from the N-terminus to the C-terminus. In some embodiments, the adaptor polypeptide comprises one or more furin cleavage sites and an F2A adaptor polypeptide from the N-terminus to the C-terminus. In some embodiments, the adaptor polypeptide comprises an F2A adaptor polypeptide and one or more furin cleavage sites from the N-terminus to the C-terminus.

[0155] In some aspects, the isolated polypeptide comprising a collagen α-1 (VII) chain polypeptide comprises the amino acid sequence of SEQ ID NO: 2. The isolated polypeptide may also comprise a collagen α-1 (VII) chain polypeptide having an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 2.

[0156] In some aspects, the isolated polypeptide comprising the lysyl hydroxylase 3 polypeptide comprises the amino acid sequence of SEQ ID NO: 4. The isolated polypeptide may also comprise the lysyl hydroxylase 3 polypeptide, the amino acid sequence of which has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 4.

[0157] In some aspects, the isolated polypeptide comprising the keratin type I cytoskeleton 17 polypeptide comprises the amino acid sequence of SEQ ID NO: 30. The isolated polypeptide may also comprise the keratin type I cytoskeleton 17 polypeptide, the amino acid sequence of which has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 30.

[0158] In some aspects, the chimeric polypeptide comprises a collagen α-1 (VII) chain polypeptide containing the amino acid sequence of SEQ ID NO: 2. The chimeric polypeptide may also comprise a collagen α-1 (VII) chain polypeptide having an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2.

[0159] In some aspects, the chimeric polypeptide comprises a lysyl hydroxylase 3 polypeptide containing the amino acid sequence of SEQ ID NO: 4. The chimeric polypeptide may also comprise a lysyl hydroxylase 3 polypeptide having an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 4.

[0160] In some aspects, the chimeric polypeptide comprises a linker polypeptide containing the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. The chimeric polypeptide may also comprise a linker polypeptide whose amino acid sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.

[0161] In some respects, the chimeric polypeptide is an amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28. The chimeric polypeptide may also be an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28.

[0162] host cells

[0163] Certain aspects of this disclosure relate to one or more host cells that contain a vector comprising the polynucleotides described herein. In some embodiments, the vector is any isolated recombinant herpes simplex virus vector described herein. In some embodiments, the host cell is a bacterial cell (e.g., *Escherichia coli* cells, etc.). In some embodiments, the host cell is a fungal cell (e.g., *Saccharomyces cerevisiae* cells, etc.). In some embodiments, the host cell is an insect cell (e.g., S2 cells, etc.). In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a cell derived from a cell line. Examples of suitable host cells or cell lines may include, but are not limited to, 293, HeLa, SH-Sy5y, Hep G2, CACO-2, A549, L929, 3T3, K562, CHO-K1, MDCK, HUVEC, Vero, N20, COS-7, PSN1, VCaP, CHO cells, etc. In some embodiments, the vector is an adenovirus vector, an adeno-associated virus vector, a retroviral vector, a lentiviral vector, a herpes simplex virus vector, a vaccinia virus vector, or any hybrid viral vector thereof. In some embodiments, the vector is a herpes simplex virus vector. In some embodiments, the vector is an HSV-1 amplicon or an HSV-1 heterozygous amplicon. In some embodiments, the host cell contains a helper virus. In some embodiments, the host cell containing the helper virus is contacted with the vector described herein. In some embodiments, contacting the host cell containing the helper virus with the HSV-1 amplicon or HSV-1 heterozygous amplicon described herein results in the production of a virus containing one or more vectors described herein. In some embodiments, the virus is collected from the supernatant of the contacted host cell. Methods for generating a virus by contacting the host cell containing the helper virus with the HSV-1 amplicon or HSV-1 / heterozygous amplicon are known in the art. In some embodiments, the host cell is a complementary host cell. In some embodiments, the complementary host cell expresses one or more genes inactivated in any viral vector described herein. In some embodiments, the complementary host cell is contacted with the recombinant herpes simplex virus genome described herein. In some embodiments, contacting a complementary host cell with the recombinant herpes simplex virus genome described herein results in the production of a virus comprising one or more vectors described herein. In some embodiments, the virus is collected from the supernatant of the contacted host cell. Methods for generating a virus by contacting a complementary host cell with recombinant herpes simplex virus are generally described in WO2015 / 009952.

[0164] Products or medicine boxes

[0165] Certain aspects of this disclosure relate to an article of manufacture or a package containing the pharmaceutical composition described herein. In some embodiments, the article of manufacture or package includes instructions for administering the pharmaceutical composition to provide preventative, astringent, or therapeutic relief of a wound, symptom, or disease of the skin in a subject.

[0166] In some embodiments, a delivery medium containing one or more polynucleotides described herein and a pharmaceutically acceptable carrier are contained in the same or separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers can be formed from a variety of materials such as glass, plastics (such as polyvinyl chloride or polyolefins), or metal alloys (such as stainless steel or Hastelloy). In some embodiments, the container includes a label on or associated with the container, wherein the label indicates the direction of use. The article or kit may also include other materials that are commercially and user-desirable, including additional buffers, diluents, filters, etc.

[0167] This specification is intended to enable those skilled in the art to practice the invention. Various modifications to the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0168] Example

[0169] This disclosure will be more fully understood by referring to the following embodiments. However, it should not be construed as limiting the scope of this disclosure. It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and modifications or changes to these embodiments and implementations will be conceived by those skilled in the art and are included within the spirit and limits of this application and the scope of the appended claims.

[0170] Example 1: Generation of modified herpes simplex virus vector, and production / isolation of virus containing the vector.

[0171] To prepare a modified herpes simplex virus genome vector capable of expressing one or more transgenes in target mammalian cells, the herpes simplex virus genome was modified ( Figure 1A This process inactivates the immediate early genes ICP0, ICP4, and ICP27, while modifying the immediate early gene ICP22 to include a heterologous inducible promoter. This reduces the toxicity of the genome in mammalian cells. Next, the cassette is inserted into the modified herpesvirus genome via restriction cloning. This cassette contains a heterologous promoter capable of expressing the transgene in target mammalian cells. The promoter is operatively linked to a nucleic acid sequence encoding a collagen α-1 (VII) chain polypeptide and downstream regulatory elements. Figure 1BThis ensures the correct production of mRNA. Alternatively, the kit includes two transgenes, each with its own heterologous promoter, which is operatively linked to a nucleic acid encoding either a collagen α-1(VII) chain polypeptide or a lysine hydroxylase 3 polypeptide. These transgenes encode ( ) on the same DNA strand. Figure 1C ), or encoded in an antisense orientation on the opposite DNA strand ( Figure 1D Linking each transgene to its own promoter and regulatory elements allows each coding sequence to be expressed independently on a separate mRNA transcript. Expressing transgenes from different promoters enables the operative linking of coding sequences to different promoter types, which can drive transgene expression at different levels, at different times in the cell cycle, in different cell types, or under the control of different inducers or repressors.

[0172] A modified herpesvirus genome was also constructed, comprising a cassette expressing a single mRNA encoding a collagen α-1 (VII) chain polypeptide separated by an internal ribosome entry site and a lysine hydroxylase 3 polypeptide. Figure 1E This allows each polypeptide to be produced approximately equimolarly when expressed in target cells. Finally, a modified herpesvirus genome comprising a cassette expressing the chimeric polypeptide was constructed. This chimeric polypeptide comprises, from the N-terminus to the C-terminus, a collagen α-1 (VII) chain polypeptide, a cleavable peptide linker, and a lysine hydroxylase 3 polypeptide. Figure 1F ).

[0173] A modified herpesvirus genome was constructed, comprising two cassettes, each expressing a collagen α-1(VII) chain polypeptide, wherein each cassette is inserted into the ICP4 locus of the wild-type herpes simplex virus genome. Figures 2B-2G In the copy of () Figure 2A These additional recombinant herpesvirus genomes constructed have various combinations of herpesvirus gene deletions / modifications.

[0174] The constructed recombinant herpesvirus genome contained deletions of two copies of the coding sequence for the ICP4 gene, as well as deletions of the coding sequences for the ICP27 and UL55 genes. These recombinant viruses were further modified to contain inactivation mutations in the promoter regions of the ICP22 and ICP47 genes, preventing the ICP22 and ICP47 genes from being expressed kinetically. Figure 2B ).

[0175] Another recombinant herpes simplex virus was constructed, incorporating an expression cassette for the collagen α-1 (VII) chain polypeptide into two loci of the herpes ICP4 gene. These recombinant viruses included viruses with the following deletions: deletion of the coding sequence of the ICP22 gene and deletion of the coding sequence of two copies of the ICP4 gene. Figure 2C); Deletion of the coding sequence of the ICP0 gene and the coding sequences of two copies of the ICP4 gene ( Figure 2D ); Deletion of the coding sequences of the ICP0 and ICP22 genes and the coding sequences of two copies of the ICP4 gene ( Figure 2E ); Deletion of the coding sequences of ICP0, ICP22 and ICP27 genes and two copies of the coding sequence of ICP4 gene ( Figure 2F ); and deletions of the coding sequences of the ICP0, ICP22, ICP27 and UL55 genes, and two copies of the coding sequence of the ICP4 gene. Figure 2G ).based on Figures 2C-2G The vector shown can be used to construct another vector, which further contains one or more transgenes encoding one or more additional effectors (e.g., LH3, KRT17) at the ICP0 and / or UL41 loci.

[0176] These modified herpes simplex virus genome vectors were transfected into engineered Vero cells modified to express herpes simplex virus genes. These engineered Vero cells secreted supernatants containing replication-defective herpes simplex virus with the modified genome. The supernatants were then collected, concentrated, and sterilely filtered through a 5 μm filter.

[0177] Example 2: Rescuing Col7 expression using replication-defective HSV-1

[0178] The following examples describe the construction of a replication-deficient type 1 herpes simplex virus modified to express the human COL7A1 gene, and the use of this viral vector to rescue several defects observed in cells isolated from RDEB patients.

[0179] method

[0180] Cells and cell culture

[0181] As previously described, normal and RDEB human skin fibroblasts and keratinocytes were isolated (NG, YZ et al. (2012) Cancer Res. 72: 3522-3534; Rheinwald, JG and Green, H. (1975) Cell 6: 331-42). Cells were cultured according to standard techniques.

[0182] KB103 Construction

[0183] The KB103 vector was generated from D3GFP, a replication-defective HSV-1 vector backbone in which GFP replaced the viral ICP4. Using a transfer plasmid, the coding sequence of human COL7A1 was substituted for the GFP sequence in D3GFP by cloning COL7A1 into the EcoRI site of the ICP4 recombinant plasmid pSASB3. Co-transfection / infection with the transfer plasmid containing COL7A1 and the D3GFP vector was performed on VeroD cells. Plaques that did not express GFP were isolated, and Col7 protein expression was tested by Western blotting.

[0184] Virus purification

[0185] Purify the KB103 virus according to standard techniques (see Diefenbach, R. and Fraefel, C. Herpes Simplex Virus. New York: Humana Press, 2014).

[0186] Viral infection

[0187] One day prior to viral infection, cells were seeded in duplicate or triplicate at 50% confluence into six-well plates. Additional wells were seeded in parallel for cell counting and MOI determination. Twenty-four hours post-seeding, cells in one well were trypsinized and counted to calculate the MOI. The viral stock solution was thawed and diluted in cell culture medium to achieve the desired MOI. Culture medium was aspirated from each well for infection, and 500 μL of KB103-containing medium (or control medium) was added to each well. The plates were incubated at 37°C and 5–7.5% CO2 for 1.5–2 hours, with intermittent shaking every 15–20 minutes. Then, 1.5–2 mL of complete cell culture medium was added to each well, and the plates were incubated at 37°C for 24–72 hours. After incubation, cells and supernatant were harvested and processed for analysis.

[0188] mRNA quantification

[0189] Following the manufacturer's instructions, Col7 transcripts were amplified from RNA isolated from primary RDEB keratinocytes post-infection using the SYBR PCR assay (Sybr Select Master Mix, LifeTechnologies). Col7 transcript levels were normalized to β-actin transcript levels.

[0190] Western blot analysis

[0191] Cell lysates were generated from the cells 48 hours post-infection and Western blotted using the following antibodies according to standard techniques: rabbit anti-human Col7 polyclonal antibody (Sigma, Cat.# HPA042420), mouse anti-human GAPDH antibody (SantaCruz Biotechnology, Cat.# sc-365062), rabbit anti-LH3 antibody (Protein Tech, Cat.# 11027-1-AP), and mouse anti-TSP1 antibody (SantaCruz Biotechnology, Cat.# sc-59887).

[0192] Immunofluorescence

[0193] Cells were seeded onto coverslips before infection, fixed 48 hours post-infection, and stained with primary rabbit anti-human Col7 polyclonal antibody (Sigma, Cat.# HPA042420). After washing, cells were further stained with fluorescently labeled anti-rabbit secondary antibody (Invitrogen, Cat.3 A11012). Cell nuclei were stained with DAPI using standard techniques.

[0194] Cell adhesion

[0195] Spread 96-well plates overnight at 4°C with 10, 20, or 50 μg / mL rat tail collagen 1 (Marathon Laboratory Supply) or human fibronectin (Sigma-Aldrich) in a reaction volume of 100 μL, then wash with PBS and block with PBS + 0.1% BSA at 37°C for 1 hour. Simulated (control) or KB103-infected RDEB keratinocytes (2.4 x 10⁻⁶ cells) in 100 μL of DMEM / HamF12 + 0.1% BSA... 4 Add cells to the plate and incubate at 37°C for 40–90 minutes. Wash the wells three times with PBS to remove any unbound cells, and fix the adherent cells with PFE for 20 minutes. Then treat the fixed cells with 70% ethanol, stain with crystal violet, decompose in 100% ethanol, and quantify by measuring absorbance at 630 nm.

[0196] Skin equivalent (SE) organoid culture

[0197] Skin-equivalent organoid cultures consisting of RDEB fibroblasts and keratinocytes were used to assess Col7 expression in the basement membrane zone (BMZ). Briefly, RDEB fibroblasts (2 x 10⁶ cells per well) were used. 5100 RDEB keratinocytes were embedded in a fibrin gel matrix in a six-well plate and incubated for 24 hours at 37°C and 5% CO2 in DMEM containing ascorbic acid and aprotinin. 6 Cells were seeded onto a substrate and grown to confluence in DMEM / F-12 keratinocyte medium containing 50 mg / mL ascorbic acid, and cultured at the gas-liquid interface. After two days of culture, KB103 virus was added to the culture (MOI 3) and incubated for 1.5 hours. After incubation, the culture was washed and incubated for 5–14 days to promote stratification and differentiation into epithelial cells. Skin equivalents (SEs) were manually separated from the plates and embedded in compounds at the optimal cutting temperature, frozen in liquid nitrogen, and cut into 6 mm sections for immunofluorescence staining with monoclonal anti-Col7 antibody.

[0198] result

[0199] Pharmacology of KB103 in normal and RDEB cells

[0200] Numerous ex vivo methods have been employed to deliver the human COL7A1 gene into primary cells isolated from RDEB patients in an attempt to correct for Col7 deficiency (Ortiz-Urda, S. et al. (2003) J. Clin. Invest. 111(2) 251-5; Woodley, DT et al. (2003) J. Invest. Dematol. 121(5) 1021-8). Despite the successful achievement of durable correction for key disease features, ex vivo gene delivery strategies for treating epidermolysis bullosa have several major drawbacks, including high cost, poor grafting, complex surgical debridement, bandaging and wound care, and a high risk of postoperative infection. An attractive alternative for gene therapy is the use of viral or non-viral vectors to deliver gene products. However, non-viral vectors using plasmid DNA have very low gene transfer efficiency when injected or administered topically, while the most widely used viral vectors in human gene therapy trials (retroviral vectors) do not infect non-dividing cells. This poses a problem for gene delivery to the skin, as retroviral gene therapy requires manipulation of tissue (such as wounds) to generate a sufficient population of dividing cells. High-capacity adenoviral vectors can deliver genome-sized transcription units and survive for extended periods in transduced cells, but the toxicity and immunogenicity of adenoviral particles, as well as the need for helper viruses during vector preparation, remain significant obstacles to their use in human gene therapy strategies. While replication-deficient HSV vectors have been used as delivery media in many preclinical studies, there is currently no reported preclinical evidence supporting the use of HSV-based viral vectors for epidermolysis bullosa or other dermatological applications.

[0201] To this end, a replication-deficient herpes simplex virus type 1 (HSV-1) encoding the human COL7A1 gene was developed as a novel vector for gene therapy in patients with DEB. The HSV-1 virus was modified to completely delete the viral ICP4, ICP27, and UL55 genes. ICP4 deletion resulted in the removal of the upstream promoter sequence, driving the transcription of the immediate early viral genes ICP22 and ICP47. The virus was further modified to include a human COL7A1 expression cassette driven by the human cytomegalovirus (HCMV) immediate early promoter, which encodes the human COL7A1 gene in two copies at the deleted ICP4 locus, resulting in a replication-deficient HSV-1 vector (designated KB103) suitable for delivering human COL7A1 to target cells. Figure 3 ).

[0202] To test the ability of KB103 to deliver and express Col7 in human cells, and to rescue Col7 deficiency in RDEB patients, patient-derived human skin fibroblasts and keratinocytes were isolated from healthy individuals and individuals with RDEB. These primary cells were then infected with KB103 at various MOIs. 24–72 hours post-infection, COL7A1 gene expression was measured in transduced cells by real-time PCR, while Col7 protein expression was analyzed in parallel by Western blotting and immunofluorescence.

[0203] RDEB keratinocytes infected with KB103 ( Figure 4A ) and fibroblasts ( Figure 4B A dose-dependent increase in COL7A1 gene expression was observed in KB103 infection. KB103 infection increased COL7A1 gene expression in RDEB keratinocytes infected with MOIs of 0.3, 1, and 3 by approximately 7.5-fold, 12.5-fold, and 25-fold, respectively. Figure 4A Surprisingly, more dramatic changes in COL7A1 gene expression were observed in infected RDEB fibroblasts. While infection at MOIs of 0.1 and 0.3 showed a moderate increase in COL7A1 gene expression, measurements showed an approximately 30-fold increase in COL7A1 gene expression in RDEB fibroblasts infected at an MOI of 1, and a 60-fold increase was observed in this cell type infected at an MOI of 3. This data indicates a significant upregulation of COL7A1 gene expression in primary RDEB cells following infection with KB103.

[0204] Therefore, robust Col7 protein expression was also observed in cells infected with KB103. At 48 hours post-infection with KB103 at MOIs of 0.3, 1, and 3, robust Col7 protein expression was observed in both normal and RDEB keratinocytes (…). Figure 5A ) and fibroblasts ( Figure 5BCol7 protein expression was detected in all samples, with an apparent dose-dependent increase in Col7 protein expression observed at higher viral titers. Col7 expression was observed in both the supernatant and cell lysate from infected cells. Surprisingly, RDEB fibroblasts infected with 0.3 MOI showed higher levels of Col7 than those observed in uninfected normal fibroblasts. Figure 5B This indicates that KB103 completely rescues Col7 expression in RDEB fibroblasts, even at low viral titers. High viral doses (MOI 3) had no significant effect on cell morphology. Furthermore, as determined by GAPDH expression, no negative impact of high-dose KB103 on fibroblast or keratinocyte proliferation was shown in these experiments.

[0205] Consistent with the experiments described above, as demonstrated by immunofluorescence assays of Col7 protein expression, robust and dose-dependent increases in Col7 protein expression were confirmed in both normal and RDEB cells infected with KB103. Figure 6 As expected, Col7 protein was not detected in uninfected RDEB human skin fibroblasts or keratinocytes; limited Col7 protein was detected in uninfected normal keratinocytes and fibroblasts. However, infection with KB103 rescued Col7 protein expression in RDEB fibroblasts and keratinocytes, bringing it to levels equal to or higher than those observed in uninfected normal cells. Furthermore, based on assessments by three or more independent groups of each infected replica, the infection efficiency of KB103 (MOI 3) was calculated to be ≥95%, indicating that KB103 effectively delivers and expresses the COL7A1 expression cassette. In conclusion, these data demonstrate that KB103 is capable of delivering and expressing COL7A1 in both normal and RDEB primary cells, and that KB103 is well tolerated by human skin fibroblasts and keratinocytes.

[0206] Functional assessment of KB103 in RDEB cells

[0207] Next, the function of human Col7 protein expressed by KB103 was investigated in human skin fibroblasts and keratinocytes. First, the effect of Col7 expression on lysyl hydroxylase 3 (LH3) levels was tested in KB103-infected cells. LH3 is essential for extracellular matrix deposition and tissue organization, and its levels have been reported to be reduced in RDEB skin (Watt, SA et al. (2015) PLoS One 10(9): p. e0137639). Little or no LH3 was observed in uninfected RDEB keratinocytes compared to normal keratinocytes. Figure 7The lane ratio was 1 to 5, consistent with previous studies. However, unexpectedly, in RDEB keratinocytes infected with KB103, a dose-dependent increase in LH3 levels was observed alongside increased Col7 protein expression. Figure 7 This indicates that KB103 can not only rescue Col7 protein expression in RDEB cells, but also LH3 expression.

[0208] Next, the effect of Col7 expression on TSP-1 levels was tested. TSP-1 is a negative regulator of angiogenesis and has been reported to be increased in RDEB fibroblasts (Ng, YZ et al. (2012) Cancer Res. 72(14): 3522-34). Consistent with previous studies, higher levels of TSP-1 were observed in uninfected RDEB compared to normal human skin fibroblasts. Figure 8 Lanes 1 and 4). Surprisingly, TSP-1 protein expression was strongly suppressed after infection of normal or RDEB fibroblasts with KB103 (lanes 1 and 4). Figure 8 This data indicates that KB103 can not only increase the levels of Col7 and LH3 in infected cells, but also promote angiogenesis by inhibiting the negative regulator TSP-1.

[0209] Finally, KB103 was tested to increase the ability of RDEB keratinocytes to adhere to collagen 1 or fibronectin. A dose-dependent increase in cell adhesion to collagen 1 and fibronectin was observed in RDEB keratinocytes infected with KB103 at various MOIs. Figure 9A and Figure 9B RDEB-infected keratinocytes, performed at all MOIs, showed significantly higher adhesion to pores treated with both substrates at all concentrations compared to uninfected (control) cells. In summary, these data indicate that the human Col7 protein expressed by KB103 is functional in transduced cells. This protein's functionality is indicated by its ability to increase LH3 protein levels, decrease TSP-1 protein levels, and improve cell adhesion with collagen 1 and fibronectin relative to the simulated infection sample.

[0210] Pharmacology and toxicity of KB103 in RDEB organoid cultures

[0211] Skin equivalent (SE) organoid cultures composed of RDEB fibroblasts and keratinocytes were used to assess Col7 protein expression in the basement membrane zone (BMZ) by KB103. RDEB fibroblasts and keratinocytes were either infected with KB103 at an MOI of 3 or mimicked infection and incubated for 5 days to promote stratification and differentiation into epithelial cells. The resulting skin equivalents (SEs) were isolated, sectioned, and stained for immunofluorescence analysis to detect Col7 protein expression. Col7 expression was detected in these organoid cultures from cells infected with KB103, and Col7 protein deposition was observed in the BMZ compared to the mimicked infection control. Figure 10 This data indicates that not only can KB103 effectively deliver COL7A1 and express the Col7 protein, but the Col7 protein also begins to organize in organoid cultures with an organization pattern similar to the expected organization pattern of the Col7 protein in vivo.

[0212] In summary, these experiments demonstrate for the first time that replication-defective HSV-1 vectors can be used as mediators for delivering the COL7A1 expression cassette into primary cells isolated from patients with epidermolysis bullosa. Furthermore, these data reveal that the Col7 protein can be expressed at high levels by this expression cassette in two distinct human cell types: healthy individuals and those with dermatological conditions. Finally, the Col7 protein was demonstrated to be functional, as it increases LH3 expression, decreases TSP-1 expression, increases cell adhesion with collagen 1 and fibronectin, and can be organized in organoid cultures with a tissue pattern similar to that of Col7 in vivo. Without being bound by theory, the data presented in this paper suggest that KB103 and other HSV-1 vectors can be used as novel in vivo therapeutic strategies for epidermolysis bullosa and / or other dermatological applications.

[0213] Example 3: In vivo Col7 expression using replication-defective HSV-1

[0214] The following examples describe the use of replication-defective type 1 herpes simplex virus (modified to contain human COL7A1 transgene) as a delivery medium for in vivo expression of human Col7 protein.

[0215] method

[0216] Construction and purification of KB103

[0217] The KB103 virus was constructed and purified as described in Example 2 above.

[0218] Viral infection

[0219] The following intradermal injection method was used to deliver KB103 virus to wild-type Balb / c or skh1-elite mice: 1 x 10⁻⁶ cells were injected into each animal at 2-4 sites in the flank region. 8 One plaque-forming unit (PFU) of virus / site, in a volume of 50 μL. Animals were sacrificed 48 hours after KB103 administration, and the injection site was harvested and processed for real-time qPCR or immunofluorescence analysis.

[0220] For qPCR analysis, skin tissue was incised down to the fascia using a 6mm drill biopsy tool. The biopsy sample was divided into two pieces, each of which was rapidly frozen in liquid nitrogen. Total RNA and DNA were isolated from half of the biopsy sample using the Qiagen AllPrep DNA / RNA Kit.

[0221] For immunofluorescence analysis, a circular area approximately 1 cm in diameter is cut from the skin at the injection site, cut in half, and placed in an OCT chamber with the center of the circular area facing upwards. The prepared sample is then frozen and placed in isopentane cooled by liquid nitrogen and stored at -80°C.

[0222] mRNA quantification

[0223] Col7 transcripts were amplified from RNA isolated from mouse skin tissue following KB103 injection using the following two-step protocol: 1) cDNA synthesis was performed using the Superscript III First Strand Synthesis Kit (Thermofisher, Cat. # 18-080-051), and 2) qPCR amplification was performed using the Quantiect Probe PCR Kit (Qiagen, Cat. # 204345) according to the manufacturer's specifications. 100 ng of cDNA was used in each reaction. Col7 transcript levels were normalized to GAPDH transcript levels.

[0224] Genome copy quantification

[0225] The copy number of the KB103 viral genome in mice injected with KB103 was amplified and quantified by qPCR using the Quantiect Probe PCR kit (Qiagen Cat.# 204345). 100 ng of mouse genomic DNA was used in each reaction, with mouse genomic GAPDH used as a control.

[0226] Immunofluorescence

[0227] Tissue sections from mice injected with KB103 were fixed, stained with primary rabbit anti-human Col7 polyclonal antibody (Sigma, Cat.# HPA042420), washed, and then further stained with fluorescently labeled anti-rabbit secondary antibody (Invitrogen, Cat.3A11012). Cell nuclei were stained with DAPI using standard techniques.

[0228] result

[0229] To test the ability of KB103 to successfully deliver and express human Col7 protein in vivo, KB103 virus was administered intradermally to mice. Viral genome copy number in infected mouse tissues was assessed, and high levels of KB103 viral genome delivery (>1,000,000 viral genome copies / 100 ng mouse DNA) were observed in mice. Figure 11 Next, the ability of the virus to express human Col7 in vivo was examined. The quantification of human Col7 transcripts in KB103-infected mice was measured and evaluated compared with the expression of the housekeeping gene in control mice. High levels of human Col7 transcripts were observed in infected mouse tissues. Figure 11 This indicates that the delivered viral genome was able to successfully express its human gene cargo. Finally, the ability of KB103 to express Col7 protein was tested in infected mice. Skin tissue was excised from mice after infection, and Col7 protein expression was assessed by immunohistochemical staining of the mouse tissue. High levels of human Col7 protein were detected after tissue staining. Figure 12 Surprisingly, not only did the KB103 virus express human Col7 protein in the dermis of mice, but human Col7 was also observed to begin depositing in the basement membrane region of KB103-infected mice. Figure 12 Without being bound by theory, these data indicate that: 1) the KB103 virus can successfully infect relevant tissues in vivo, providing these tissues with high genome copy numbers; 2) delivery of the KB103 virus to relevant tissues results in significant expression of the encoded human gene on the virus; and 3) KB103 not only successfully expresses the human Col7 protein in vivo, but this protein is also able to initiate tissue formation in a certain way (e.g., in the basement membrane region), suggesting that KB103 can rescue endogenous Col7 deficiency in affected individuals.

[0230] This disclosure relates to the following implementation plan.

[0231] 1. A pharmaceutical composition comprising:

[0232] a) A virus comprising a vector, wherein the vector comprises one or more transgenic peptides encoding a polypeptide selected from the group consisting of: collagen α-1 (VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, and chimeric polypeptides thereof; and

[0233] b) Pharmaceutically acceptable carriers.

[0234] 2. The pharmaceutical composition as described in Embodiment 1, wherein the virus is an adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, herpes simplex virus, vaccinia virus, or any hybrid virus thereof.

[0235] 3. The pharmaceutical composition as described in Embodiment 1, wherein the virus is herpes simplex virus (HSV).

[0236] 4. The pharmaceutical composition according to any one of embodiments 1 to 3, wherein the virus is a replication-defective type.

[0237] 5. The pharmaceutical composition as described in Embodiment 3, wherein the herpes simplex virus is herpes simplex virus type 1, herpes simplex virus type 2, or any derivative thereof.

[0238] 6. The pharmaceutical composition of embodiment 3, wherein the herpes simplex virus comprises a modified envelope.

[0239] 7. The pharmaceutical composition of embodiment 6, wherein the modified envelope alters the tissue tropism of the herpes simplex virus relative to wild-type herpes simplex virus.

[0240] 8. The pharmaceutical composition of embodiment 6, wherein the modified envelope comprises a mutated herpes simplex virus glycoprotein.

[0241] 9. The pharmaceutical composition as described in Embodiment 1, wherein the carrier is an HSV-1 amplicon or an HSV-1 heterozygous amplicon.

[0242] 10. The pharmaceutical composition of embodiment 9, wherein the HSV-1 heterozygous amplicon is an HSV / AAV heterozygous amplicon, an HSV / EBV heterozygous amplicon, an HSV / EBV / RV heterozygous amplicon, or an HSV / Sleeping Beauty heterozygous amplicon.

[0243] 11. The pharmaceutical composition as described in Embodiment 1, wherein the vector is a recombinant herpes simplex virus genome.

[0244] 12. The pharmaceutical composition of embodiment 11, wherein the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof.

[0245] 13. The pharmaceutical composition of embodiment 11 or 12, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the immediate early herpes simplex virus gene.

[0246] 14. The pharmaceutical composition of embodiment 13, wherein the herpes simplex virus gene is selected from the group consisting of: ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41 and UL55.

[0247] 15. The pharmaceutical composition of any one of embodiments 11 to 14, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4 and ICP22 genes.

[0248] 16. The pharmaceutical composition of any one of embodiments 11 to 14, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes.

[0249] 17. The pharmaceutical composition of embodiment 15 or embodiment 16, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP0, ICP4 and ICP22 genes.

[0250] 18. The pharmaceutical composition of any one of embodiments 15 to 17, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22 and ICP27 genes.

[0251] 19. The pharmaceutical composition of any one of embodiments 15 to 18, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, ICP27 and UL55 genes.

[0252] 20. The pharmaceutical composition of any one of embodiments 15 to 18, wherein the inactivation mutation is a deletion of the coding sequence of the gene.

[0253] 21. The pharmaceutical composition according to any one of embodiments 15 to 20, further comprising an inactivation mutation in the ICP47 gene.

[0254] 22. The pharmaceutical composition according to any one of embodiments 11 to 21, further comprising an inactivating mutation in the UL41 gene.

[0255] 23. The pharmaceutical composition of any one of embodiments 11 to 22, wherein the recombinant herpes simplex virus genome contains one or more transgenes within one or more viral loci.

[0256] 24. The pharmaceutical composition of any one of embodiments 11 to 23, wherein the recombinant herpes simplex virus genome contains one or more transgenes within one or more ICP4 viral loci.

[0257] 25. The pharmaceutical composition of any one of embodiments 11 to 24, wherein the recombinant herpes simplex virus genome contains one or more transgenes within the UL41 viral locus.

[0258] 26. The pharmaceutical composition of embodiment 1, wherein the carrier is capable of replicating within the target cells upon delivery to the target cells.

[0259] 27. The pharmaceutical composition of embodiment 1, wherein the pharmaceutically acceptable carrier is suitable for topical or transdermal administration.

[0260] 28. The pharmaceutical composition of embodiment 1, wherein the one or more transgenes comprises a miRNA binding site.

[0261] 29. The pharmaceutical composition of embodiment 1, wherein the one or more transgenes are operatively linked to one or more heterologous promoters.

[0262] 30. The pharmaceutical composition of embodiment 29, wherein the one or more heterologous promoters are selected from the group consisting of: human cytomegalovirus (HCMV) immediate early promoter, elongation factor-1 (EF1) promoter, and any combination thereof.

[0263] 31. The pharmaceutical composition of embodiment 1, wherein the carrier comprises a transgene encoding a collagen α-1 (VII) chain polypeptide.

[0264] 32. The pharmaceutical composition of embodiment 1, wherein the carrier comprises two transgenes, each transgene encoding a collagen α-1 (VII) chain polypeptide.

[0265] 33. The pharmaceutical composition of embodiment 1, wherein the collagen α-1 (VII) chain polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 2.

[0266] 34. The pharmaceutical composition of embodiment 1, wherein the collagen α-1 (VII) chain polypeptide is a fragment, wherein the fragment has at least 100 consecutive amino acids of SEQ ID NO: 2.

[0267] 35. The pharmaceutical composition of embodiment 1, wherein when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of the subject, the polypeptide enhances, increases, strengthens and / or reinforces the anchoring fibrillation of the subject.

[0268] 36. The pharmaceutical composition of embodiment 1, wherein when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens and / or reinforces the epithelial basement membrane tissue and / or epithelial basement adhesion of the subject.

[0269] 37. The pharmaceutical composition of embodiment 1, wherein the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO:4.

[0270] 38. The pharmaceutical composition of embodiment 1, wherein the lysyl hydroxylase 3 polypeptide is a fragment, wherein the fragment has at least 100 consecutive amino acids of SEQ ID NO: 4.

[0271] 39. The pharmaceutical composition of embodiment 1, wherein when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of a subject, the lysyl hydroxylase 3 polypeptide enhances, increases, strengthens and / or reinforces the formation of hydroxylysine residues on one or more collagen polypeptides of the subject.

[0272] 40. The pharmaceutical composition of embodiment 1, wherein when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens and / or reinforces anchoring fibrillation, epithelial basement membrane tissue and / or epithelial basement adhesion of the subject.

[0273] 41. The pharmaceutical composition of embodiment 1, wherein the carrier comprises at least a first transgene and a second transgene.

[0274] 42. The pharmaceutical composition of embodiment 41, wherein the first transgene encodes a collagen α-1 (VII) chain polypeptide and the second transgene encodes a lysine hydroxylase 3 polypeptide.

[0275] 43. The pharmaceutical composition of embodiment 1, wherein the carrier comprises a polycistronic transgene.

[0276] 44. The pharmaceutical composition of embodiment 43, wherein the polycistronic transgene encodes a collagen α-1 (VII) chain polypeptide in a first open reading frame (ORF) and a lysine hydroxylase 3 polypeptide in a second open reading frame (ORF).

[0277] 45. The pharmaceutical composition of embodiment 44, wherein the first ORF and the second ORF are separated by an internal ribosome entry site (IRES).

[0278] 46. ​​The pharmaceutical composition according to any one of embodiments 42 to 45, wherein the peptides are in an approximately equimolar ratio when the collagen α-1 (VII) chain peptide and the lysyl hydroxylase 3 peptide are expressed in one or more target cells of the subject.

[0279] 47. The pharmaceutical composition of any one of embodiments 42 to 45, wherein when the collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide are expressed in one or more target cells of the subject, the polypeptide enhances, increases, strengthens and / or reinforces the anchoring fibrillation, epithelial basement membrane tissue and / or epithelial basement adhesion of the subject.

[0280] 48. The pharmaceutical composition of embodiment 1, wherein the chimeric polypeptide comprises a linker polypeptide between the collagen α-1(VII) chain polypeptide and the lysine hydroxylase 3 polypeptide.

[0281] 49. The pharmaceutical composition of embodiment 48, wherein the adapter peptide is a T2A, P2A, E2A or F2A adapter peptide.

[0282] 50. The pharmaceutical composition of embodiment 48 or 49, wherein the adapter polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12.

[0283] 51. The pharmaceutical composition of any one of embodiments 48 to 50, wherein the chimeric polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28.

[0284] 52. The pharmaceutical composition of any one of embodiments 48 to 51, wherein when the chimeric polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens and / or reinforces the subject's anchoring fibrillation, epithelial basement membrane tissue and / or epithelial basement adhesion.

[0285] 53. A method for providing preventive, palliative, or therapeutic relief of skin wounds, conditions, or diseases in a subject, the method comprising applying, topically or transdermally, a pharmaceutical composition capable of enhancing, increasing, strengthening, and / or reinforcing the levels of collagen α-1 (VII) chain polypeptides and / or lysyl hydroxylase 3 polypeptides in one or more cells of the subject.

[0286] 54. The method of embodiment 53, wherein the pharmaceutical composition comprises:

[0287] a) A virus comprising a vector, wherein the vector comprises one or more transgenic peptides encoding a polypeptide selected from the group consisting of: collagen α-1 (VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, and chimeric polypeptides thereof; and

[0288] b) Pharmaceutically acceptable carriers.

[0289] 55. The method of embodiment 54, wherein the virus is an adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, herpes simplex virus, vaccinia virus, or any hybrid virus thereof.

[0290] 56. The method of embodiment 54, wherein the virus is herpes simplex virus (HSV).

[0291] 57. The method of any one of embodiments 54 to 56, wherein the virus is replication-defective.

[0292] 58. The method of embodiment 56, wherein the herpes simplex virus is herpes simplex virus type 1, herpes simplex virus type 2, or any derivative thereof.

[0293] 59. The method of embodiment 56, wherein the herpes simplex virus comprises a modified envelope.

[0294] 60. The method of embodiment 59, wherein the modified envelope alters the tissue tropism of the herpes simplex virus relative to wild-type herpes simplex virus.

[0295] 61. The method of embodiment 59, wherein the modified envelope comprises a mutated herpes simplex virus glycoprotein.

[0296] 62. The method of embodiment 54, wherein the vector is an HSV-1 amplicon or an HSV-1 heterozygous amplicon.

[0297] 63. The method of embodiment 62, wherein the HSV-1 heterozygous amplicon is an HSV / AAV heterozygous amplicon, an HSV / EBV heterozygous amplicon, an HSV / EBV / RV heterozygous amplicon, or an HSV / Sleeping Beauty heterozygous amplicon.

[0298] 64. The method of embodiment 54, wherein the vector is a recombinant herpes simplex virus genome.

[0299] 65. The method of embodiment 64, wherein the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof.

[0300] 66. The method of embodiment 64 or 65, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the immediate early herpes simplex virus gene.

[0301] 67. The method of embodiment 66, wherein the herpes simplex virus gene is selected from the group consisting of: ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41 and UL55.

[0302] 68. The method of any one of embodiments 64 to 67, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP4 and ICP22 genes.

[0303] 69. The method of any one of embodiments 64 to 67, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes.

[0304] 70. The method of embodiment 68 or embodiment 69, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP0, ICP4 and ICP22 genes.

[0305] 71. The method of any one of embodiments 68 to 70, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22 and ICP27 genes.

[0306] 72. The method of any one of embodiments 68 to 71, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, ICP27 and UL55 genes.

[0307] 73. The method of any one of embodiments 68 to 72, wherein the inactivation mutation is a deletion of the coding sequence of the gene.

[0308] 74. The method as described in any one of embodiments 68 to 73, further comprising an inactivation mutation in the ICP47 gene.

[0309] 75. The method as described in any one of embodiments 64 to 74, further comprising an inactivation mutation in the UL41 gene.

[0310] 76. The method of any one of embodiments 64 to 75, wherein the recombinant herpes simplex virus genome contains one or more transgenes within one or more viral loci.

[0311] 77. The method of any one of embodiments 64 to 76, wherein the recombinant herpes simplex virus genome contains one or more transgenes within one or more ICP4 viral loci.

[0312] 78. The method of any one of embodiments 64 to 77, wherein the recombinant herpes simplex virus genome contains one or more transgenes within the UL41 viral locus.

[0313] 79. The method of embodiment 54, wherein the vector is capable of replicating within the target cell upon delivery to the target cell.

[0314] 80. The method of embodiment 54, wherein the pharmaceutically acceptable carrier is suitable for topical or transdermal administration.

[0315] 81. The method of embodiment 54, wherein the one or more transgenes comprises a miRNA binding site.

[0316] 82. The method of embodiment 54, wherein the one or more transgenes are operatively linked to one or more heterologous promoters.

[0317] 83. The method of embodiment 82, wherein the one or more heterologous promoters are selected from the group consisting of: human cytomegalovirus (HCMV) immediate early promoter, elongation factor-1 (EF1) promoter, and any combination thereof.

[0318] 84. The method of embodiment 54, wherein the vector comprises a transgene encoding a collagen α-1 (VII) chain polypeptide.

[0319] 85. The method of embodiment 54, wherein the vector comprises two transgenes, each transgene encoding a collagen α-1 (VII) chain polypeptide.

[0320] 86. The method of embodiment 54, wherein the collagen α-1 (VII) chain polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 2.

[0321] 87. The method of embodiment 54, wherein the collagen α-1 (VII) chain polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 2.

[0322] 88. The method of embodiment 54, wherein when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of the subject, the polypeptide enhances, increases, strengthens and / or reinforces the anchoring fibrillation of the subject.

[0323] 89. The method of embodiment 54, wherein when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of the subject, the polypeptide enhances, increases, strengthens and / or reinforces the epithelial basement membrane tissue and / or epithelial basement adhesion of the subject.

[0324] 90. The method of embodiment 54, wherein the lysine hydroxylase 3 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 4.

[0325] 91. The method of embodiment 54, wherein the lysyl hydroxylase 3 polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 4.

[0326] 92. The method of embodiment 54, wherein when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of the subject, the lysyl hydroxylase 3 polypeptide enhances, increases, strengthens and / or reinforces the formation of hydroxylysine residues on one or more collagen polypeptides of the subject.

[0327] 93. The method of embodiment 54, wherein when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of the subject, the polypeptide enhances, increases, strengthens and / or reinforces the subject's anchoring fibrillation, epithelial basement membrane tissue and / or epithelial basement adhesion.

[0328] 94. The method of embodiment 54, wherein the vector comprises at least a first transgene and a second transgene.

[0329] 95. The method of embodiment 94, wherein the first transgene encodes a collagen α-1 (VII) chain polypeptide and the second transgene encodes a lysine hydroxylase 3 polypeptide.

[0330] 96. The method of embodiment 54, wherein the vector comprises a polycistronic transgene.

[0331] 97. The method of embodiment 96, wherein the polycistronic transgene encodes a collagen α-1 (VII) chain polypeptide in a first open reading frame (ORF) and a lysine hydroxylase 3 polypeptide in a second open reading frame (ORF).

[0332] 98. The method of embodiment 97, wherein the first ORF and the second ORF are separated by an internal ribosome entry site (IRES).

[0333] 99. The method of any one of embodiments 95 to 98, wherein the peptides are in approximately an equimolar ratio when the collagen α-1 (VII) chain peptide and the lysyl hydroxylase 3 peptide are expressed in one or more target cells of the subject.

[0334] 100. The method of any one of embodiments 95 to 98, wherein when the collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide are expressed in one or more target cells of the subject, the polypeptides enhance, increase, strengthen and / or reinforce the subject's anchoring fibrillation, epithelial basement membrane tissue and / or epithelial basement adhesion.

[0335] 101. The method of embodiment 54, wherein the chimeric polypeptide comprises a linker polypeptide between the collagen α-1 (VII) chain polypeptide and the lysine hydroxylase 3 polypeptide.

[0336] 102. The method of embodiment 101, wherein the adapter polypeptide is a T2A, P2A, E2A or F2A adapter polypeptide.

[0337] 103. The method as described in embodiment 101 or 102, wherein the adapter polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12.

[0338] 104. The method of any one of embodiments 101 to 103, wherein the chimeric polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28.

[0339] 105. The method of any one of embodiments 101 to 104, wherein when the chimeric polypeptide is expressed in one or more target cells of the subject, the polypeptide enhances, increases, strengthens and / or reinforces the subject's anchoring fibrillation, epithelial basement membrane tissue and / or epithelial basement adhesion.

[0340] 106. The method of embodiment 53, wherein the pharmaceutical composition is applied once, twice, three times, four times, five times or more daily.

[0341] 107. The method of embodiment 53, wherein the pharmaceutical composition is applied to one or more affected and / or unaffected areas of the subject.

[0342] 108. The method of embodiment 53, wherein the skin disease or condition is one or more of the following: epidermolysis bullosa, skin cancer, psoriasis, lichen planus, lupus, rosacea, eczema, cutaneous candidiasis, cellulitis, impetigo, bedsores, erysipelas, ichthyosis vulgaris, dermatomyositis, acrodermatitis, stasis dermatitis, Natherton's syndrome, epidermolysis bullosa simplex (LAMB3 gene), autosomal recessive congenital ichthyosis, xeroderma pigmentosum, and bullous pemphigoid.

[0343] 109. An isolated chimeric polypeptide, wherein the isolated chimeric polypeptide comprises;

[0344] a) Collagen α-1 (VII) chain polypeptide;

[0345] b) Lysyl hydroxylase 3 polypeptide; and

[0346] c) Connector peptides;

[0347] The collagen α-1 (VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide are separated by the linker polypeptide.

[0348] 110. The isolated chimeric polypeptide as described in embodiment 109, wherein the collagen α-1 (VII) chain polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 2.

[0349] 111. The isolated chimeric polypeptide as described in embodiment 109, wherein the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 4.

[0350] 112. The isolated chimeric polypeptide as described in the embodiment or 109, wherein the adapter polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12.

[0351] 113. The isolated chimeric polypeptide as described in any one of embodiments 109 to 112, wherein the isolated chimeric polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28.

[0352] 114. A polynucleotide encoding a chimeric polypeptide as described in any one of embodiments 109 to 113.

[0353] 115. A vector comprising a polynucleotide as described in embodiment 114.

[0354] 116. The vector as described in embodiment 115, wherein the vector is an HSV-1 amplicon or an HSV-1 heterozygous amplicon.

[0355] 117. The vector as described in embodiment 116, wherein the HSV-1 heterozygous amplicon is an HSV / AAV heterozygous amplicon, an HSV / EBV heterozygous amplicon, an HSV / EBV / RV heterozygous amplicon, or an HSV / Sleeping Beauty heterozygous amplicon.

[0356] 118. The vector as described in embodiment 115, wherein the vector is a recombinant herpes simplex virus genome.

[0357] 119. The vector as described in embodiment 118, wherein the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof.

[0358] 120. The vector as described in embodiment 118 or 119, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in the immediate early herpes simplex virus gene.

[0359] 121. The vector as described in embodiment 120, wherein the herpes simplex virus gene is selected from the group consisting of: ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41 and UL55.

[0360] 122. The vector as described in any one of embodiments 118 to 121, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP4 and ICP22 genes.

[0361] 123. The vector as described in any one of embodiments 118 to 121, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes.

[0362] 124. The vector as described in embodiment 122 or embodiment 123, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP0, ICP4 and ICP22 genes.

[0363] 125. The vector as described in any one of embodiments 122 to 124, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP0, ICP4, ICP22 and ICP27 genes.

[0364] 126. The vector as described in any one of embodiments 122 to 125, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP0, ICP4, ICP22, ICP27 and UL55 genes.

[0365] 127. The vector as described in any one of embodiments 122 to 126, wherein the inactivation mutation is a deletion of the coding sequence of the gene.

[0366] 128. The vector as described in any one of embodiments 122 to 127 further comprises an inactivation mutation in the ICP47 gene.

[0367] 129. The vector as described in any one of embodiments 118 to 128 further comprises an inactivation mutation in the UL41 gene.

[0368] 130. The vector as described in any one of embodiments 118 to 129, wherein the recombinant herpes simplex virus genome contains the polynucleotide within one or more viral loci.

[0369] 131. The vector as described in any one of embodiments 118 to 130, wherein the recombinant herpes simplex virus genome contains the polynucleotide within one or more of the ICP4 viral loci.

[0370] 132. The vector as described in any one of embodiments 118 to 131, wherein the recombinant herpes simplex virus genome contains the polynucleotide within the UL41 viral locus.

[0371] 133. A vector comprising one or more polynucleotides encoding a collagen α-1 (VII) chain polypeptide, a lysine hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, or any combination thereof, wherein the vector is a recombinant herpes simplex virus genome.

[0372] 134. The vector as described in embodiment 133, wherein the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof.

[0373] 135. The vector as described in embodiment 133 or embodiment 134, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in the immediate early herpes simplex virus gene.

[0374] 136. The vector as described in embodiment 135, wherein the herpes simplex virus gene is selected from the group consisting of: ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41 and UL55.

[0375] 137. The vector as described in any one of embodiments 133 to 136, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP4 and ICP22 genes.

[0376] 138. The vector as described in any one of embodiments 133 to 136, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes.

[0377] 139. The vector as described in embodiment 137 or embodiment 138, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP0, ICP4 and ICP22 genes.

[0378] 140. The vector as described in any one of embodiments 137 to 139, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22 and ICP27 genes.

[0379] 141. The vector as described in any one of embodiments 137 to 140, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP0, ICP4, ICP22, ICP27 and UL55 genes.

[0380] 142. The vector as described in any one of embodiments 137 to 141, wherein the inactivation mutation is a deletion of the coding sequence of the gene.

[0381] 143. The vector as described in any one of embodiments 137 to 142 further comprises an inactivation mutation in the ICP47 gene.

[0382] 144. The vector as described in any one of embodiments 133 to 143 further comprises an inactivation mutation in the UL41 gene.

[0383] 145. The vector as described in any one of embodiments 133 to 144, wherein the recombinant herpes simplex virus genome contains one or more polynucleotides within one or more viral loci.

[0384] 146. The vector as described in any one of embodiments 133 to 145, wherein the recombinant herpes simplex virus genome contains one or more of the polynucleotides within one or more ICP4 viral loci.

[0385] 147. The vector as described in any one of embodiments 133 to 146, wherein the recombinant herpes simplex virus genome contains one or more of the polynucleotides within the UL41 viral locus.

[0386] 148. The vector as described in any one of embodiments 133 to 147, wherein the vector comprises a polynucleotide encoding a collagen α-1 (VII) chain polypeptide.

[0387] 149. The vector as described in any one of embodiments 133 to 147, wherein the vector comprises two polynucleotides encoding collagen α-1 (VII) chain polypeptides.

[0388] 150. A host cell comprising a vector as described in any one of embodiments 115 to 149.

[0389] 151. A method for collecting herpes simplex virus, wherein a target vector is encapsulated within the herpes simplex virus, the method comprising:

[0390] a) To bring host cells into contact with a vector encoding a helper virus;

[0391] b) Contact the host cell with the carrier as described in any one of embodiments 115 to 117; and

[0392] c) Collect the herpes simplex virus produced by the host cells.

[0393] 152. A method for collecting herpes simplex virus, wherein a target vector is encapsulated within the herpes simplex virus, the method comprising:

[0394] a) Contacting the complementary host cell with the vector as described in any one of embodiments 118 to 149; and

[0395] b) Collect the herpes simplex virus produced by the complementary host cell.

[0396] 153. The method as described in embodiment 151 or 152, wherein the herpes simplex virus is herpes simplex virus type 1, herpes simplex virus type 2, or any derivative thereof.

[0397] 154. A medicine box comprising:

[0398] a) the pharmaceutical composition as described in any one of embodiments 1 to 52; and

[0399] b) Instructions for use of the pharmaceutical composition.

[0400] 155. A pharmaceutical composition comprising:

[0401] a) A virus comprising a vector, wherein the vector comprises one or more transgenic peptides encoding a polypeptide selected from the group consisting of: collagen α-1 (VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, keratin type I cytoskeleton 17 polypeptide, and chimeric polypeptides thereof; and

[0402] b) Pharmaceutically acceptable carriers.

[0403] 156. The pharmaceutical composition of embodiment 155, wherein the virus is an adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, herpes simplex virus, vaccinia virus, or any hybrid virus thereof.

[0404] 157. The pharmaceutical composition of embodiment 155, wherein the virus is herpes simplex virus (HSV).

[0405] 158. The pharmaceutical composition according to any one of embodiments 155 to 157, wherein the virus is a replication-defective type.

[0406] 159. The pharmaceutical composition according to any one of embodiments 155 to 158, wherein the herpes simplex virus is herpes simplex virus type 1, herpes simplex virus type 2, or any derivative thereof.

[0407] 160. The pharmaceutical composition according to any one of embodiments 155 to 159, wherein the carrier is an HSV-1 amplicon or an HSV-1 heterozygous amplicon.

[0408] 161. The pharmaceutical composition of embodiment 160, wherein the HSV-1 heterozygous amplicon is an HSV / AAV heterozygous amplicon, an HSV / EBV heterozygous amplicon, an HSV / EBV / RV heterozygous amplicon, or an HSV / Sleeping Beauty heterozygous amplicon.

[0409] 162. The pharmaceutical composition according to any one of embodiments 155 to 159, wherein the vector is a recombinant herpes simplex virus genome.

[0410] 163. The pharmaceutical composition of embodiment 162, wherein the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof.

[0411] 164. The pharmaceutical composition of embodiment 162 or 163, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the immediate early herpes simplex virus gene.

[0412] 165. The pharmaceutical composition of embodiment 164, wherein the herpes simplex virus gene is selected from the group consisting of: ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41 and UL55.

[0413] 166. The pharmaceutical composition of any one of embodiments 162 to 165, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4 and ICP22 genes.

[0414] 167. The pharmaceutical composition of any one of embodiments 162 to 165, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes.

[0415] 168. The pharmaceutical composition of embodiment 166 or embodiment 167, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4 and ICP22 genes.

[0416] 169. The pharmaceutical composition of any one of embodiments 166 to 168, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22 and ICP27 genes.

[0417] 170. The pharmaceutical composition of any one of embodiments 166 to 169, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, ICP27 and UL55 genes.

[0418] 171. The pharmaceutical composition of any one of embodiments 166 to 170, wherein the inactivation mutation is a deletion of the coding sequence of the gene.

[0419] 172. The pharmaceutical composition according to any one of embodiments 166 to 171, further comprising an inactivating mutation in the ICP47 gene.

[0420] 173. The pharmaceutical composition according to any one of embodiments 162 to 172, further comprising an inactivating mutation in the UL41 gene.

[0421] 174. The pharmaceutical composition of any one of embodiments 162 to 173, wherein the recombinant herpes simplex virus genome contains one or more transgenes within one or more viral loci.

[0422] 175. The pharmaceutical composition of any one of embodiments 162 to 174, wherein the recombinant herpes simplex virus genome contains one or more transgenes within one or more ICP4 viral loci.

[0423] 176. The pharmaceutical composition of any one of embodiments 162 to 175, wherein the recombinant herpes simplex virus genome contains one or more transgenes within the UL41 viral locus.

[0424] 177. The pharmaceutical composition of embodiment 155, wherein the carrier is capable of replicating within the target cells upon delivery to the target cells.

[0425] 178. The pharmaceutical composition of embodiment 155, wherein the pharmaceutically acceptable carrier is suitable for topical or transdermal administration.

[0426] 179. The pharmaceutical composition of embodiment 155, wherein the pharmaceutically acceptable carrier is suitable for subcutaneous or intradermal administration.

[0427] 180. The pharmaceutical composition of embodiment 155, wherein the one or more transgenes comprises a miRNA binding site.

[0428] 181. The pharmaceutical composition of any one of embodiments 155 to 180, wherein the carrier comprises a transgene encoding a collagen α-1 (VII) chain polypeptide.

[0429] 182. The pharmaceutical composition of any one of embodiments 155 to 180, wherein the carrier comprises a transgene encoding a lysylhydroxylase 3 polypeptide.

[0430] 183. The pharmaceutical composition of any one of embodiments 155 to 180, wherein the carrier comprises a transgene encoding a polypeptide of keratin type I cytoskeleton 17.

[0431] 184. The pharmaceutical composition of any one of embodiments 155 to 180, wherein the collagen α-1 (VII) chain polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 2.

[0432] 185. The pharmaceutical composition of any one of embodiments 155 to 180, wherein the collagen α-1 (VII) chain polypeptide is a fragment, wherein the fragment has at least 100 consecutive amino acids of SEQ ID NO: 2.

[0433] 186. The pharmaceutical composition of any one of embodiments 155 to 180, wherein when the collagen α-1(VII) chain polypeptide is expressed in one or more target cells of the subject, the polypeptide enhances, increases, strengthens and / or reinforces the anchoring fibrillation of the subject.

[0434] 187. The pharmaceutical composition of any one of embodiments 155 to 180, wherein when the collagen α-1(VII) chain polypeptide is expressed in one or more target cells of the subject, the polypeptide enhances, increases, strengthens and / or reinforces the epithelial basement membrane tissue and / or epithelial basement adhesion of the subject.

[0435] 188. The pharmaceutical composition of any one of embodiments 155 to 180, wherein the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 4.

[0436] 189. The pharmaceutical composition of any one of embodiments 155 to 180, wherein the lysyl hydroxylase 3 polypeptide is a fragment, wherein the fragment has at least 100 consecutive amino acids of SEQ ID NO: 4.

[0437] 190. The pharmaceutical composition according to any one of embodiments 155 to 180, wherein when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of the subject, the lysyl hydroxylase 3 polypeptide enhances, increases, strengthens and / or reinforces the formation of hydroxylysine residues on one or more collagen polypeptides of the subject.

[0438] 191. The pharmaceutical composition according to any one of embodiments 155 to 180, wherein when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of a subject, the polypeptide enhances, increases, strengthens and / or reinforces anchoring fibrillation, epithelial basement membrane tissue and / or epithelial basement adhesion of the subject.

[0439] 192. The pharmaceutical composition of any one of embodiments 155 to 180, wherein the keratin type I cytoskeleton 17 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 30.

[0440] 193. The pharmaceutical composition of any one of embodiments 155 to 180, wherein the keratin type I cytoskeleton 17 polypeptide is a fragment, wherein the fragment has at least 100 consecutive amino acids of SEQ ID NO: 30.

[0441] 194. The pharmaceutical composition of any one of embodiments 155 to 180, wherein the keratin type I cytoskeleton 17 polypeptide enhances, increases, strengthens and / or reinforces wound healing in the subject.

[0442] 195. The pharmaceutical composition according to any one of embodiments 155 to 194, wherein the carrier comprises at least a first transgene and a second transgene.

[0443] 196. The pharmaceutical composition of embodiment 195, wherein the first transgene and the second transgene each encode a collagen α-1 (VII) chain polypeptide.

[0444] 197. The pharmaceutical composition of embodiment 195, wherein the first transgene encodes a collagen α-1(VII) chain polypeptide and the second transgene encodes a lysine hydroxylase 3 polypeptide.

[0445] 198. The pharmaceutical composition of embodiment 195, wherein the first transgene encodes a collagen α-1(VII) chain polypeptide and the second transgene encodes a keratin type I cytoskeleton 17 polypeptide.

[0446] 199. The pharmaceutical composition of embodiment 195, wherein the first transgene encodes a lysyl hydroxylase 3 polypeptide and the second transgene encodes a keratin type I cytoskeleton 17 polypeptide.

[0447] 200. The pharmaceutical composition of embodiment 155, wherein the carrier comprises at least a first transgene, a second transgene, and a third transgene.

[0448] 201. The pharmaceutical composition of embodiment 200, wherein the first transgene encodes a collagen α-1(VII) chain polypeptide, the second transgene encodes a lysine hydroxylase 3 polypeptide, and the third transgene encodes a keratin type I cytoskeleton 17 polypeptide.

[0449] 202. A method for providing preventive, palliative, or therapeutic relief of a wound, condition, or disease of the skin in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a carrier, wherein the carrier is a recombinant herpes simplex virus genome, and wherein the pharmaceutical composition is capable of enhancing, increasing, strengthening, and / or amplifying the levels of collagen α-1 (VII) chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeleton 17 polypeptide in one or more cells of the subject.

[0450] 203. The method of embodiment 202, wherein the pharmaceutical composition comprises:

[0451] a) A virus comprising the vector, wherein the vector comprises one or more transgenic peptides encoding a polypeptide selected from the group consisting of: collagen α-1 (VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, keratin type I cytoskeleton 17 polypeptide, and chimeric polypeptides thereof; and

[0452] b) Pharmaceutically acceptable carriers.

[0453] 204. The method as described in embodiment 203, wherein the virus is an adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, herpes simplex virus, vaccinia virus, or any hybrid virus thereof.

[0454] 205. The method as described in embodiment 203, wherein the virus is herpes simplex virus (HSV).

[0455] 206. The method of any one of embodiments 203 to 205, wherein the virus is replication-defective.

[0456] 207. The method of any one of embodiments 203 to 206, wherein the herpes simplex virus is herpes simplex virus type 1, herpes simplex virus type 2, or any derivative thereof.

[0457] 208. The method of any one of embodiments 202 to 207, wherein the recombinant herpes simplex virus genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivative thereof.

[0458] 209. The method of any one of embodiments 202 to 208, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the immediate early herpes simplex virus gene.

[0459] 210. The method of embodiment 209, wherein the herpes simplex virus gene is selected from the group consisting of: ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41 and UL55.

[0460] 211. The method of any one of embodiments 202 to 210, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP4 and ICP22 genes.

[0461] 212. The method of any one of embodiments 202 to 210, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes.

[0462] 213. The method of embodiment 211 or embodiment 212, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP0, ICP4 and ICP22 genes.

[0463] 214. The method of any one of embodiments 211 to 213, wherein the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22 and ICP27 genes.

[0464] 215. The method of any one of embodiments 211 to 214, wherein the recombinant herpes simplex virus genome comprises inactivation mutations in the ICP0, ICP4, ICP22, ICP27 and UL55 genes.

[0465] 216. The method of any one of embodiments 211 to 215, wherein the inactivation mutation is a deletion of the coding sequence of the gene.

[0466] 217. The method of any one of embodiments 211 to 216, further comprising an inactivation mutation in the ICP47 gene.

[0467] 218. The method of any one of embodiments 202 to 217, further comprising an inactivation mutation in the UL41 gene.

[0468] 219. The method of any one of embodiments 202 to 218, wherein the recombinant herpes simplex virus genome contains one or more transgenes within one or more viral loci.

[0469] 220. The method of any one of embodiments 202 to 219, wherein the recombinant herpes simplex virus genome contains one or more transgenes within one or more ICP4 viral loci.

[0470] 221. The method of any one of embodiments 202 to 220, wherein the recombinant herpes simplex virus genome contains one or more transgenes within the UL41 viral locus.

[0471] 222. The method of embodiment 202, wherein the vector is capable of replicating within the target cell upon delivery to the target cell.

[0472] 223. The method of embodiment 203, wherein the pharmaceutically acceptable carrier is suitable for topical or transdermal administration.

[0473] 224. The method of embodiment 203, wherein the pharmaceutically acceptable carrier is suitable for subcutaneous or intradermal administration.

[0474] 225. The method of embodiment 203, wherein the one or more transgenes comprises a miRNA binding site.

[0475] 226. The method of any one of embodiments 202 to 225, wherein the vector comprises a transgene encoding a collagen α-1(VII) chain polypeptide.

[0476] 227. The method of any one of embodiments 202 to 225, wherein the vector comprises a transgene encoding a lysine hydroxylase 3 polypeptide.

[0477] 228. The method of any one of embodiments 202 to 225, wherein the vector comprises a transgene encoding a polypeptide of keratin type I cytoskeleton 17.

[0478] 229. The method of any one of embodiments 202 to 225, wherein the collagen α-1 (VII) chain polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 2.

[0479] 230. The method of any one of embodiments 202 to 225, wherein the collagen α-1 (VII) chain polypeptide is a fragment having at least 100 consecutive amino acids of SEQ ID NO: 2.

[0480] 231. The method of any one of embodiments 202 to 225, wherein when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of the subject, the polypeptide enhances, increases, strengthens and / or reinforces the anchoring fibrillation of the subject.

[0481] 232. The method of any one of embodiments 202 to 225, wherein when the collagen α-1 (VII) chain polypeptide is expressed in one or more target cells of the subject, the polypeptide enhances, increases, strengthens and / or reinforces the epithelial basement membrane tissue and / or epithelial basement adhesion of the subject.

[0482] 233. The method of any one of embodiments 202 to 225, wherein the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 4.

[0483] 234. The method of any one of embodiments 202 to 225, wherein the lysyl hydroxylase 3 polypeptide is a fragment, wherein the fragment has at least 100 consecutive amino acids of SEQ ID NO: 4.

[0484] 235. The method of any one of embodiments 202 to 225, wherein when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of the subject, the lysyl hydroxylase 3 polypeptide enhances, increases, strengthens and / or reinforces the formation of hydroxylysine residues on one or more collagen polypeptides of the subject.

[0485] 236. The method of any one of embodiments 202 to 225, wherein when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of the subject, the polypeptide enhances, increases, strengthens and / or reinforces the anchoring fibrillation, epithelial basement membrane tissue and / or epithelial basement adhesion of the subject.

[0486] 237. The method of any one of embodiments 202 to 225, wherein the keratin type I cytoskeleton 17 polypeptide has at least 80% sequence identity with the sequence of SEQ ID NO: 30.

[0487] 238. The method of any one of embodiments 202 to 225, wherein the keratin type I cytoskeleton 17 polypeptide is a fragment, wherein the fragment has at least 100 consecutive amino acids of SEQ ID NO: 30.

[0488] 239. The method of any one of embodiments 202 to 225, wherein the keratin type I cytoskeleton 17 polypeptide enhances, increases, strengthens and / or reinforces wound healing in the subject.

[0489] 240. The method of any one of embodiments 202 to 239, wherein the vector comprises at least a first transgene and a second transgene.

[0490] 241. The method of embodiment 240, wherein the first transgene and the second transgene each encode a collagen α-1 (VII) chain polypeptide.

[0491] 242. The method of embodiment 240, wherein the first transgene encodes a collagen α-1 (VII) chain polypeptide and the second transgene encodes a lysine hydroxylase 3 polypeptide.

[0492] 243. The method of embodiment 240, wherein the first transgene encodes a collagen α-1 (VII) chain polypeptide and the second transgene encodes a keratin type I cytoskeleton 17 polypeptide.

[0493] 244. The method of embodiment 240, wherein the first transgene encodes a lysyl hydroxylase 3 polypeptide and the second transgene encodes a keratin type I cytoskeleton 17 polypeptide.

[0494] 245. The method of any one of embodiments 202 to 239, wherein the vector comprises at least a first transgene, a second transgene, and a third transgene.

[0495] 246. The method of embodiment 245, wherein the first transgene encodes a collagen α-1 (VII) chain polypeptide, the second transgene encodes a lysine hydroxylase 3 polypeptide, and the third transgene encodes a keratin type I cytoskeleton 17 polypeptide.

[0496] 247. The method of any one of embodiments 202 to 246, wherein the pharmaceutical composition is applied to the subject in a local or transdermal manner.

[0497] 248. The method of any one of embodiments 202 to 246, wherein the pharmaceutical composition is administered to the subject subcutaneously or intradermally.

[0498] 249. The method of any one of embodiments 202 to 248, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times or more daily.

[0499] 250. The method of any one of embodiments 202 to 249, wherein the pharmaceutical composition is applied to one or more affected and / or unaffected areas of the subject.

[0500] 251. The method according to any one of embodiments 202 to 250, wherein the disease or condition of the skin is one or more of epidermolysis bullosa, skin cancer, psoriasis, lichen planus, lupus, acne erythematosus, eczema, cutaneous candidiasis, cellulitis, impetigo, bedsores, erysipelas, ichthyosis vulgaris, dermatomyositis, acrodermatitis, stasis dermatitis, Natherton's syndrome, epidermolysis bullosa simplex (LAMB3 gene), autosomal recessive congenital ichthyosis, xeroderma pigmentosum, and bullous pemphigoid.

Claims

1. A pharmaceutical composition comprising: a) A herpes simplex virus comprising a recombinant herpes simplex virus (HSV) genome, wherein the recombinant herpes simplex virus genome comprises one or more transgenes encoding a polypeptide selected from the group consisting of: collagen α-1 (VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, keratin type I cytoskeleton 17 polypeptide, and chimeric polypeptides thereof; and b) Pharmaceutically acceptable carriers.

2. The pharmaceutical composition of claim 1, wherein the recombinant HSV genome comprises an inactivating mutation in a herpes simplex virus gene selected from the group consisting of: ICP0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41, and UL55.

3. A pharmaceutical composition comprising: a) A replication-defective herpes simplex virus type 1 (HSV-1) containing a recombinant HSV-1 genome, wherein the recombinant HSV-1 genome contains one or more polynucleotides encoding a transgene; and b) Pharmaceutically acceptable carriers, The recombinant HSV-1 genome contains an inactivating mutation in one or both copies of the infecting cell protein 4 (ICP4) herpes simplex virus gene.

4. The pharmaceutical composition of claim 3, wherein the recombinant HSV-1 genome comprises an inactivating mutation in a herpes simplex virus gene selected from the group consisting of: ICP0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41, and UL55.

5. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for providing preventive, alleviating or therapeutic relief to a subject’s skin wounds, conditions or diseases.

6. The use according to claim 5, wherein the pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, or intradermally.

7. The use according to claim 5, wherein the skin disease or condition is selected from the group consisting of: epidermolysis bullosa, skin cancer, psoriasis, lichen planus, lupus, acne erythematosus, eczema, cutaneous candidiasis, cellulitis, impetigo, bedsores, erysipelas, ichthyosis vulgaris, dermatomyositis, acrodermatitis, stasis dermatitis, Natherton's syndrome, epidermolysis bullosa simplex, autosomal recessive congenital ichthyosis, xeroderma pigmentosum, and bullous pemphigoid.

8. Use of a pharmaceutical composition in the preparation of a medicament for skin delivery of a transgenic substance to a subject, said pharmaceutical composition comprising: a) A herpes simplex virus comprising a recombinant herpes simplex virus genome, wherein the recombinant herpes simplex virus genome contains one or more polynucleotides encoding the transgene; and b) Pharmaceutically acceptable carriers; The recombinant herpes simplex virus genome contains an inactivating mutation in one or both copies of the ICP4 herpes simplex virus gene. The pharmaceutically acceptable carriers described herein are suitable for topical, transdermal, or intradermal administration, and The pharmaceutical composition is adapted to deliver one or more polynucleotides encoding the genetic material to one or more target cells in the epidermis and / or dermis of the subject.

9. The use according to claim 8, wherein the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP22 herpes simplex virus gene.

10. Use of a pharmaceutical composition in the preparation of a medicament for delivering a transgenic substance to the eye of a subject, said pharmaceutical composition comprising: a) A replication-defective herpes simplex virus comprising a recombinant herpes simplex virus (HSV) genome, wherein the recombinant herpes simplex virus genome contains one or more polynucleotides encoding the transgene; and b) Pharmaceutically acceptable carriers; The pharmaceutical composition is applied topically or by injection to the subject's eye.

11. The use according to claim 10, wherein the pharmaceutical composition is applied to one or more areas of the eye affected by an eye condition or disease.

12. The use according to claim 11, wherein the condition or disease is vision loss.

13. Use of a pharmaceutical composition in the preparation of a medicament for correcting visual loss in a subject, said pharmaceutical composition comprising: a) A replication-defective herpes simplex virus comprising a recombinant herpes simplex virus (HSV) genome, wherein the recombinant herpes simplex virus genome contains one or more polynucleotides encoding a transgene; and b) Pharmaceutically acceptable carriers, The pharmaceutical composition is applied topically or by injection to the subject's eye.

14. The use according to claim 13, wherein the subject is diagnosed with dystrophic epidermolysis bullosa.

15. The use according to claim 10 or claim 13, wherein the injection is an intravitreal injection.

16. The use according to claim 10 or claim 13, wherein the replication-defective HSV is adapted to deliver one or more polynucleotides encoding the transgene to one or more target cells in the eye of the subject.

17. The use according to claim 10 or claim 13, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in one or both copies of the ICP4 herpes simplex virus gene.

18. The use according to claim 10 or claim 13, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 herpes simplex virus gene.

19. A kit comprising: a) A pharmaceutical composition comprising: (i) A herpes simplex virus containing a recombinant herpes simplex virus (HSV) genome, wherein the recombinant herpes simplex virus genome contains one or more transgenes encoding a polypeptide selected from the group consisting of: collagen α-1 (VII) chain polypeptide, lysine hydroxylase 3 polypeptide, keratin type I cytoskeleton 17 polypeptide and their chimeric polypeptides. as well as (ii) A pharmaceutically acceptable carrier; and b) Instructions for use of the pharmaceutical composition.

20. The kit of claim 19, wherein the recombinant HSV genome comprises an inactivating mutation in a herpes simplex virus gene selected from the group consisting of: ICP0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41, and UL55.

21. A kit comprising: a) A pharmaceutical composition comprising: (i) a replication-deficient HSV-1 containing a recombinant herpes simplex virus type 1 (HSV-1) genome, wherein the recombinant HSV-1 genome contains one or more polynucleotides encoding a transgene; and (ii) A pharmaceutically acceptable carrier; and b) Instructions for use of the pharmaceutical composition, The recombinant HSV-1 genome contains an inactivating mutation in one or both copies of the infecting cell protein 4 (ICP4) herpes simplex virus gene.

22. The kit of claim 21, wherein the recombinant HSV-1 genome further comprises an inactivating mutation in a herpes simplex virus gene selected from the group consisting of: ICP0, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41, and UL55.

23. The kit according to claim 19 or claim 21, wherein the pharmaceutically acceptable carrier is suitable for topical application, transdermal application, subcutaneous injection, intradermal injection, or any combination thereof.

24. The kit according to claim 19 or claim 21, wherein the pharmaceutical composition is adapted to deliver one or more polynucleotides encoding the transgene to a wound, symptom, or disease on the skin of a subject.

25. The kit according to claim 19 or claim 21, wherein the pharmaceutical composition comprises an ointment, paste, cream, suspension, emulsion, fat ointment, gel, powder, lotion, solution, spray, patch, or microneedle array.

26. The kit according to claim 19 or claim 21, wherein the pharmaceutical composition comprises hydroxypropyl methylcellulose.

27. A vector comprising one or more polynucleotides encoding a collagen α-1 (VII) chain polypeptide, a lysine hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, or any combination thereof, wherein the vector is a recombinant herpes simplex virus genome.

28. A host cell comprising the vector according to claim 27.

29. Use of the vector of claim 27 in the preparation of a medicament for contacting a host cell to generate herpes simplex virus, wherein the vector is encapsulated within the herpes simplex virus.

30. The use according to claim 29, wherein the host cell comprises a helper virus or is in contact with a vector encoding the helper virus, or wherein the host cell is a complementary host cell.

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