Compositions and methods for treatment of wounds, disorders, and diseases of skin

Abstract

To provide compositions and methods for treating skin wounds, disorders and diseases.SOLUTION: The disclosure relates, in part, to a pharmaceutical composition comprising one or more polynucleotide species suitable for enhancing, increasing, augmenting, and / or supplementing the 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 disclosure also relates, in part, to pharmaceutical compositions and methods of use for providing prophylactic, palliative, or therapeutic relief of a wound, disorder, or disease of the skin in a subject, including a subject having, or at risk of developing, one or more symptoms of epidermolysis bullosa.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 320,316, filed April 8, 2016, which is incorporated herein by reference in its entirety.

[0002] Submission of sequence listing as an ASCII text file The contents of the following submission, in an ASCII text file, are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (File Name: 761342000140SEQLIST.txt, Date Recorded: December 28, 2016, Size: 394 KB).

[0003] The present disclosure relates, in part, to pharmaceutical compositions and methods of use for providing preventative, palliative, or therapeutic relief of wounds, disorders, or diseases in the skin of a subject, including subjects having or at risk of developing one or more symptoms of epidermolysis bullosa. [Background technology]

[0004] Many serious disease-related skin conditions are associated with one or more genetic disorders in patients suffering from these diseases. One such disease, epidermolysis bullosa (EB), is a group of genetic disorders in which affected individuals' skin and mucous membranes blister and erode in response to minor injury or friction, such as scraping, rubbing, or scratching. Dystrophic epidermolysis bullosa (DEB) is one of the major forms of EB. The signs and symptoms of this condition vary greatly among affected individuals, ranging from mild (blistering may affect only the hands, feet, knees, and elbows) to severe (widespread blistering and scarring, potentially resulting in vision loss, disfigurement, and other serious, sometimes fatal, health conditions).

[0005] Dystrophic epidermolysis bullosa is divided into three main types. Autosomal dominant dystrophic epidermolysis bullosa (referred to as dominant dystrophic epidermolysis bullosa or DDEB) is typically the mildest type, with blistering often limited to the hands, feet, knees, and elbows. The other two types of dystrophic epidermolysis bullosa, Hallopeau-Siemens recessive dystrophic epidermolysis bullosa and non-Hallopeau-Siemens recessive epidermolysis bullosa (collectively referred to as recessive dystrophic epidermolysis bullosa or RDEB), are more severe. RDEB is most often characterized by extensive blistering and scarring of the skin and mucous membranes. Blisters are routinely present throughout the body, including on mucous membranes (such as those of the oral cavity and digestive tract), and healing of these blisters results in extensive scarring. Damage to the oral cavity and esophagus can make chewing and swallowing difficult, leading to chronic malnutrition and poor growth. Complications of extensive scarring can include fused fingers and toes, joint deformities, and ophthalmia resulting in vision loss. Furthermore, patients with RDEB are at increased risk of developing squamous cell carcinoma, a disease that can be highly aggressive and often life-threatening in this patient population. While the three types of dystrophic epidermolysis bullosa vary in severity, they share many common characteristics and are caused by the same genetic mutations.

[0006] Dystrophic epidermolysis bullosa is caused by mutations in the Col7a1 gene, which encodes the collagen alpha-1(VII) chain protein (collagen 7). Over 240 distinct mutations in this gene have been identified in DEB patients. Furthermore, a significant decrease in the expression of the PLOD3 gene, which encodes the collagen-modifying lysyl hydroxylase 3 enzyme (LH3), has also been observed in patients with dystrophic epidermolysis bullosa. While collagen alpha-1(VII) chain protein functions to strengthen and stabilize the skin, lysyl hydroxylase 3 plays a critical role in the synthesis and secretion of functional collagen alpha-1(VII) chain protein. Briefly, Col7a1 transcripts are translated, and the resulting peptides are post-translationally modified by hydroxylation of their proline residues (by prolyl hydroxylases) and their lysyl residues (by lysyl hydroxylases such as LH3). The hydroxylysine residues can then be glycosylated, and the three glycosylated peptides subsequently form a triple helix known as procollagen and are secreted from cells. The secreted procollagen can then associate into higher-order structures and form anchoring fibers. The anchoring fibers are then available to help organize, stabilize, and support epithelial basement membrane adhesion. The epithelial basement membrane is involved in anchoring the epithelium to the underlying loose connective tissue and is essential for dermal-epidermal stability (integrity of the epidermal-dermal junction). Mutations in the Col7a1 gene and reduced levels of PLOD3 expression impair the ability of collagen alpha-1(VII) chain protein to properly connect the epidermis and dermis in patients with dystrophic epidermolysis bullosa, causing fragile skin.

[0007] Treatment options for patients with epidermolysis bullosa are limited, and current care focuses on managing the symptoms of the disease, including providing medications to control pain and itching, administering oral antibiotics to avoid infections resulting from open wounds on the skin and mucous membranes, and surgical strategies to address scarring and deformity. Investigational methods for treating the underlying cause of epidermolysis bullosa include administering purified collagen 7, fibroblasts containing collagen 7, or viral vectors encoding collagen 7 via intradermal injection. Because many DEB patients have multiple wounds spanning many traumatic sites (such as the sacrum, buttocks, feet, lower back, and hands), any treatment involving intradermal injections would be highly invasive, as all of these large wound areas would need to be injected, possibly repeatedly, for uncertain intervals between injections.

[0008] Therefore, there is a clear need for less invasive / minimally invasive / non-invasive treatment options for epidermolysis bullosa patients that can address the deficiency of collagen alpha-1(VII) chain protein, as well as the deficiency of lysyl hydroxylase 3 protein, observed in this patient population.

[0009] All references cited herein, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot accession numbers, are incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0010] To meet these needs, the present disclosure relates, in part, to pharmaceutical compositions and methods of use for providing preventative, palliative, or therapeutic relief of wounds, disorders, or diseases in the skin of subjects, particularly subjects having or at risk of developing one or more symptoms of epidermolysis bullosa. In particular, the present disclosure relates, in part, to methods of treating individuals by administering (e.g., topically or transdermally) a pharmaceutical composition comprising one or more polynucleotides encoding a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, and / or chimeric polypeptides thereof.

[0011] Thus, certain aspects of the present disclosure relate to pharmaceutical compositions comprising a vector, i.e., a virus comprising a vector containing one or more transgenes encoding a collagen alpha-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, an adeno-associated virus, a retrovirus, a lentivirus, a Sendai virus, a herpes simplex virus, a vaccinia virus, or any hybrid virus thereof. In some embodiments, the virus is replication-deficient. In some embodiments, the virus is a herpes simplex virus (HSV). In some embodiments, the herpes simplex virus is a herpes simplex type 1 virus, a herpes simplex type 2 virus, 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 compared to a 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 hybrid amplicon. In some embodiments, the HSV-1 hybrid amplicon is an HSV / AAV hybrid amplicon, an HSV / EBV hybrid amplicon, an HSV / EBV / RV hybrid amplicon, or an HSV / Sleeping Beauty hybrid 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 comprises an inactivating mutation in an 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 comprises inactivating mutations in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises 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 comprises inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises 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 further comprises an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes in one or more viral loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes in one or more of the ICP4 viral loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes in the UL41 viral locus. In some embodiments, the vector, when delivered into a target cell, is capable of replicating in the target cell. In some embodiments, the pharmaceutically acceptable carrier is suitable for topical or transdermal administration.In some embodiments, the one or more transgenes comprise an miRNA binding site. In some embodiments, the one or more transgenes are operably 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, the elongation factor-1 (EF1) promoter, and / or any combination thereof. In some embodiments, the vector comprises a transgene encoding a collagen alpha-1(VII) chain polypeptide. In some embodiments, the vector comprises two transgenes, each transgene encoding a collagen alpha-1(VII) chain polypeptide. In some embodiments, the collagen alpha-1(VII) chain polypeptide has at least 80% sequence identity to the sequence of SEQ ID NO:2. In some embodiments, the collagen alpha-1(VII) chain polypeptide is a fragment, the fragment having at least 100 consecutive amino acids of SEQ ID NO:2. In some embodiments, the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements tethering fibril formation in a subject when the polypeptide is expressed in one or more target cells of the subject. In some embodiments, the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements epithelial basement membrane organization and / or epithelial basal adhesion in a subject when the polypeptide is expressed in one or more target cells in the subject. In some embodiments, the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity to the sequence of SEQ ID NO:4. In some embodiments, the lysyl hydroxylase 3 polypeptide is a fragment, wherein the fragment has at least 100 consecutive amino acids of SEQ ID NO:4. In some embodiments, the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements the formation of hydroxylysine residues on one or more collagen polypeptides of a subject when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells in the subject.In some embodiments, the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or complements anchoring fiber formation, epithelial basement membrane organization, and / or epithelial basal adhesion in a subject when the polypeptide is expressed in one or more target cells of the subject. In some embodiments, the vector comprises at least a first transgene and a second transgene. In some embodiments, the first transgene encodes a collagen alpha-1(VII) chain polypeptide and the second transgene encodes a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector comprises a polycistronic transgene. In some embodiments, the polycistronic transgene encodes a collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF) and a lysyl hydroxylase 3 polypeptide on 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, the collagen alpha-1(VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide are in about equimolar ratios when the polypeptides are expressed in one or more target cells of the subject. In some embodiments, the collagen alpha-1(VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide enhance, increase, augment, and / or supplement anchoring fibril formation, epithelial basement membrane organization, and / or epithelial basal adhesion in a subject when the polypeptides are expressed in one or more target cells in the subject. In some embodiments, the chimeric polypeptide comprises a linker polypeptide between the collagen alpha-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 to 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 polypeptide has at least 80% sequence identity to 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. In some embodiments, the chimeric polypeptide enhances, increases, augments, and / or supplements anchoring fiber formation, epithelial basement membrane organization, and / or epithelial basal adhesion in a subject when the polypeptide is expressed in one or more target cells in the subject.

[0012] Another aspect of the present disclosure relates to a method for providing preventative, palliative, or therapeutic relief of a wound, injury, or disease in the skin of a subject, comprising topically or transdermally administering a pharmaceutical composition capable of enhancing, increasing, augmenting, and / or supplementing the level of a collagen alpha-1(VII) chain polypeptide and / or a lysyl hydroxylase 3 polypeptide in one or more cells of the subject. In some embodiments, the pharmaceutical composition comprises a vector, i.e., a virus comprising a vector containing one or more transgenes encoding a collagen alpha-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, an adeno-associated virus, a retrovirus, a lentivirus, a Sendai virus, a herpes simplex virus, a vaccinia virus, or any hybrid virus thereof. In some embodiments, the virus is replication-deficient. In some embodiments, the virus is a herpes simplex virus (HSV). In some embodiments, the herpes simplex virus is herpes simplex type 1 virus, herpes simplex type 2 virus, 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 compared 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 hybrid amplicon. In some embodiments, the HSV-1 hybrid amplicon is an HSV / AAV hybrid amplicon, an HSV / EBV hybrid amplicon, an HSV / EBV / RV hybrid amplicon, or an HSV / Sleeping Beauty hybrid 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 comprises an inactivating mutation in an 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 comprises an inactivating mutation in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4, ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutations in the ICP4, ICP27, and UL55 genes are deletions in 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 comprises inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutations are deletions of the coding sequences of the genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes within one or more viral loci, hi some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes within one or more of the ICP4 viral loci.In some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes within the UL41 viral locus. In some embodiments, the vector is capable of replicating within a target cell when delivered thereto. In some embodiments, the pharmaceutically acceptable carrier is suitable for topical or transdermal administration. In some embodiments, the one or more transgenes comprise an miRNA binding site. In some embodiments, the one or more transgenes are operably 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, the elongation factor-1 (EF1) promoter, and / or any combination thereof. In some embodiments, the vector comprises a transgene encoding a collagen alpha-1(VII) chain polypeptide. In some embodiments, the vector comprises two transgenes, each encoding a collagen alpha-1(VII) chain polypeptide. In some embodiments, the collagen alpha-1(VII) chain polypeptide has at least 80% sequence identity to the sequence of SEQ ID NO:2. In some embodiments, the collagen alpha-1(VII) chain polypeptide is a fragment, the fragment having at least 100 consecutive amino acids of SEQ ID NO:2. In some embodiments, the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements anchoring fibril formation in a subject when the polypeptide is expressed in one or more target cells in the subject. In some embodiments, the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements epithelial basement membrane organization and / or epithelial basal adhesion in a subject when the polypeptide is expressed in one or more target cells in the subject. In some embodiments, the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity to the sequence of SEQ ID NO:4. In some embodiments, the lysyl hydroxylase 3 polypeptide is a fragment, the fragment having at least 100 consecutive amino acids of SEQ ID NO:4.In some embodiments, the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or complements the formation of hydroxylysine residues on one or more collagen polypeptides in a subject when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells of the subject. In some embodiments, the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or complements anchoring fiber formation, epithelial basement membrane organization, and / or epithelial basal adhesion in a subject when the polypeptide is expressed in one or more target cells of the subject. In some embodiments, the vector comprises at least a first transgene and a second transgene. In some embodiments, the first transgene encodes a collagen alpha-1(VII) chain polypeptide and the second transgene encodes a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector comprises a polycistronic transgene. In some embodiments, the polycistronic transgene encodes a collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF) and a lysyl hydroxylase 3 polypeptide on 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, the collagen alpha-1(VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide are in an approximately equimolar ratio when the polypeptides are expressed in one or more target cells of a subject. In some embodiments, the collagen alpha-1(VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide enhance, increase, augment, and / or supplement anchoring fibril formation, epithelial basement membrane organization, and / or epithelial basal adhesion in a subject when the polypeptides are expressed in one or more target cells of a subject. In some embodiments, the chimeric polypeptide comprises a linker polypeptide between the collagen alpha-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 to 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 polypeptide has at least 80% sequence identity to 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. In some embodiments, the chimeric polypeptide enhances, increases, augments, and / or supplements anchoring fibril formation, epithelial basement membrane organization, and / or epithelial basal adhesion in a subject when the polypeptide is expressed in one or more target cells in the subject. In some embodiments, the pharmaceutical composition is administered once, twice, three times, four times, five times, or more times per day. In some embodiments, the pharmaceutical composition is administered to one or more affected and / or unaffected sites in a subject. In some embodiments, the skin disease or disorder is one or more of epidermolysis bullosa, skin cancer, psoriasis, lichen planus, lupus, rosacea, eczema, cutaneous candidiasis, cellulitis, impetigo, decubitus ulcers, erysipelas, ichthyosis vulgaris, dermatomyositis, acrodermatitis, stasis dermatitis, Netherton syndrome, epidermolysis bullosa simplex (LAMB3 gene), autosomal recessive congenital ichthyosis, xeroderma pigmentosum, and pemphigoid.

[0013] Other aspects of the present disclosure relate to an isolated chimeric polypeptide, i.e., an isolated chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and a linker polypeptide, wherein the collagen alpha-1(VII) chain polypeptide and the lysyl hydroxylase 3 polypeptide are separated by a linker polypeptide; a polynucleotide encoding the same; a vector comprising the polynucleotide; and a host cell comprising the vector. In some embodiments, the vector is an HSV-1 amplicon or an HSV-1 hybrid amplicon. In some embodiments, the HSV-1 hybrid amplicon is an HSV / AAV hybrid amplicon, an HSV / EBV hybrid amplicon, an HSV / EBV / RV hybrid amplicon, or an HSV / Sleeping Beauty hybrid 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 comprises an inactivating mutation in an 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 comprises an inactivating mutation in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4, ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutations in the ICP4, ICP27, and UL55 genes are deletions in 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 comprises inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutations are deletions of the coding sequences of the genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome comprises a polynucleotide within one or more viral loci. In some embodiments, the recombinant herpes simplex virus genome comprises a polynucleotide within one or more of the ICP4 viral loci. In some embodiments, the recombinant herpes simplex virus genome comprises a polynucleotide within the UL41 viral locus.

[0014] Other aspects of the present disclosure relate to vectors comprising one or more polynucleotides encoding a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, or any combination thereof, the vector being a recombinant herpes simplex virus genome, as well as host cells comprising 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 comprises an inactivating mutation in an 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 comprises an inactivating mutation in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises 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 comprises inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutations are deletions of the coding sequences of the genes.In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides within one or more viral loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides within one or more of the ICP4 viral loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides within the UL41 viral locus. In some embodiments, the vector comprises one polynucleotide encoding a collagen alpha-1(VII) chain polypeptide. In some embodiments, the vector comprises two polynucleotides encoding a collagen alpha-1(VII) chain polypeptide.

[0015] Other aspects of the present disclosure relate to methods for harvesting herpes simplex virus, wherein a vector of interest is packaged within the herpes simplex virus. In some embodiments, the method comprises contacting a host cell with a vector encoding a helper virus, contacting the host cell with an HSV-1 amplicon or an HSV-1 hybrid amplicon comprising one or more polynucleotides described herein, and harvesting the herpes simplex virus produced by the host cell. In some embodiments, the method comprises contacting a complementing host cell with a recombinant herpes simplex virus genome vector comprising one or more polynucleotides described herein, and harvesting the herpes simplex virus produced by the complementing host cell. In some embodiments, the harvested herpes simplex virus is herpes simplex type 1 virus, herpes simplex type 2 virus, or any derivative thereof.

[0016] Another aspect of the present disclosure relates to a kit comprising a pharmaceutical composition described herein and instructions for administering the pharmaceutical composition.

[0017] Another aspect of the present disclosure relates to pharmaceutical compositions comprising a vector, i.e., a virus comprising a vector containing one or more transgenes encoding a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, or a chimeric polypeptide thereof, and a pharmaceutically acceptable carrier. In some embodiments, the virus is an adenovirus, an adeno-associated virus, a retrovirus, a lentivirus, a Sendai virus, a herpes simplex virus, a vaccinia virus, or any hybrid virus thereof. In some embodiments, the virus is replication-deficient. In some embodiments, the virus is a herpes simplex virus (HSV). In some embodiments, the herpes simplex virus is a herpes simplex type 1 virus, a herpes simplex type 2 virus, or any derivative thereof. In some embodiments, the vector is an HSV-1 amplicon or an HSV-1 hybrid amplicon. In some embodiments, the HSV-1 hybrid amplicon is an HSV / AAV hybrid amplicon, an HSV / EBV hybrid amplicon, an HSV / EBV / RV hybrid amplicon, or an HSV / Sleeping Beauty hybrid 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 comprises an inactivating mutation in an 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 comprises an inactivating mutation in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises 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 in 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 comprises inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutation is a deletion in the coding sequence of the gene. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes in one or more viral loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes in one or more of the ICP4 viral loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes in the UL41 viral locus. In some embodiments, the vector is capable of replicating in a target cell when delivered thereto. 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 comprise an miRNA binding site. In some embodiments, the vector comprises a transgene encoding a collagen alpha-1(VII) chain polypeptide.In some embodiments, the vector comprises a transgene encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector comprises a transgene encoding a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the vector comprises two transgenes, each transgene encoding a collagen alpha-1(VII) chain polypeptide. In some embodiments, the collagen alpha-1(VII) chain polypeptide has at least 80% sequence identity to the sequence of SEQ ID NO:2. In some embodiments, the collagen alpha-1(VII) chain polypeptide is a fragment, the fragment having at least 100 consecutive amino acids of SEQ ID NO:2. In some embodiments, the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements anchoring fibril formation in a subject when the polypeptide is expressed in one or more target cells of the subject. In some embodiments, the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements epithelial basement membrane organization and / or epithelial basal adhesion in a subject when the polypeptide is expressed in one or more target cells of the subject. In some embodiments, the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity to the sequence of SEQ ID NO:4. In some embodiments, the lysyl hydroxylase 3 polypeptide is a fragment, the fragment having at least 100 consecutive amino acids of SEQ ID NO:4. In some embodiments, the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements the formation of hydroxylysine residues on one or more collagen polypeptides in a subject when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells in the subject. In some embodiments, the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements anchoring fiber formation, epithelial basement membrane organization, and / or epithelial basal adhesion in a subject when the polypeptide is expressed in one or more target cells in the subject. In some embodiments, the keratin type I cytoskeleton 17 polypeptide has at least 80% sequence identity to the sequence of SEQ ID NO:30.In some embodiments, the keratin type I cytoskeletal 17 polypeptide is a fragment, the fragment having at least 100 consecutive amino acids of SEQ ID NO:30. In some embodiments, the keratin type I cytoskeletal 17 polypeptide enhances, increases, augments, and / or supplements wound healing in a subject. In some embodiments, the vector comprises at least a first transgene and a second transgene. In some embodiments, the first transgene and the second transgene each encode a collagen alpha-1(VII) chain polypeptide. In some embodiments, the first transgene encodes a collagen alpha-1(VII) chain polypeptide and the second transgene encodes a lysyl hydroxylase 3 polypeptide. In some embodiments, the first transgene encodes a collagen alpha-1(VII) chain polypeptide and the second transgene encodes a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the first transgene encodes a lysyl hydroxylase 3 polypeptide and the second transgene encodes a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the vector comprises at least a first transgene, a second transgene, and a third transgene. In some embodiments, the first transgene encodes a collagen alpha-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.

[0018] Another aspect of the present disclosure relates to a method for providing preventative, palliative, or therapeutic relief of a wound, injury, or disease in a subject's skin, the method comprising administering to the subject a pharmaceutical composition comprising a vector, wherein the vector is a recombinant herpes simplex virus genome, and the pharmaceutical composition is capable of enhancing, increasing, augmenting, and / or supplementing the level of collagen alpha-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 vector, i.e., a virus comprising a vector containing one or more transgenes encoding a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, or a chimeric polypeptide 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 a herpes simplex virus (HSV). In some embodiments, the herpes simplex virus is a herpes simplex type 1 virus, a herpes simplex type 2 virus, 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 comprises an inactivating mutation in an 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 comprises an inactivating mutation in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4, ICP22, ICP27, ICP47, and UL55 genes.In some embodiments, the inactivating mutations in the ICP4, ICP27, and UL55 genes are deletions in 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 comprises inactivating mutations in the ICP4 and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4, ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutation is a deletion in the coding sequence of the gene. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP47 gene, an inactivating mutation in the UL41 gene, or an inactivating mutation in the ICP47 and UL41 genes. In some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes in one or more viral loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes in one or more of the ICP4 viral loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more transgenes in the UL41 viral locus. In some embodiments, the vector is capable of replicating in a target cell when delivered thereto. 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 comprise an miRNA binding site. In some embodiments, the vector comprises a transgene encoding a collagen alpha-1(VII) chain polypeptide.In some embodiments, the vector comprises a transgene encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector comprises a transgene encoding a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the collagen alpha-1(VII) chain polypeptide has at least 80% sequence identity to the sequence of SEQ ID NO:2. In some embodiments, the collagen alpha-1(VII) chain polypeptide is a fragment, the fragment having at least 100 consecutive amino acids of SEQ ID NO:2. In some embodiments, the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements anchoring fibril formation in a subject when the polypeptide is expressed in one or more target cells of the subject. In some embodiments, the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements epithelial basement membrane organization and / or epithelial basal adhesion in a subject when the polypeptide is expressed in one or more target cells of the subject. In some embodiments, the lysyl hydroxylase 3 polypeptide has at least 80% sequence identity to the sequence of SEQ ID NO:4. In some embodiments, the lysyl hydroxylase 3 polypeptide is a fragment, the fragment having at least 100 consecutive amino acids of SEQ ID NO:4. In some embodiments, the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements the formation of hydroxylysine residues on one or more collagen polypeptides in a subject when the lysyl hydroxylase 3 polypeptide is expressed in one or more target cells in the subject. In some embodiments, the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements anchoring fiber formation, epithelial basement membrane organization, and / or epithelial basal adhesion in a subject when the polypeptide is expressed in one or more target cells in the subject. In some embodiments, the keratin type I cytoskeleton 17 polypeptide has at least 80% sequence identity to the sequence of SEQ ID NO:30. In some embodiments, the keratin type I cytoskeleton 17 polypeptide is a fragment, the fragment having at least 100 consecutive amino acids of SEQ ID NO:30.In some embodiments, the keratin type I cytoskeletal 17 polypeptide enhances, increases, augments, and / or supplements wound healing in a subject. In some embodiments, the vector comprises at least a first transgene and a second transgene. In some embodiments, the first transgene and the second transgene each encode a collagen alpha-1(VII) chain polypeptide. In some embodiments, the first transgene encodes a collagen alpha-1(VII) chain polypeptide and the second transgene encodes a lysyl hydroxylase 3 polypeptide. In some embodiments, the first transgene encodes a collagen alpha-1(VII) chain polypeptide and the second transgene encodes a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the first transgene encodes a lysyl hydroxylase 3 polypeptide and the second transgene encodes a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the vector comprises at least a first transgene, a second transgene, and a third transgene. In some embodiments, the first transgene encodes a collagen alpha-1(VII) chain polypeptide, the second transgene encodes a lysyl hydroxylase 3 polypeptide, and the third transgene encodes a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the pharmaceutical composition is administered topically or transdermally to a subject. In some embodiments, the pharmaceutical composition is administered subcutaneously or intradermally to a subject. In some embodiments, the pharmaceutical composition is administered once, twice, three times, four times, five times, or more times per day. In some embodiments, the pharmaceutical composition is administered to one or more affected and / or unaffected areas of a subject. In some embodiments, the skin disease or disorder is one or more of epidermolysis bullosa, skin cancer, psoriasis, lichen planus, lupus, rosacea, eczema, cutaneous candidiasis, cellulitis, impetigo, decubitus ulcers, erysipelas, ichthyosis vulgaris, dermatomyositis, acrodermatitis, stasis dermatitis, Netherton syndrome, epidermolysis bullosa simplex (LAMB3 gene), autosomal recessive congenital ichthyosis, xeroderma pigmentosum, and pemphigoid. [Brief explanation of the drawings]

[0019] [Figure 1] 1A-1F show schematic diagrams of wild-type and modified herpes simplex virus genomes. [Figure 1-1] Figure 1A shows a wild-type herpes simplex virus genome, and Figure 1B shows a modified herpes simplex virus genome containing a transgene encoding a collagen alpha-1(VII) chain polypeptide. [Figure 1-2] Figure 1C shows a modified herpes simplex virus genome containing two transgenes, one encoding a collagen alpha-1(VII) chain polypeptide and the other encoding a lysyl hydroxylase 3 polypeptide, encoded on the same strand of DNA. Figure 1D shows a modified herpes simplex virus genome containing two transgenes, one encoding a collagen alpha-1(VII) chain polypeptide and the other encoding a lysyl hydroxylase 3 polypeptide, encoded in antisense orientation on opposite strands of DNA. [Figure 1-3] Figure 1E shows a modified herpes simplex virus genome containing a polycistronic transgene encoding a collagen alpha-1(VII) chain polypeptide and a lysyl hydroxylase 3 polypeptide separated by an internal ribosome entry site (IRES). Figure 1F shows a modified herpes simplex virus genome containing a transgene encoding a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a linker polypeptide, and a lysyl hydroxylase 3 polypeptide. [Figure 2] 2A-2G show additional schematic diagrams of wild-type and modified herpes simplex virus genomes. [Figure 2-1] Figure 2A shows a wild-type herpes simplex virus genome. Figure 2B shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP4 (both copies), ICP27, and UL55, as well as deletions of the promoter sequences for ICP22 and ICP47, and two transgenes encoding collagen alpha-1(VII) chain polypeptides integrated into the ICP4 locus. [Figure 2-2]Figure 2C shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP4 (both copies) and ICP22, with two transgenes encoding collagen alpha-1(VII) chain polypeptides integrated into the ICP4 locus. Figure 2D shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP0 and ICP4 (both copies), with two transgenes encoding collagen alpha-1(VII) chain polypeptides integrated into the ICP4 locus. [Figure 2-3] Figure 2E shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP0, ICP4 (both copies), and ICP22, with two transgenes encoding collagen alpha-1(VII) chain polypeptides integrated into the ICP4 locus. Figure 2F shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP0, ICP4 (both copies), ICP22, and ICP27, with two transgenes encoding collagen alpha-1(VII) chain polypeptides integrated into the ICP4 locus. [Figure 2-4] Figure 2G shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP0, ICP4 (both copies), ICP22, ICP27, and UL55, with two transgenes encoding collagen alpha-1(VII) chain polypeptides integrated into the ICP4 locus. [Figure 3] 1 shows a schematic diagram of "KB103," a replication-deficient herpes simplex type 1 virus (HSV-1) carrying a human collagen 7 (COL7A1) expression cassette. [Figure 4] Figures 4A-4B show a dose-dependent increase in COL7 transcript levels from KB103-infected RDEB human skin keratinocytes (Figure 4A) and RDEB human skin fibroblasts (Figure 4B). Transcripts were quantified relative to β-actin levels and normalized to expression in uninfected cells. [Figure 5]Figures 5A-5B show human Col7 protein expression detected in KB103-infected cells. Figure 5A shows human Col7 protein expression in uninfected normal and RDEB fibroblasts, as well as fibroblasts infected with KB103, at the indicated multiplicity of infection (MOI). Figure 5B shows human Col7 protein expression in uninfected normal and RDEB keratinocytes, as well as keratinocytes infected with KB103, at the indicated multiplicity of infection (MOI). Human GAPDH protein expression is shown as a loading control. [Figure 6] 1 shows human COL7A1 protein expression in uninfected (control) or KB103-infected (C7, MOI 3) RDEB human dermal fibroblasts (EB HDF), normal human dermal keratinocytes (normal HDK), and RDEB human dermal keratinocytes (RDEB HDK), as assessed by immunofluorescence. [Figure 7] Human Col7 and LH3 protein expression is shown in uninfected normal and RDEB human skin keratinocytes, as well as keratinocytes infected with KB103, at the indicated MOI. Human GAPDH protein expression is shown as a loading control. [Figure 8] Human TSP-1 protein expression is shown in uninfected normal and RDEB human skin fibroblasts, as well as fibroblasts infected with KB103, at the indicated MOI. Human GAPDH protein expression is shown as a loading control. [Figure 9] Figures 9A-9B show cell adhesion of uninfected (control) RDEB human skin keratinocytes and KB103-infected keratinocytes at the indicated MOI to wells treated with increasing concentrations of rat tail collagen 1 (Figure 9A) and human fibronectin (Figure 9B). [Figure 10] Immunofluorescence shows Col7 deposition in the basement membrane zone (BMZ) in KB103-infected skin equivalent organotypic cultures. [Figure 11] Quantification of viral genome copy number and human Col7 transcript levels in tissues isolated from KB103-infected mice is shown. [Figure 12]Figure 1 shows human Col7 protein expression in skin tissue from KB103-infected mice by immunofluorescence, including the initiation of human Col7 deposition in the basement membrane zone (BMZ). DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure relates, in part, to pharmaceutical compositions comprising one or more polynucleotides encoding collagen alpha-1(VII) chain polypeptides, lysyl hydroxylase 3 polypeptides, keratin type I cytoskeletal 17 polypeptides, and / or chimeric polypeptides thereof. In some embodiments, the pharmaceutical compositions comprise vectors, wherein the vectors comprise one or more transgenes encoding collagen alpha-1(VII) chain polypeptides, lysyl hydroxylase 3 polypeptides, keratin type I cytoskeletal 17 polypeptides, and / or chimeric polypeptides thereof. In some embodiments, the vectors comprise one or more transgenes suitable for enhancing, increasing, augmenting, and / or supplementing the levels of collagen alpha-1(VII) chain polypeptides and / or lysyl hydroxylase 3 polypeptides and / or keratin type I cytoskeletal 17 polypeptides in one or more cells of a subject. The present disclosure also relates, in part, to methods of providing preventative, palliative, or therapeutic relief of a wound, disorder, or disease (e.g., dystrophic epidermolysis bullosa) in a subject's skin by administering (e.g., topically or transdermally) a pharmaceutical composition described herein.

[0021] The following description sets forth example methods, parameters, etc. However, it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but rather is provided as a description of example embodiments.

[0022] general technology The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and may be performed by conventional methods, e.g., as described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds., (2003)), Series: Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J.MacPherson, B.D.Hames and G.R.Taylor eds. (1995)), Harlow and Lane, eds. (1988), Oligonucleotide Synthesis (M.J.Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (J.E.Cellis, ed., 1998) Academic Press, Animal Cell Culture (RIFreshney), ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons, Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994) and Short Protocols in Molecular Biology (Wiley and Sons, 1999), and are commonly used.

[0023] definition Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0024] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0025] As used herein, the term "about" refers to a normal range of error for each value that is readily apparent to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter per se.

[0026] As used herein, the terms "polynucleotide," "nucleic acid sequence," "nucleic acid," and variations thereof are intended to be general to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and other polymers containing a non-nucleotide backbone, provided that the polymer contains the nucleobases in an arrangement that allows for base pairing and base stacking as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, such as substitution of one or more naturally occurring nucleotides with an analog, and internucleotide modifications.

[0027] As used herein, a nucleic acid is "operatively linked" or "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous.

[0028] As used herein, the term "vector" refers to a separate element used to introduce heterologous nucleic acid into cells for either cellular expression or replication. Expression vectors include vectors capable of expressing a nucleic acid operably linked to a regulatory sequence, such as a promoter region, which can effect expression of such a nucleic acid. Thus, an expression vector can refer to a DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector that, upon introduction into an appropriate host cell, results in expression of a nucleic acid. Suitable expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic cells and those that remain episomal or that integrate into the host cell genome.

[0029] As used herein, "open reading frame" or "ORF" refers to a continuous stretch of nucleic acid, i.e., either DNA or RNA, that encodes a protein or polypeptide. Typically, the nucleic acid includes a translation initiation signal or start codon, such as ATG or AUG, and a stop codon.

[0030] As used herein, "internal ribosome entry site" or "IRES" refers to a nucleotide sequence in the middle of an mRNA sequence, e.g., after its first start codon, that allows translation initiation.

[0031] As used herein, "untranslated region" or "UTR" refers to untranslated nucleic acid at the 5' and / or 3' end of an open reading frame. The inclusion of one or more UTRs in a polynucleotide may affect post-transcriptional regulation, mRNA stability, and / or translation of the polynucleotide.

[0032] As used herein, the term "transgene" refers to a polynucleotide that can be introduced into a cell and then transcribed into RNA and translated and / or expressed under appropriate conditions. In some embodiments, a transgene confers a desired characteristic on the cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic result.

[0033] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably and may refer to a polymer of two or more amino acids.

[0034] As used herein, "subject," "host," or "individual" refers to any animal classified as a mammal, including humans, domestic and livestock animals, and zoo, sport, or pet animals such as dogs, horses, cats, cows, etc., as well as animals used in research, such as mice and rats. In some embodiments, the mammal is a human.

[0035] As used herein, "topical administration" or "administering topically" refers to the delivery of a composition to a subject by directly or otherwise contacting a formulation containing the composition with all or a portion of the subject's skin. The term encompasses several routes of administration, including, but not limited to, topical and transdermal routes. Topical administration is used as a means to deliver a composition to the epidermis or dermis of a subject, or to specific layers thereof.

[0036] As used herein, an "effective amount" is at least the minimum amount required to cause measurable improvement or prevention of one or more symptoms of a particular disorder. An effective amount is also an amount in which any toxic or adverse effects of treatment are outweighed by the therapeutically beneficial effects. For preventive use, beneficial or desired results include results such as eliminating or reducing the risk, reducing the severity, or delaying the onset of the disease, its complications, and intermediate pathological phenotypes that appear during the onset of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms caused by the disease, improving the quality of life of patients suffering from the disease, delaying the progression of the disease, and / or prolonging survival. An effective amount can be administered in one or more administrations.

[0037] Pharmaceutical Composition Polynucleotides In one aspect, provided herein are pharmaceutical compositions comprising one or more polynucleotides encoding a collagen alpha-1(VII) chain (Col7) polypeptide, a lysyl hydroxylase 3 (LH3) polypeptide, a keratin type I cytoskeletal 17 (KRT17) polypeptide, and / or chimeric polypeptides thereof. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a collagen alpha-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 cytoskeletal 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 alpha-1(VII) chain polypeptide and a lysyl hydroxylase 3 polypeptide. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a collagen alpha-1(VII) chain polypeptide and a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a lysyl hydroxylase 3 polypeptide and a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the pharmaceutical composition comprises one or more polynucleotides encoding a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and a keratin type I cytoskeletal 17 polypeptide.

[0038] In some embodiments, the pharmaceutical composition comprises a vector, the vector encoding one or more transgenes comprising a polynucleotide described herein. In some embodiments, the pharmaceutical composition comprises a vector, the vector comprising one or more transgenes encoding a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeletal 17 polypeptide, and / or chimeric polypeptides thereof. In some embodiments, the vector comprises one or more transgenes encoding a collagen alpha-1(VII) chain polypeptide. In some embodiments, the vector comprises one or more transgenes encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector comprises one or more transgenes encoding a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the vector comprises one or more transgenes encoding a chimeric polypeptide. In some embodiments, the vector comprises one or more transgenes encoding a collagen alpha-1(VII) chain polypeptide and one or more transgenes encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector comprises one or more transgenes encoding a collagen alpha-1(VII) chain polypeptide and one or more transgenes encoding a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the vector comprises one or more transgenes encoding a lysyl hydroxylase 3 polypeptide and one or more transgenes encoding a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the vector comprises one or more transgenes encoding a collagen alpha-1(VII) chain polypeptide, one or more transgenes encoding a lysyl hydroxylase 3 polypeptide, and one or more transgenes encoding a keratin type I cytoskeletal 17 polypeptide.

[0039] In some embodiments, the pharmaceutical composition comprises synthetic RNA, wherein the synthetic RNA encodes one or more transgenes comprising a polynucleotide described herein. In some embodiments, the pharmaceutical composition comprises synthetic RNA, wherein the synthetic RNA comprises one or more transgenes encoding a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeletal 17 polypeptide, and / or chimeric polypeptides thereof. In some embodiments, the synthetic RNA comprises one or more transgenes encoding a collagen alpha-1(VII) chain polypeptide. In some embodiments, the synthetic RNA comprises one or more transgenes encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the synthetic RNA comprises one or more transgenes encoding a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the synthetic RNA comprises one or more transgenes encoding a chimeric polypeptide. In some embodiments, the synthetic RNA comprises one or more transgenes encoding a collagen alpha-1(VII) chain polypeptide and one or more transgenes encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the synthetic RNA comprises one or more transgenes encoding a collagen alpha-1(VII) chain polypeptide and one or more transgenes encoding a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the synthetic RNA comprises one or more transgenes encoding lysyl hydroxylase 3 polypeptides and one or more transgenes encoding keratin type I cytoskeletal 17 polypeptides. In some embodiments, the synthetic RNA comprises one or more transgenes encoding collagen alpha-1(VII) chain polypeptides, one or more transgenes encoding lysyl hydroxylase 3 polypeptides, and one or more transgenes encoding keratin type I cytoskeletal 17 polypeptides.

[0040] Collagen alpha-1(VII) chain In some embodiments, polynucleotides of the present disclosure encode collagen alpha-1(VII) chain polypeptides. One example of a polynucleotide encoding a collagen alpha-1(VII) chain polypeptide is SEQ ID NO: 1. Polynucleotides of the present 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 to the sequence of SEQ ID NO: 1.

[0041] In some embodiments, the polynucleotide encoding a collagen alpha-1(VII) chain polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of a collagen alpha-1(VII) chain polypeptide. Polynucleotides encoding N-terminal truncations, C-terminal truncations, or fragments of a collagen alpha-1(VII) chain polypeptide include 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, at least 7500, but fewer than 8835 contiguous nucleotides of SEQ ID NO:1.

[0042] In some embodiments, the polynucleotide encoding a collagen alpha-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 a collagen alpha-1(VII) chain polypeptide is a polynucleotide encoding a polypeptide having 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 to the sequence of SEQ ID NO:2. In some embodiments, the present disclosure relates to polynucleotides encoding polypeptides that are homologs of H. sapiens collagen alpha-1(VII) chain polypeptides. Methods for identifying polypeptides that are homologs of a polypeptide of interest are well known to those of skill in the art.

[0043] In some embodiments, the polynucleotide encoding the collagen alpha-1(VII) chain polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 2. The N-terminal truncation, C-terminal truncation, or fragment 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 fewer than 2944 consecutive amino acids of SEQ ID NO: 2.

[0044] In some embodiments, a polynucleotide encoding a collagen alpha-1(VII) chain polypeptide expresses the collagen alpha-1(VII) chain polypeptide when the polynucleotide is delivered into one or more target cells of a subject. In some embodiments, expression of the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements the level of the collagen alpha-1 chain polypeptide in one or more target cells. In some embodiments, expression of the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements the function of the collagen alpha-1 chain polypeptide in one or more target cells. In some embodiments, expression of the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements the activity of the collagen alpha-1 chain polypeptide in one or more target cells. In some embodiments, expression of the collagen alpha-1(VII) chain polypeptide enhances, increases, augments, and / or supplements tethering fibril formation in a subject. In some embodiments, expression of collagen alpha-1(VII) chain polypeptides enhances, increases, augments, and / or supplements epithelial basement membrane organization and / or epithelial basal adhesion in a subject. In some embodiments, expression of collagen alpha-1(VII) chain polypeptides enhances, increases, augments, and / or supplements the integrity of the epidermal-dermal junction in a subject.

[0045] Lysyl hydroxylase 3 In some embodiments, polynucleotides of the disclosure encode a lysyl hydroxylase 3 polypeptide. One example of a polynucleotide encoding a lysyl hydroxylase 3 polypeptide is SEQ ID NO: 3. Polynucleotides of the 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 to the sequence of SEQ ID NO: 3.

[0046] In some embodiments, the polynucleotide encoding the lysyl hydroxylase 3 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the lysyl hydroxylase 3 polypeptide. Polynucleotides encoding N-terminal truncations, C-terminal truncations, or fragments of the lysyl hydroxylase 3 polypeptide include 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 fewer than 2217, contiguous nucleotides of SEQ ID NO:3.

[0047] 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 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. In some embodiments, the present disclosure relates to polynucleotides encoding polypeptides that are homologs of H. sapiens lysyl hydroxylase 3 polypeptides. Methods for identifying polypeptides that are homologs of a polypeptide of interest are well known to those of skill in the art.

[0048] In some embodiments, the polynucleotide encoding a lysyl hydroxylase 3 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 4. The N-terminal truncation, C-terminal truncation, or fragment 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 fewer than 738 contiguous amino acids of SEQ ID NO: 4.

[0049] In some embodiments, a polynucleotide encoding a lysyl hydroxylase 3 polypeptide expresses the lysyl hydroxylase 3 polypeptide when the polynucleotide is delivered into one or more target cells of a subject. In some embodiments, expression of the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements the level of the lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, expression of the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements the function of the lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, expression of the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements the activity of the lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, expression of the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements the formation of hydroxylysine residues on one or more collagen polypeptides of the subject. In some embodiments, expression of the lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements tethered fiber formation in the subject. In some embodiments, expression of a lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements epithelial basement membrane organization and / or epithelial basal adhesion in a subject, hi some embodiments, expression of a lysyl hydroxylase 3 polypeptide enhances, increases, augments, and / or supplements the integrity of the epidermal-dermal junction in a subject.

[0050] In some embodiments, a polynucleotide encoding a collagen alpha-1(VII) chain polypeptide and a polynucleotide encoding a lysyl hydroxylase 3 polypeptide are delivered to the same cell of a subject. In some embodiments, a polynucleotide encoding a collagen alpha-1(VII) chain polypeptide and a polynucleotide encoding a lysyl hydroxylase 3 polypeptide express a collagen alpha-1(VII) chain polypeptide and a lysyl hydroxylase 3 polypeptide when the polynucleotides are delivered to the same cell of a subject. In some embodiments, a polynucleotide encoding a collagen alpha-1(VII) chain polypeptide and a polynucleotide encoding a lysyl hydroxylase 3 polypeptide express a collagen alpha-1(VII) chain polypeptide and a lysyl hydroxylase 3 polypeptide in an equimolar ratio.

[0051] Keratin type I cytoskeleton 17 In some embodiments, polynucleotides of the present disclosure encode keratin type I cytoskeletal 17 polypeptides. One example of a polynucleotide encoding a keratin type I cytoskeletal 17 polypeptide is SEQ ID NO: 29. Polynucleotides of the present 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 to the sequence of SEQ ID NO: 29.

[0052] In some embodiments, the polynucleotide encoding the keratin type I cytoskeletal 17 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the keratin type I cytoskeletal 17 polypeptide. Polynucleotides encoding N-terminal truncations, C-terminal truncations, or fragments of the collagen alpha-1(VII) chain polypeptide include 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 1250, but less than 1299 contiguous nucleotides of SEQ ID NO:29.

[0053] In some embodiments, the polynucleotide encoding the keratin type I cytoskeletal 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 cytoskeletal 17 polypeptide is a polynucleotide encoding a 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: 30. In some embodiments, the present disclosure relates to polynucleotides encoding polypeptides that are homologs of H. sapiens keratin type I cytoskeletal 17 polypeptides. Methods for identifying polypeptides that are homologs of a polypeptide of interest are well known to those of skill in the art.

[0054] In some embodiments, the polynucleotide encoding the keratin type I cytoskeletal 17 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 30. The N-terminal truncation, C-terminal truncation, or fragment 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 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 425, but fewer than 432 consecutive amino acids of SEQ ID NO: 30.

[0055] In some embodiments, a polynucleotide encoding a keratin type I cytoskeletal 17 polypeptide expresses the keratin type I cytoskeletal 17 polypeptide when the polynucleotide is delivered into one or more target cells of a subject. In some embodiments, the expression of the keratin type I cytoskeletal 17 polypeptide enhances, increases, increases, and / or supplements the level of the keratin type I cytoskeletal 17 polypeptide in one or more target cells. In some embodiments, the expression of the keratin type I cytoskeletal 17 polypeptide enhances, increases, increases, and / or supplements the function of the keratin type I cytoskeletal 17 polypeptide in one or more target cells. In some embodiments, the expression of the keratin type I cytoskeletal 17 polypeptide enhances, increases, increases, and / or supplements the activity of the keratin type I cytoskeletal 17 polypeptide in one or more target cells. In some embodiments, the expression of the keratin type I cytoskeletal 17 polypeptide enhances, increases, increases, and / or supplements wound healing in a subject.

[0056] Chimeric polypeptides containing linkers In some embodiments, a polynucleotide of the present disclosure encodes a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide and a lysyl hydroxylase 3 polypeptide. In some embodiments, the polynucleotide encoding the chimeric polypeptide further comprises a polynucleotide encoding a linker polypeptide. In some embodiments, the polynucleotide encoding the linker polypeptide is a polynucleotide encoding a cleavable linker polypeptide. Examples of polynucleotides encoding cleavable linker polypeptides may include, but are not limited to, polynucleotides encoding T2A, P2A, E2A, or F2A linker polypeptides. In some embodiments, the polynucleotide encoding the linker polypeptide is a polynucleotide encoding a T2A linker polypeptide. In some embodiments, the polynucleotide encoding the linker polypeptide is a polynucleotide encoding a P2A linker polypeptide. In some embodiments, the polynucleotide encoding the linker polypeptide is a polynucleotide encoding an E2A linker polypeptide. In some embodiments, the polynucleotide encoding the linker polypeptide is a polynucleotide encoding an F2A linker polypeptide.

[0057] In some embodiments, polynucleotides of the present disclosure encode linker polypeptides. Examples of polynucleotides encoding linker polypeptides are SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11. Polynucleotides of the present 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 to the sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11.

[0058] In some embodiments, the polynucleotide encoding the linker polypeptide is a polynucleotide encoding an N-terminal truncated, C-terminal truncated, or fragment of the linker polypeptide. Polynucleotides encoding N-terminal truncated, C-terminal truncated, or fragments of the linker polypeptide include 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 fewer than 66, contiguous nucleotides of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11.

[0059] In some embodiments, the polynucleotide encoding the linker polypeptide is a polynucleotide that encodes 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 linker polypeptide is a polynucleotide that encodes a polypeptide having an amino acid sequence that 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 to the sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12.

[0060] In some embodiments, the polynucleotide encoding the linker polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of 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 truncation, C-terminal truncation, or fragment 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 fewer than 22, consecutive amino acids of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12.

[0061] In some embodiments, the polynucleotide encoding the linker polypeptide further comprises a polynucleotide encoding one or more furin cleavage sites. In some embodiments, the polynucleotide encoding the one or more furin cleavage sites encodes an amino acid sequence that is the same as or substantially similar to the sequence of a classical furin cleavage site (Arg-X-(Arg / Lys)-Arg). In some embodiments, the one or more furin cleavage sites are encoded upstream of the linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the T2A linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the T2A linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the P2A linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the P2A linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the E2A linker polypeptide. In some embodiments, one or more furin cleavage sites are encoded downstream of the E2A linker polypeptide. In some embodiments, one or more furin cleavage sites are encoded upstream of the F2A linker polypeptide. In some embodiments, one or more furin cleavage sites are encoded downstream of the F2A linker polypeptide.

[0062] In some embodiments, the polynucleotide encoding the chimeric polypeptide encodes a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a linker polypeptide, and a lysyl hydroxylase 3 polypeptide. In some embodiments, the polynucleotide encoding the chimeric polypeptide comprises, from 5' to 3', a polynucleotide encoding a collagen alpha-1(VII) chain polypeptide, a polynucleotide encoding the linker polypeptide, and a polynucleotide encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the polynucleotide encoding the chimeric polypeptide comprises, from 5' to 3', a polynucleotide encoding a lysyl hydroxylase 3 polypeptide, a polynucleotide encoding the linker polypeptide, and a polynucleotide encoding a collagen alpha-1(VII) chain polypeptide.

[0063] Examples of polynucleotides encoding chimeric polypeptides comprising a collagen alpha-1(VII) chain polypeptide, a linker polypeptide, and a 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. Polynucleotides of the present 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 to 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.

[0064] In some embodiments, the polynucleotide encoding the chimeric polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the chimeric polypeptide. The polynucleotide encoding the N-terminal truncation, C-terminal truncation, or fragment of the chimeric polypeptide is 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 600, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, 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 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 1000, at least 1250, at least 1500, at least 175 ...500, at least 1000, at least 1250, at least 1500, at least 1 Polynucleotides having 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 fewer than 11121 contiguous nucleotides.

[0065] In some embodiments, the polynucleotide encoding a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a linker polypeptide, and a lysyl 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 the chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a linker polypeptide, and a lysyl hydroxylase 3 polypeptide is a polynucleotide encoding a polypeptide having 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 to 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.

[0066] In some embodiments, the polynucleotide encoding the chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a linker polypeptide, and a lysyl hydroxylase 3 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of 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. N-terminal truncations, C-terminal truncations, or fragments may comprise 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 fewer than 3706, contiguous amino acids 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.

[0067] In some embodiments, a polynucleotide encoding a chimeric polypeptide expresses the chimeric polypeptide when the polynucleotide is delivered into one or more target cells of a subject. In some embodiments, the chimeric polypeptide is cleaved after being expressed in one or more target cells. In some embodiments, the chimeric polypeptide is cleaved within the 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 alpha-1(VII) chain polypeptide and the other containing a lysyl hydroxylase 3 polypeptide. In some embodiments, expression of the chimeric polypeptide enhances, increases, augments, and / or supplements the level of collagen alpha-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, expression of the chimeric polypeptide enhances, increases, augments, and / or supplements the function of collagen alpha-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, expression of the chimeric polypeptide enhances, increases, augments, and / or complements the activity of a collagen alpha-1 chain polypeptide and / or a lysyl hydroxylase 3 polypeptide in one or more target cells. In some embodiments, expression of the chimeric polypeptide enhances, increases, augments, and / or complements the formation of hydroxylysine residues on one or more collagen polypeptides in a subject. In some embodiments, expression of the chimeric polypeptide enhances, increases, augments, and / or complements anchoring fiber formation in a subject. In some embodiments, expression of the chimeric polypeptide enhances, increases, augments, and / or complements epithelial basement membrane organization and / or epithelial-basal adhesion in a subject. In some embodiments, expression of the chimeric polypeptide enhances, increases, augments, and / or complements the integrity of the epidermal-dermal junction in a subject.

[0068] Polynucleotides of the present disclosure may be codon-optimized. In some embodiments, polynucleotides of the present disclosure are codon-optimized for human cells. In some embodiments, polynucleotides of the present disclosure are codon-optimized for mouse cells. In some embodiments, polynucleotides of the present disclosure are codon-optimized for rat cells. In some embodiments, polynucleotides of the present disclosure are codon-optimized for hamster cells. In some embodiments, polynucleotides of the present disclosure are codon-optimized for dog cells. In some embodiments, polynucleotides of the present disclosure are codon-optimized for yeast cells. In some embodiments, polynucleotides of the present disclosure are codon-optimized for bacterial cells. Polynucleotides of the present disclosure may be DNA polynucleotides, RNA polynucleotides, or a combination of one or more DNA polynucleotides and one or more RNA polynucleotides.

[0069] vector In some aspects, the present disclosure relates to vectors, preferably expression vectors, containing one or more polynucleotides described herein. In some embodiments, the vector is a DNA vector. Generally, any vector suitable for maintaining, propagating, or expressing a polynucleotide to produce one or more polypeptides in a subject may be used. Examples of suitable vectors include, but are not limited to, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus, vaccinia virus, Sindbis virus, measles virus, herpes virus, lentivirus, retrovirus, adeno-associated virus vectors, etc.). In some embodiments, the vector is capable of autonomous replication in a host cell. In some embodiments, the vector is not capable of autonomous replication in a host cell. In some embodiments, the vector is capable of integration into host DNA. Methods for generating vectors containing one or more polynucleotides of interest are well known to those skilled in the art.

[0070] 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 of making the vectors, are generally 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 hybrid amplicon. Examples of HSV-1 hybrid amplicons may include, but are not limited to, an HSV / AAV hybrid amplicon, an HSV / EBV hybrid amplicon, an HSV / EBV / RV hybrid amplicon, and an HSV / Sleeping Beauty hybrid amplicon. In some embodiments, the vector is an HSV / AAV hybrid amplicon. In some embodiments, the vector is an HSV / EBV hybrid amplicon. In some embodiments, the vector is an HSV / EBV / RV hybrid amplicon. In some embodiments, the vector is an HSV / Sleeping Beauty hybrid amplicon.

[0071] In some embodiments, the herpes simplex virus vector is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is engineered to reduce or eliminate expression of one or more toxic herpes simplex virus genes. Methods for engineering recombinant herpes simplex virus genomes are generally described in WO 2015 / 009952. In some embodiments, the recombinant herpes simplex virus genome comprises 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 comprises 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 comprises inactivating mutations in the ICP0 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies) gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises 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.

[0072] In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4 (one or both copies), ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4 (one or both copies), ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutations in the ICP4 (one or both copies), ICP27, and / or UL55 genes are deletions in the coding sequences of the ICP4 (one or both 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 not transcriptionally active). In some embodiments, the recombinant herpes simplex virus genome comprises deletions in the coding sequences of the ICP4 (one or both 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 comprises 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.

[0073] In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4 (one or both copies) and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 and ICP4 (one or both copies) genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4 (one or both copies), and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4 (one or both copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4 (one or both copies), ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutations in the ICP0, ICP4 (one or both copies), ICP22, ICP27, and / or UL55 genes comprise deletions in the coding sequences of the ICP0, ICP4 (one or both 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 both copies), ICP22, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP47 gene. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome further comprises inactivating mutations in the ICP47 gene and 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.

[0074] In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4 (one or both copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4 (one or both copies), ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0, ICP4 (one or both 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.

[0075] In some embodiments, the recombinant herpes simplex virus genome comprises 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 ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41 and UL55 herpes simplex virus loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or more of the viral ICP4 loci (e.g., a recombinant virus having a polynucleotide encoding Col7 at one or both ICP4 loci, a recombinant virus having a polynucleotide encoding LH3 at one or both ICP4 loci, a recombinant virus having a polynucleotide encoding KRT17 at one or both ICP4 loci, a recombinant virus having a polynucleotide encoding Col7 at one ICP4 locus and a polynucleotide encoding KRT17 at the other ICP4 locus, a recombinant virus having a polynucleotide encoding Col7 at one ICP4 locus and a polynucleotide encoding LH3 at the other ICP4 locus, a recombinant virus having a polynucleotide encoding LH3 at one ICP4 locus and a polynucleotide encoding KRT17 at the other ICP4 locus, etc.). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral UL41 locus. In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral ICP47 locus.In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure in one or more of the viral ICP4 loci and one or more polynucleotides of the present disclosure in the viral UL41 locus (e.g., a recombinant virus having a polynucleotide encoding Col7 at one or both of the ICP4 loci and a polynucleotide encoding LH3 at the UL41 locus, a recombinant virus having a polynucleotide encoding Col7 at one or both of the ICP4 loci and a polynucleotide encoding Col7 at the UL41 locus, a recombinant virus having a polynucleotide encoding Col7 at one or both of the ICP4 loci and a polynucleotide encoding KRT17 at the UL41 locus, a recombinant virus having a polynucleotide encoding LH3 at one or both of the ICP4 loci and a polynucleotide encoding LH3 at the UL41 locus, recombinant viruses having a polynucleotide encoding LH3 at one or both ICP4 loci and a polynucleotide encoding Col7 at the UL41 locus; recombinant viruses having a polynucleotide encoding LH3 at one or both ICP4 loci and a polynucleotide encoding KRT17 at the UL41 locus; recombinant viruses having a polynucleotide encoding KRT17 at one or both ICP4 loci and a polynucleotide encoding LH3 at the UL41 locus; recombinant viruses having a polynucleotide encoding KRT17 at one or both ICP4 loci and a polynucleotide encoding Col7 at the UL41 locus; recombinant viruses having a polynucleotide encoding KRT17 at one or both ICP4 loci and a polynucleotide encoding KRT17 at the UL41 locus; recombinant viruses having a polynucleotide encoding KRT17 at one or both ICP4 loci and a polynucleotide encoding KRT17 at the UL41 locus; recombinant viruses having a polynucleotide encoding KRT17 at one or both ICP4 loci and a polynucleotide encoding KRT17 at the UL41 locus

[0076] A vector may contain a polynucleotide of the present disclosure in a form suitable for expression of the polynucleotide in a host cell. An expression vector may contain one or more regulatory sequences operably 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 include, but are not limited to, enhancer sequences from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, insulin, etc.) and enhancer sequences from eukaryotic viruses (e.g., the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, etc.). Examples of promoters suitable for transcription in mammalian host cells may include, but are not limited to, promoters obtained from the genome of viruses (such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40)), or heterologous mammalian promoters (such as from actin promoters, immunoglobulin promoters, heat shock promoters), provided that such promoters are compatible with the host cell. In some embodiments, the polynucleotides of the present disclosure are operably 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 a constitutive promoter, a tissue-specific promoter, a temporal promoter, a spatial promoter, an inducible promoter, and a repressible promoter. Regulatory sequences may include those that direct constitutive expression of a 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 to be contacted with a polynucleotide of the present disclosure, the expression level of the desired protein, etc. The expression vector of the present disclosure can be introduced into a host cell to produce a protein or polypeptide (e.g., collagen alpha-1(VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, keratin type I cytoskeleton 17 polypeptide, chimeric polypeptide, etc.) encoded by a polynucleotide as described herein.

[0077] In some embodiments, a vector of the present disclosure comprises one or more transgenes comprising one or more polynucleotides described herein. The one or more transgenes may be inserted in any orientation within the vector. When a vector comprises two or more transgenes (e.g., two or more, three or more, etc.), the transgenes may be inserted in the same or opposite orientation relative to each other. Without wishing to be bound by theory, introducing two transgenes into a vector in antisense orientation may help avoid read-through and ensure proper expression of each transgene. In some embodiments, a vector comprises one or more transgenes encoding a polypeptide selected from the group consisting of a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, and / or chimeric polypeptides thereof. In some embodiments, a vector comprises a single transgene encoding a collagen alpha-1(VII) chain polypeptide. In some embodiments, a vector comprises two transgenes, each encoding a collagen alpha-1(VII) chain polypeptide. In some embodiments, a vector comprises three transgenes, each encoding a collagen alpha-1(VII) chain polypeptide. In some embodiments, the vector comprises a single transgene encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector comprises two transgenes, each encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector comprises three transgenes, each encoding a lysyl hydroxylase 3 polypeptide. In some embodiments, the vector comprises a single transgene encoding a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the vector comprises two transgenes, each encoding a keratin type I cytoskeletal 17 polypeptide. In some embodiments, the vector comprises three transgenes, each encoding a keratin type I cytoskeletal 17 polypeptide.In some embodiments, the vector comprises a single transgene encoding a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeletal 17 polypeptide.

[0078] In some embodiments, the vector comprises 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 alpha-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 alpha-1(VII) chain polypeptide. In some embodiments, at least a first transgene encodes a collagen alpha-1(VII) chain polypeptide and at least a second transgene encodes a keratin type I cytoskeletal 17 polypeptide. In some embodiments, at least a first transgene encodes a keratin type I cytoskeletal 17 polypeptide and at least a second transgene encodes a collagen alpha-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 cytoskeletal 17 polypeptide. In some embodiments, at least a first transgene encodes a keratin type I cytoskeletal 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 alpha-1(VII) chain polypeptide and at least a second transgene encodes a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeletal 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 alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeletal 17 polypeptide.In some embodiments, at least a first transgene encodes a keratin type I cytoskeletal 17 polypeptide and at least a second transgene encodes a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeletal 17 polypeptide.

[0079] In some embodiments, the vector comprises at least three transgenes (e.g., three, four, five, six, seven, or more transgenes), in which at least a first transgene encodes a collagen alpha-1(VII) chain polypeptide, at least a second transgene encodes a lysyl hydroxylase 3 polypeptide, and at least a third transgene encodes a keratin type I cytoskeleton 17 polypeptide.

[0080] In some embodiments, the vector comprises a polycistronic transgene. In some embodiments, the polycistronic transgene encodes a collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF) and a lysyl hydroxylase 3 polypeptide on a second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a lysyl hydroxylase 3 polypeptide on a first open reading frame (ORF) and a collagen alpha-1(VII) chain polypeptide on a second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF) and a keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a keratin type I cytoskeletal polypeptide on a first open reading frame (ORF) and a collagen alpha-1(VII) chain polypeptide on a second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a lysyl hydroxylase 3 polypeptide on a first open reading frame (ORF) and a keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a keratin type I cytoskeletal polypeptide on a first open reading frame (ORF) and a lysyl hydroxylase 3 polypeptide on a second open reading frame (ORF).

[0081] In some embodiments, the polycistronic transgene encodes a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF) and a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeletal 17 polypeptide on a first open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF). In some embodiments, the first and second ORFs are separated by an internal ribosome entry site (IRES).

[0082] In some embodiments, the polycistronic transgene encodes a collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF), a keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF), and a lysyl hydroxylase 3 polypeptide on a third open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF), a lysyl hydroxylase 3 polypeptide on a second open reading frame (ORF), and a keratin type I cytoskeletal 17 polypeptide on a third open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a lysyl hydroxylase 3 polypeptide on a first open reading frame (ORF), a collagen alpha-1(VII) chain polypeptide on a second open reading frame (ORF), and a keratin type I cytoskeletal 17 polypeptide on a third open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a lysyl hydroxylase 3 polypeptide on a first open reading frame (ORF), a keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF), and a collagen alpha-1(VII) chain polypeptide on a third open reading frame (ORF). In some embodiments, the polycistronic transgene encodes a keratin type I cytoskeletal 17 polypeptide on a first open reading frame (ORF), a lysyl hydroxylase 3 polypeptide on a second open reading frame (ORF), and a collagen alpha-1(VII) chain polypeptide on a third open reading frame (ORF).In some embodiments, the polycistronic transgene encodes a keratin type I cytoskeletal 17 polypeptide on a first open reading frame (ORF), a collagen alpha-1(VII) chain polypeptide on a second open reading frame (ORF), and a lysyl hydroxylase 3 polypeptide on a third open reading frame (ORF). In some embodiments, the first, second, and third ORFs are separated by an internal ribosome entry site (IRES).

[0083] Examples of suitable IRES include IRES derived from viruses (e.g., IRES derived from poliovirus, rhinovirus, encephalomyocarditis virus, foot-and-mouth disease virus, hepatitis C virus, classical swine cholera virus, Rous sarcoma virus, human immunodeficiency virus, cricket paralysis virus, Kaposi's sarcoma-associated herpesvirus, etc.) and IRES derived from cellular mRNAs (e.g., IRES derived from growth factor mRNAs such as fibroblast growth factor 2, platelet-derived growth factor B, and vascular endothelial growth factor, IRES derived from transcription factor mRNAs such as antennapedia, ultrapithorax, and NF-κB inhibitor, IRES derived from oncogene mRNAs such as c-myc, pim-1, and protein kinase p58, etc.). PITSLRE Examples of suitable IRES include, but are not limited to, IRESs derived from

[0084] The vectors of the present disclosure may 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 polypeptide tags, introns, 5' and 3' UTRs, etc. Examples of suitable polypeptide tags may include, but are not limited to, purification tags such as his tags, flag tags, maltose-binding protein, and glutathione-S-transferase tags, detection tags such as tags that may be photometrically detected (e.g., red fluorescent protein) and tags with detectable enzymatic activity (e.g., alkaline phosphatase), secretion sequences, leader sequences, and / or stabilization sequences, tags containing protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites), and any combination thereof. In some embodiments, the 5' and / or 3' UTRs increase the stability, localization, and / or translation efficiency of the polynucleotide. In some embodiments, the 5' and / or 3' UTRs are modified to increase the stability, localization, and / or translation efficiency of one or more polynucleotides. 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 contain elements (e.g., one or more miRNA binding sites) that may block or reduce off-target transgene expression (e.g., inhibiting expression in particular cell types (e.g., neuronal cells), at particular times in the cell cycle, at particular developmental stages, etc.). In some embodiments, the 5' and / or 3' UTRs contain elements (e.g., one or more miRNA binding sites) that may enhance transgene expression in particular cell types.

[0085] Synthetic RNA polynucleotides In some aspects, the present 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 adjacent to 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' UTRs contain elements (e.g., miRNA binding sites, etc.) that may limit off-target expression (e.g., inhibit expression in specific cell types (e.g., neuronal cells), at specific times in the cell cycle, at specific developmental stages, etc.). In some embodiments, the 5' UTR contains a Kozak sequence. In some embodiments, the Kozak sequence is the same as or substantially similar to a Kozak consensus sequence. Methods for generating synthetic mRNA polynucleotides containing one or more polynucleotides of interest are well known to those of skill in the art.

[0086] In some embodiments, the synthetic mRNA polynucleotides of the present disclosure comprise one or more modified ribonucleotides. Examples of modified ribonucleotides include 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-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, 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-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio N4-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidineN4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine , N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-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-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 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-N 4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine,2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methyl Cytidine, 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-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N 4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza- These may include, but are not limited to, 8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.

[0087] In some embodiments, the polynucleotide encoding the collagen alpha-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 alpha-1(VII) chain polypeptide and the polynucleotide encoding the keratin type I cytoskeletal 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 cytoskeletal 17 polypeptide are contained within two separate synthetic mRNA polynucleotides. In some embodiments, the polynucleotide encoding the collagen alpha-1(VII) chain polypeptide, the polynucleotide encoding the lysyl hydroxylase 3 polypeptide, and the polynucleotide encoding the keratin type I cytoskeletal 17 polypeptide are contained within three separate synthetic mRNA polynucleotides.

[0088] In some embodiments, the polynucleotide encoding the collagen alpha-1(VII) chain polypeptide, the polynucleotide encoding the lysyl hydroxylase 3 polypeptide, and / or the polynucleotide encoding the keratin type I cytoskeletal 17 polypeptide are a single contiguous polynucleotide contained within a single synthetic mRNA polynucleotide. In some embodiments, the single contiguous polynucleotide encodes the collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF) and the lysyl hydroxylase 3 polypeptide on a second open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the single contiguous polynucleotide encodes the lysyl hydroxylase 3 polypeptide on a first open reading frame (ORF) and the collagen alpha-1(VII) chain polypeptide on a second open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the single contiguous polynucleotide encodes the collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF) and the keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the single contiguous polynucleotide encodes a keratin type I cytoskeletal polypeptide on a first open reading frame (ORF) and a collagen alpha-1(VII) chain polypeptide on a second open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the single contiguous polynucleotide encodes a lysyl hydroxylase 3 polypeptide on a first open reading frame (ORF) and a keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the single contiguous polynucleotide encodes a keratin type I cytoskeletal polypeptide on a first open reading frame (ORF) and a lysyl hydroxylase 3 polypeptide on a second open reading frame (ORF) in a single synthetic mRNA.In some embodiments, the single contiguous polynucleotide encodes a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF) and a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the single contiguous polynucleotide encodes a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeletal 17 polypeptide on a first open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the single contiguous polynucleotide encodes a chimeric polypeptide comprising a keratin type I cytoskeletal 17 polypeptide on a first open reading frame (ORF) and a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the two ORFs are separated by an IRES.

[0089] In some embodiments, the single contiguous polynucleotide encodes a collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF), a keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF), and a lysyl hydroxylase 3 polypeptide on a third open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the single contiguous polynucleotide encodes a collagen alpha-1(VII) chain polypeptide on a first open reading frame (ORF), a lysyl hydroxylase 3 polypeptide on a second open reading frame (ORF), and a keratin type I cytoskeletal 17 polypeptide on a third open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the single contiguous polynucleotide encodes a lysyl hydroxylase 3 polypeptide on a first open reading frame (ORF), a collagen alpha-1(VII) chain polypeptide on a second open reading frame (ORF), and a keratin type I cytoskeletal 17 polypeptide on a third open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the single contiguous polynucleotide encodes a lysyl hydroxylase 3 polypeptide on a first open reading frame (ORF), a keratin type I cytoskeletal 17 polypeptide on a second open reading frame (ORF), and a collagen alpha-1(VII) chain polypeptide on a third open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the single contiguous polynucleotide encodes a keratin type I cytoskeletal 17 polypeptide on a first open reading frame (ORF), a lysyl hydroxylase 3 polypeptide on a second open reading frame (ORF), and a collagen alpha-1(VII) chain polypeptide on a third open reading frame (ORF) in a single synthetic mRNA.In some embodiments, the single contiguous polynucleotide encodes a keratin type I cytoskeletal 17 polypeptide on a first open reading frame (ORF), a collagen alpha-1(VII) chain polypeptide on a second open reading frame (ORF), and a lysyl hydroxylase 3 polypeptide on a third open reading frame (ORF) in a single synthetic mRNA. In some embodiments, the first, second, and third ORFs are separated by an internal ribosome entry site (IRES).

[0090] Examples of suitable IRES include IRES derived from viruses (e.g., IRES derived from poliovirus, rhinovirus, encephalomyocarditis virus, foot-and-mouth disease virus, hepatitis C virus, classical swine cholera virus, Rous sarcoma virus, human immunodeficiency virus, cricket paralysis virus, Kaposi's sarcoma-associated herpesvirus, etc.) and IRES derived from cellular mRNAs (e.g., IRES derived from growth factor mRNAs such as fibroblast growth factor 2, platelet-derived growth factor B, and vascular endothelial growth factor, IRES derived from transcription factor mRNAs such as antennapedia, ultrapithorax, and NF-κB inhibitor, IRES derived from oncogene mRNAs such as c-myc, pim-1, and protein kinase p58, etc.). PITSLRE Examples of suitable IRES include, but are not limited to, IRESs derived from

[0091] In some embodiments, a polynucleotide encoding any of the chimeric polypeptides comprising a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, and / or a keratin type I cytoskeletal 17 polypeptide described herein is encoded on a single ORF within a synthetic mRNA polynucleotide.

[0092] The synthetic mRNA polynucleotides of the present disclosure may 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, purification tags such as his tags, flag tags, maltose-binding protein, and glutathione-S-transferase tags, detection tags such as tags that may be detected photometrically (e.g., red fluorescent protein) and tags with detectable enzymatic activity (e.g., alkaline phosphatase), secretion sequences, leader sequences, and / or stabilization sequences, tags containing protease cleavage sites (e.g., furin cleavage sites), and any combination thereof.

[0093] Delivery Vehicle Certain aspects of the present disclosure relate to pharmaceutical compositions comprising a delivery vehicle comprising one or more polynucleotides described herein. In some embodiments, the delivery vehicle is suitable for delivering one or more polynucleotides into one or more target cells.

[0094] In some embodiments, the delivery vehicle is a virus. Examples of viral delivery vehicles may 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-deficient. In some embodiments, the virus is replication-competent. In some embodiments, the virus is modified to change its tissue tropism compared to the tissue tropism of unmodified wild-type virus. Methods for producing viruses containing one or more polynucleotides are well known to those skilled in the art.

[0095] In some embodiments, the viral delivery vehicle is a herpes simplex virus. The herpes simplex virus delivery vehicle may be made, for example, by the process disclosed 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) mutant 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 compared to wild-type herpes simplex virus. In some embodiments, the herpes simplex virus is a herpes simplex type 1 virus, a herpes simplex type 2 virus, or any derivative thereof. In some embodiments, the virus is a herpes simplex type 1 virus. In some embodiments, the virus is a herpes simplex type 2 virus.

[0096] In some embodiments, the delivery vehicle is a non-viral delivery vehicle. In some embodiments, the non-viral delivery vehicle is a chemical delivery vehicle (chemical delivery reagent). Examples of chemical delivery vehicles may include, but are not limited to, calcium phosphate, dendrimers, liposomes (cationic liposomes, non-cationic liposomes, and mixtures), exosomes, charged lipids, and cationic polymers (DEAE-dextran, polyethyleneimine, etc.). In some embodiments, the non-viral delivery vehicle is a non-chemical delivery vehicle. Examples of non-chemical delivery vehicles may include, but are not limited to, electroporation, nucleofection, sonoporation, optical transfection, and particle-based vehicles (gene gun, magnetically assisted transfection, impale infection, particle bombardment, etc.). In some embodiments, the non-viral delivery vehicle is a dendrimer, liposome, exosome, charged lipid, or cationic polymer. In some embodiments, the non-viral delivery vehicle is a dendrimer. In some embodiments, the non-viral delivery vehicle is a liposome. In some embodiments, the non-viral delivery vehicle is an exosome. In some embodiments, the non-viral delivery vehicle is a charged lipid. In some embodiments, the non-viral delivery vehicle is a cationic polymer. Methods for preparing one or more polynucleotides of interest in complex with a non-viral delivery vehicle are well known to those skilled in the art.

[0097] Pharmaceutically acceptable carrier Certain aspects of the present disclosure relate to pharmaceutical compositions comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is sufficient for topical and / or transdermal administration / application. In some embodiments, the pharmaceutically acceptable carrier is 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 nontoxic to recipients at the dosages and concentrations employed and may include buffers, such as phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or the like. These may include, but are not limited to, immunoglobulins and the like; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, 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 thorough discussion of pharmaceutically acceptable carriers is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991).

[0098] In some embodiments, the pharmaceutically acceptable carrier is suitable for topical or transdermal application / administration. Examples of carriers suitable for use in topical or transdermal application / administration may include, but are not limited to, ointments, pastes, creams, suspensions, emulsions, fatty ointments, gels, powders, lotions, solutions, sprays, patches, microneedle arrays, and inhalants. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of an ointment, paste, cream, suspension, emulsion, fatty ointment, gel, powder, lotion, solution, spray, and inhalant. In some embodiments, the pharmaceutically acceptable carrier comprises an ointment. In some embodiments, the pharmaceutically acceptable carrier comprises a paste. In some embodiments, the pharmaceutically acceptable carrier comprises a cream. In some embodiments, the pharmaceutically acceptable carrier comprises a suspension. In some embodiments, the pharmaceutically acceptable carrier comprises an emulsion. In some embodiments, the pharmaceutically acceptable carrier comprises a gel. In some embodiments, the pharmaceutically acceptable carrier comprises a powder. In some embodiments, the pharmaceutically acceptable carrier comprises a lotion. In some embodiments, the pharmaceutically acceptable carrier comprises a solution. In some embodiments, the pharmaceutically acceptable carrier comprises a propellant. In some embodiments, the pharmaceutically acceptable carrier comprises an inhalant. In some embodiments, the pharmaceutical carrier comprises a patch (e.g., a patch that adheres to the skin). In some embodiments, the pharmaceutically acceptable carrier comprises a microneedle array. Methods for making and using microneedle arrays suitable for delivering pharmaceutical compositions are generally known in the art (Kim Y. et al. "Microneedles for drug and vaccine delivery". Advanced Drug Delivery Reviews 2012, 64(14):1547-68).

[0099] In some embodiments, the pharmaceutically acceptable carrier comprises a combination of two, three, four, five or more different pharmaceutically acceptable carriers suitable for topical or transdermal application / administration.

[0100] In some embodiments, the pharmaceutically acceptable carrier further comprises one or more additional ingredients, such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, maize starch, polyethylene glycol, sodium benzoate). , sodium acetate, etc.), disintegrating agents (e.g., starch, sodium starch glycolate, etc.), wetting agents (e.g., sodium lauryl sulfate, etc.), saline, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, sweeteners, flavorings, fragrances, colorants, moisturizers, sunscreens, antibacterial agents, agents that can stabilize polynucleotides or prevent their degradation, etc., but are not limited to these.

[0101] Pharmaceutical compositions and preparations as described herein can be prepared by mixing the delivery vehicle comprising one or more polynucleotides as described herein with one or more pharmaceutically acceptable carriers.The preparations used for in vivo administration are generally sterile.Sterility can be easily achieved, for example, by filtering through sterile filtration membrane.

[0102] Treatment method The present disclosure relates, in part, to pharmaceutical compositions and methods of use for providing preventative, palliative, or therapeutic relief of wounds, disorders, or diseases in a subject's skin. Examples of skin diseases or disorders may include, but are not limited to, epidermolysis bullosa, skin cancer, psoriasis, lichen planus, lupus, rosacea, eczema, cutaneous candidiasis, cellulitis, impetigo, decubitus ulcers, erysipelas, ichthyosis vulgaris, dermatomyositis, acrodermatitis, stasis dermatitis, Netherton syndrome, epidermolysis bullosa simplex (LAMB3 gene), autosomal recessive congenital ichthyosis, xeroderma pigmentosum, and pemphigoid. In some embodiments, the skin disease or disorder is epidermolysis bullosa. In some embodiments, the subject has or is at risk of developing one or more symptoms of epidermolysis bullosa.

[0103] The polynucleotides and pharmaceutical compositions described herein are useful for providing preventative, palliative, or therapeutic relief of wounds, disorders, or diseases in a subject's skin, including treatment for one or more symptoms of epidermolysis bullosa (e.g., recessive dystrophic epidermolysis bullosa, dominant dystrophic epidermolysis bullosa, etc.). The pharmaceutical compositions of the present disclosure may be administered by any suitable method known in the art, including, but not limited to, oral, sublingual, buccal, topical, rectal, or inhalation administration, transdermal, subcutaneous, intradermal, intravenous (IV), intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravaginal, intravitreal, intraarticular, periarticular, local, epicutaneous, or any combination thereof. The pharmaceutical compositions may be delivered to an individual by a variety of routes, including, but not limited to, subcutaneous, intradermal, topical, transdermal, and transmucosal administration. Thus, the present disclosure also encompasses methods of delivering any of the polynucleotides or pharmaceutical compositions described herein to an individual (such as an individual having or at risk of developing epidermolysis bullosa).

[0104] In some embodiments, preventive, palliative, or therapeutic relief of a wound, disorder, or disease in a subject's skin is provided, comprising administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, enhancing, and / or supplementing the level of collagen alpha-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 and / or subcutaneously. In some embodiments, the pharmaceutical composition is administered topically and / or transdermally. In some embodiments, preventive, palliative, or therapeutic relief of a wound, disorder, or disease in a subject's skin is provided, comprising topically administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, enhancing, and / or supplementing the level of collagen alpha-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 of the pharmaceutical compositions described herein. In some embodiments, the individual is suffering from epidermolysis bullosa. In some embodiments, the individual is afflicted with dystrophic epidermolysis bullosa. In some embodiments, the individual is afflicted with dominant dystrophic epidermolysis bullosa. In some embodiments, the individual is afflicted with recessive dystrophic epidermolysis bullosa. In some embodiments, the pharmaceutical composition is administered once, twice, three times, four times, five times, or more times per day. In some embodiments, the pharmaceutical composition is administered to one or more affected areas of the individual. In some embodiments, the pharmaceutical composition is administered to one or more unaffected areas of the individual.

[0105] 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., recessive dystrophic epidermolysis bullosa, dominant 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 mucous membranes, skin scarring, mucous membrane scarring, skin erosion, fingernail and / or toenail deformities, loss of fingernails and / or toenails, internal blisters (including on the vocal cords, esophagus, and upper respiratory tract), skin thickening (especially skin thickening on the palms and soles), scalp blisters, scalp scarring, hair loss (cicatricial alopecia), thin-appearing skin, atrophic scarring, milia, dental conditions (such as tooth decay and hypoplastic enamel), joint deformities, fused fingers and toes, and swallowing difficulties.

[0106] In some embodiments, methods are provided for therapeutically treating an individual suffering from epidermolysis bullosa, comprising administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, augmenting, and / or supplementing the level of collagen alpha-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 and / or subcutaneously. In some embodiments, the pharmaceutical composition is administered topically and / or transdermally. In some embodiments, methods are provided for therapeutically treating an individual suffering from epidermolysis bullosa, comprising topically administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, augmenting, and / or supplementing the level of collagen alpha-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 of the pharmaceutical compositions described herein. In some embodiments, the individual is suffering from dystrophic epidermolysis bullosa. In some embodiments, the individual is afflicted with dominant dystrophic epidermolysis bullosa. In some embodiments, the individual is afflicted with recessive dystrophic epidermolysis bullosa. In some embodiments, the pharmaceutical composition is administered once, twice, three times, four times, five times, or more times per day. In some embodiments, the pharmaceutical composition is administered to one or more affected areas of the individual. In some embodiments, the pharmaceutical composition is administered to one or more unaffected areas of the individual.

[0107] In some embodiments, methods are provided for prophylactically treating an individual suffering from epidermolysis bullosa, comprising administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, enhancing, and / or supplementing the level of collagen alpha-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 and / or subcutaneously. In some embodiments, the pharmaceutical composition is administered topically and / or transdermally. In some embodiments, methods are provided for prophylactically treating an individual suffering from epidermolysis bullosa, comprising topically administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, enhancing, and / or supplementing the level of collagen alpha-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 of the pharmaceutical compositions described herein. In some embodiments, the individual is suffering from dystrophic epidermolysis bullosa. In some embodiments, the individual is afflicted with dominant dystrophic epidermolysis bullosa. In some embodiments, the individual is afflicted with recessive dystrophic epidermolysis bullosa. In some embodiments, the pharmaceutical composition is administered once, twice, three times, four times, five times, or more times per day. In some embodiments, the pharmaceutical composition is administered to one or more affected areas of the individual. In some embodiments, the pharmaceutical composition is administered to one or more unaffected areas of the individual.

[0108] In some embodiments, methods are provided for prophylactically treating an individual at risk of developing epidermolysis bullosa, comprising administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, enhancing, and / or supplementing the levels of collagen alpha-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 and / or subcutaneously. In some embodiments, the pharmaceutical composition is administered topically and / or transdermally. In some embodiments, methods are provided for prophylactically treating an individual at risk of developing epidermolysis bullosa, comprising topically administering an effective amount of a pharmaceutical composition capable of enhancing, increasing, enhancing, and / or supplementing the levels of collagen alpha-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 of the pharmaceutical compositions 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 dominant dystrophic epidermolysis bullosa. In some embodiments, the individual is at risk of developing recessive dystrophic epidermolysis bullosa. In some embodiments, the pharmaceutical composition is administered once, twice, three times, four times, five times, or more times per day. In some embodiments, the pharmaceutical composition is administered to one or more affected areas of the individual. In some embodiments, the pharmaceutical composition is administered to one or more unaffected areas of the individual.

[0109] In some embodiments, administering an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, augments, and / or supplements the level of collagen alpha-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeletal 17 polypeptide in one or more cells of the individual. In some embodiments, administering an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, augments, and / or supplements the function of collagen alpha-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeletal 17 polypeptide in one or more cells of the individual. In some embodiments, administering an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, augments, and / or supplements the activity of collagen alpha-1 chain polypeptide and / or lysyl hydroxylase 3 polypeptide and / or keratin type I cytoskeletal 17 polypeptide in one or more cells of the individual.

[0110] In some embodiments, administering an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, augments, and / or supplements tethering fiber formation in the individual. In some embodiments, administering an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, augments, and / or supplements epithelial basement membrane organization in the individual. In some embodiments, administering an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, augments, and / or supplements epithelial-basal adhesion in the individual. In some embodiments, administering an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, augments, and / or supplements the integrity of the epidermal-dermal junction in the individual. In some embodiments, administering an effective amount of any of the pharmaceutical compositions described herein to an individual enhances, increases, augments, and / or supplements wound healing in the individual. Without wishing to be bound by theory, it is believed that increasing, enhancing, and / or supplementing the level of collagen alpha-1(VII) chain polypeptide in one or more cells of an individual by administering one or more of the pharmaceutical compositions described herein will allow for increased production and secretion of functional collagen alpha-1(VII) chain protein in the individual. Without wishing to be bound by theory, it is believed that increasing, enhancing, and / or supplementing the level of lysyl hydroxylase 3 polypeptide in one or more cells of an individual by administering one or more of the pharmaceutical compositions described herein will increase post-translational modification of collagen alpha-1(VII) chain polypeptide, thereby enhancing the production and / or secretion of functional collagen alpha-1(VII) chain protein in the individual.Without wishing to be bound by theory, it is further believed that increasing, enhancing, and / or supplementing the levels of collagen alpha-1(VII) chain polypeptide and lysyl hydroxylase 3 polypeptide in the same cells of an individual by administering one or more of the pharmaceutical compositions described herein (by contacting the cells with two separate polynucleotides expressing the polypeptides, by contacting the cells with a single contiguous polynucleotide expressing two separate polypeptides, or by contacting the cells with a single contiguous polynucleotide expressing a chimeric polypeptide) will have an additive effect on enhancing the production and secretion of functional collagen alpha-1(VII) chain protein. Without wishing to be bound by theory, it is believed that increased production and secretion of functional collagen alpha-1(VII) chain protein will allow for improved anchoring fiber formation and help organize, stabilize, and support the adhesion of the individual's epithelial basement membrane. Without wishing to be bound by theory, it is believed that this ultimately results in increased dermal-epidermal stability in patients with epidermolysis bullosa, treating existing wounds and preventing or slowing wound reformation at the treatment site.

[0111] Isolated Polynucleotides and Polypeptides Certain aspects of the present disclosure relate to isolated polynucleotides, including polynucleotides encoding collagen alpha-1(VII) chain polypeptides. Other aspects of the present disclosure relate to isolated polynucleotides, including polynucleotides encoding lysyl hydroxylase 3 polypeptides. Other aspects of the present disclosure relate to isolated polynucleotides, including polynucleotides encoding keratin type I cytoskeleton 17 polypeptides.

[0112] Another aspect of the present disclosure relates to an isolated polynucleotide comprising a polynucleotide encoding a collagen alpha-1(VII) chain polypeptide and a polynucleotide encoding a lysyl hydroxylase 3 polypeptide, separated by a polynucleotide encoding a linker polypeptide. In some embodiments, the isolated polynucleotide encodes a chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide, a linker polypeptide, and a lysyl hydroxylase 3 polypeptide.

[0113] In some embodiments, the polynucleotide encoding the linker 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 linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the T2A linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the T2A linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the P2A linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the P2A linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the E2A linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded downstream of the E2A linker polypeptide. In some embodiments, the one or more furin cleavage sites are encoded upstream of the F2A linker polypeptide. In some embodiments, one or more furin cleavage sites are encoded downstream of the F2A linker polypeptide.

[0114] An example of a polynucleotide encoding a collagen alpha-1(VII) chain polypeptide is SEQ ID NO: 1. Polynucleotides encoding collagen alpha-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 to the sequence of SEQ ID NO: 1.

[0115] 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 to the sequence of SEQ ID NO: 3.

[0116] An example of a polynucleotide encoding a keratin type I cytoskeletal 17 polypeptide is SEQ ID NO: 29. Polynucleotides encoding keratin type I cytoskeletal 17 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 to the sequence of SEQ ID NO: 29.

[0117] Examples of polynucleotides encoding linker polypeptides are SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11. Polynucleotides encoding linker 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 to the sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11.

[0118] Examples of polynucleotides encoding chimeric polypeptides comprising a collagen alpha-1(VII) chain polypeptide, a linker polypeptide, and a 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. Polynucleotides encoding chimeric 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 to 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.

[0119] Further aspects of the present 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 adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex viral vector, a vaccinia viral vector, or any hybrid viral vector thereof. In some embodiments, the vector is a herpes simplex viral vector. In some embodiments, the vector comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) transgenes.

[0120] In some embodiments, the herpes simplex virus vector is a herpes virus amplicon vector. In some embodiments, the vector is an HSV-1 amplicon. In some embodiments, the vector is an HSV-1 hybrid amplicon. Examples of HSV-1 hybrid amplicons may include, but are not limited to, an HSV / AAV hybrid amplicon, an HSV / EBV hybrid amplicon, an HSV / EBV / RV hybrid amplicon, and an HSV / Sleeping Beauty hybrid amplicon. In some embodiments, the vector is an HSV / AAV hybrid amplicon. In some embodiments, the vector is an HSV / EBV hybrid amplicon. In some embodiments, the vector is an HSV / EBV / RV hybrid amplicon. In some embodiments, the vector is an HSV / Sleeping Beauty hybrid amplicon. A further aspect of the present disclosure relates to methods of making viral delivery vehicles containing one or more polynucleotides described herein. In some embodiments, the method comprises contacting a host cell with one or more viral vectors containing one or more isolated polynucleotides described herein and collecting the viral delivery vehicle produced by the host cell. Methods of culturing cells and contacting cells with one or more viral vectors of interest (e.g., by transduction or transfection) are well known to those of skill in the art.

[0121] 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 expression of one or more virulent herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations. Examples of inactivating mutations may include, but are not limited to, deletions (e.g., deletions of the coding sequence of a gene or deletions of one or more of the transcriptional regulatory elements of a gene), insertions, point mutations, and rearrangements. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in one or more immediate-early genes. In some embodiments, the recombinant herpes simplex virus genome comprises 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 comprises an inactivating mutation in the ICP0 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4, ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4, ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4, ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises 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.

[0122] In some embodiments, the isolated recombinant herpes simplex virus genome comprises 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 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 ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41, and UL55 herpes simplex virus loci. In some embodiments, the isolated recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or more of the viral ICP4 loci (e.g., a recombinant virus having a polynucleotide encoding Col7 at one or both ICP4 loci, a recombinant virus having a polynucleotide encoding LH3 at one or both ICP4 loci, a recombinant virus having a polynucleotide encoding KRT17 at one or both ICP4 loci, a recombinant virus having a polynucleotide encoding Col7 at one ICP4 locus and a polynucleotide encoding KRT17 at the other ICP4 locus, a recombinant virus having a polynucleotide encoding Col7 at one ICP4 locus and a polynucleotide encoding LH3 at the other ICP4 locus, a recombinant virus having a polynucleotide encoding LH3 at one ICP4 locus and a polynucleotide encoding KRT17 at the other ICP4 locus, etc.). In some embodiments, the isolated recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral UL41 locus. In some embodiments, the isolated recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral ICP47 locus.In some embodiments, the isolated recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure in one or more of the viral ICP4 loci and one or more polynucleotides of the present disclosure within the viral UL41 locus (e.g., a recombinant virus having a polynucleotide encoding Col7 at one or both of the ICP4 loci and a polynucleotide encoding LH3 at the UL41 locus, a recombinant virus having a polynucleotide encoding Col7 at one or both of the ICP4 loci and a polynucleotide encoding Col7 at the UL41 locus, a recombinant virus having a polynucleotide encoding Col7 at one or both of the ICP4 loci and a polynucleotide encoding KRT17 at the UL41 locus, a polynucleotide encoding LH3 at one or both of the ICP4 loci and a polynucleotide encoding LH3 at the UL41 locus, recombinant viruses having a polynucleotide encoding LH3 at one or both of the ICP4 loci and a polynucleotide encoding Col7 at the UL41 locus; recombinant viruses having a polynucleotide encoding LH3 at one or both of the ICP4 loci and a polynucleotide encoding KRT17 at the UL41 locus; recombinant viruses having a polynucleotide encoding KRT17 at one or both of the ICP4 loci and a polynucleotide encoding LH3 at the UL41 locus; recombinant viruses having a polynucleotide encoding KRT17 at one or both of the ICP4 loci and a polynucleotide encoding Col7 at the UL41 locus; recombinant viruses having a polynucleotide encoding KRT17 at one or both of the ICP4 loci and a polynucleotide encoding KRT1 ...

[0123] In some aspects, the isolated polynucleotides described herein are contained within a synthetic mRNA. In some embodiments, the synthetic mRNA comprises one or more modified ribonucleotides.

[0124] Certain aspects of the disclosure relate to isolated polypeptides comprising collagen alpha-1(VII) chain polypeptides. Other aspects of the disclosure relate to isolated polypeptides comprising lysyl hydroxylase 3 polypeptides. Other aspects of the disclosure relate to isolated polypeptides comprising keratin type I cytoskeleton 17 polypeptides.

[0125] Another aspect of the present disclosure relates to an isolated chimeric polypeptide comprising a collagen alpha-1(VII) chain polypeptide and a lysyl hydroxylase 3 polypeptide separated by a linker polypeptide.

[0126] In some embodiments, the linker polypeptide further comprises one or more furin cleavage sites. In some embodiments, the amino acid sequence of the furin cleavage site is the same as or substantially similar to the sequence of a classical furin cleavage site (Arg-X-(Arg / Lys)-Arg). In some embodiments, the one or more furin cleavage sites are at the N-terminus of the linker polypeptide. In some embodiments, the one or more furin cleavage sites are at the C-terminus of the linker polypeptide. In some embodiments, the linker polypeptide comprises, from N-terminus to C-terminus, one or more furin cleavage sites and a T2A linker polypeptide. In some embodiments, the linker polypeptide comprises, from N-terminus to C-terminus, a T2A linker polypeptide and one or more furin cleavage sites. In some embodiments, the linker polypeptide comprises, from N-terminus to C-terminus, one or more furin cleavage sites and a P2A linker polypeptide. In some embodiments, the linker polypeptide comprises, from N-terminus to C-terminus, a P2A linker polypeptide and one or more furin cleavage sites. In some embodiments, the linker polypeptide comprises, from its N- to C-terminus, one or more furin cleavage sites and an E2A linker polypeptide. In some embodiments, the linker polypeptide comprises, from its N- to C-terminus, an E2A linker polypeptide and one or more furin cleavage sites. In some embodiments, the linker polypeptide comprises, from its N- to C-terminus, one or more furin cleavage sites and an F2A linker polypeptide. In some embodiments, the linker polypeptide comprises, from its N- to C-terminus, an F2A linker polypeptide and one or more furin cleavage sites.

[0127] In some embodiments, the isolated polypeptide comprising a collagen alpha-1(VII) chain polypeptide comprises the amino acid sequence of SEQ ID NO: 2. The isolated polypeptide may also comprise a collagen alpha-1(VII) chain polypeptide containing 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 to the sequence of SEQ ID NO: 2.

[0128] In some embodiments, an isolated polypeptide comprising a lysyl hydroxylase 3 polypeptide comprises the amino acid sequence of SEQ ID NO: 4. Isolated polypeptides may also include lysyl hydroxylase 3 polypeptides containing 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 to the sequence of SEQ ID NO: 4.

[0129] In some embodiments, the isolated polypeptide comprising a keratin type I cytoskeletal 17 polypeptide comprises the amino acid sequence of SEQ ID NO: 30. The isolated polypeptide may also comprise a keratin type I cytoskeletal 17 polypeptide containing 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 to the sequence of SEQ ID NO: 30.

[0130] In some embodiments, the chimeric polypeptide comprises a collagen alpha-1(VII) chain polypeptide containing the amino acid sequence of SEQ ID NO: 2. The chimeric polypeptide may also comprise a collagen alpha-1(VII) chain polypeptide containing 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 to the sequence of SEQ ID NO: 2.

[0131] In some embodiments, 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 containing 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 to the sequence of SEQ ID NO: 4.

[0132] In some embodiments, 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 containing 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 to the sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12.

[0133] In some embodiments, the chimeric polypeptide is 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. The chimeric polypeptide can 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 to 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.

[0134] host cell Certain aspects of the present disclosure relate to one or more host cells comprising a vector comprising a polynucleotide described herein. In some embodiments, the vector is any of the isolated recombinant herpes simplex virus vectors described herein. In some embodiments, the host cell is a bacterial cell (e.g., an E. coli cell, etc.). In some embodiments, the host cell is a fungal cell (e.g., an S. cerevisiae cell, etc.). In some embodiments, the host cell is an insect cell (e.g., an S2 cell, etc.). In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a cell 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 retrovirus vector, a lentivirus vector, a herpes simplex virus vector, a vaccinia virus vector, or any hybrid virus 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 hybrid amplicon. In some embodiments, the host cell comprises a helper virus. In some embodiments, the host cell comprising the helper virus is contacted with a vector described herein. In some embodiments, contacting the host cell comprising the helper virus with the HSV-1 amplicon or HSV-1 hybrid amplicon 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 producing a virus by contacting a host cell comprising a helper virus with an HSV-1 amplicon or an HSV-1 / hybrid amplicon are known in the art. In some embodiments, the host cell is a complementing host cell.In some embodiments, the complementing host cells express one or more genes that are inactivated in any of the viral vectors described herein. In some embodiments, the complementing host cells are contacted with a recombinant herpes simplex virus genome described herein. In some embodiments, contacting the complementing host cells with a 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 cells. Methods for producing viruses by contacting complementing host cells with recombinant herpes simplex viruses are generally described in WO2015 / 009952.

[0135] Product or Kit Certain aspects of the present disclosure relate to articles of manufacture or kits that include the pharmaceutical compositions described herein. In some embodiments, the articles of manufacture or kits include a package insert that includes instructions for administering the pharmaceutical composition to provide preventative, palliative, or therapeutic relief of a wound, disorder, or disease in a subject's skin.

[0136] In some embodiments, a delivery vehicle comprising one or more polynucleotides described herein and a pharmaceutically acceptable carrier are in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers may be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or alloys (such as stainless steel or Hastelloy). In some embodiments, the container comprises a label on or associated with the container, the label indicating instructions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, etc.

[0137] The specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. [Example]

[0138] The present disclosure will be more fully understood by reference to the following examples. However, the examples should not be construed as limiting the scope of the present disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and should be included within the spirit and scope of this application and the scope of the appended claims.

[0139] Example 1: Generation of modified herpes simplex virus vectors and production / isolation of virus containing the vectors To generate a modified herpes simplex virus genome vector capable of expressing one or more transgenes in target mammalian cells, the herpes simplex virus genome (Figure 1A) is modified to inactivate the immediate-early genes ICP0, ICP4, and ICP27, while the immediate-early gene ICP22 is modified to contain a heterologous inducible promoter. This reduces the toxicity of the genome in mammalian cells. A cassette is then inserted into the modified herpesvirus genome by restriction cloning. The cassette contains a heterologous promoter capable of expressing the transgene in target mammalian cells. The promoter is operably linked to a nucleic acid sequence encoding a collagen alpha-1(VII) chain polypeptide, as well as downstream regulatory elements (Figure 1B), to ensure proper production of mRNA. Alternatively, the cassette contains two transgenes, each with its own heterologous promoter operably linked to a nucleic acid encoding either a collagen alpha-1(VII) chain polypeptide or a lysyl hydroxylase 3 polypeptide. The transgenes are encoded either on the same strand of DNA (Figure 1C) or in antisense orientation on opposite strands of DNA (Figure 1D). Linkage of each transgene to its own promoter and regulatory elements allows for the independent expression of each coding sequence on a separate mRNA transcript. Expressing transgenes from different promoters allows the ability to operably link 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.

[0140] The modified herpesvirus genome is also constructed to contain a cassette expressing a single mRNA encoding a collagen alpha-1(VII) chain polypeptide and a lysyl hydroxylase 3 polypeptide, separated by an internal ribosome entry site (Fig. 1E). This allows for approximately equimolar production of each polypeptide when expressed in target cells. Finally, the modified herpesvirus genome is constructed to contain a cassette expressing a chimeric polypeptide. This chimeric polypeptide comprises, from N- to C-terminus, a collagen alpha-1(VII) chain polypeptide, a cleavable peptide linker, and a lysyl hydroxylase 3 polypeptide (Fig. 1F).

[0141] Additional modified herpesvirus genomes were constructed containing two cassettes, each expressing a collagen alpha-1(VII) chain polypeptide, inserted into a copy of the ICP4 locus of the wild-type herpes simplex virus genome (Figure 2A) (Figures 2B-2G). These additional recombinant herpesvirus genomes were constructed using various combinations of herpesvirus gene deletions / modifications.

[0142] Recombinant herpesvirus genomes containing deletions of the coding sequences of both copies of the ICP4 gene, as well as the ICP27 and UL55 genes, are constructed. These recombinant viruses are further modified to contain inactivating mutations in the promoter regions of the ICP22 and ICP47 genes, such that these genes are not expressed at normal kinetics (Figure 2B).

[0143] Recombinant herpes simplex viruses were further constructed to incorporate expression cassettes for collagen alpha-1(VII) chain polypeptides into both loci of the herpes ICP4 gene, including viruses containing a deletion of the coding sequence of the ICP22 gene and both ICP4 gene copies (Figure 2C), a deletion of the coding sequence of the ICP0 gene and both ICP4 gene copies (Figure 2D), a deletion of the coding sequence of the ICP0 and ICP22 genes and both ICP4 gene copies (Figure 2E), a deletion of the coding sequence of the ICP0, ICP22, and ICP27 genes and both ICP4 gene copies (Figure 2F), and a deletion of the coding sequence of the ICP0, ICP22, ICP27, and UL55 genes and both ICP4 gene copies (Figure 2G). Based on the vectors shown in Figures 2C to 2G, additional vectors are constructed that further contain one or more transgenes encoding one or more additional effectors (e.g., LH3, KRT17) in the ICP0 and / or UL41 loci.

[0144] These modified herpes simplex virus genome vectors are transfected into engineered Vero cells that have been modified to express herpes virus genes. These engineered Vero cells secrete replication-deficient herpes simplex viruses with the modified genome packaged within the virus into the supernatant. The supernatant is then collected, concentrated, and sterile-filtered through a 5 μm filter.

[0145] Example 2: Restoration of Col7 expression using replication-deficient HSV-1 The following example describes the construction of a replication-deficient herpes simplex type 1 virus modified to express the human COL7A1 gene, and the use of such a viral vector to restore several defects observed in cells isolated from RDEB patients.

[0146] method Cells and cell cultures Normal and RDEB human skin fibroblasts and keratinocytes were isolated as previously described (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.

[0147] Building KB103 The KB103 vector was generated from D3GFP, a replication-deficient HSV-1 vector backbone carrying GFP in place of viral ICP4. The GFP sequence in D3GFP was replaced with the coding sequence for human COL7A1 by cloning COL7A1 into the EcoRI site of the ICP4 recombinant plasmid pSASB3 using a transfer plasmid. Mixed transfection / infection of the COL7A1-containing transfer plasmid and the D3GFP vector was performed on VeroD cells. The resulting plaques that did not express GFP were isolated and tested for Col7 protein expression by Western blot.

[0148] Virus purification KB103 virus was purified according to standard techniques (see Diefenbach, R. and Fraefel, C. Herpes Simplex Virus. New York: Humana Press, 2014).

[0149] Viral infection Cells were seeded in duplicate or triplicate in 6-well plates at approximately 50% confluence one day before virus infection. Additional wells were seeded in parallel for cell counting and MOI determination. 24 h after cell seeding, cells from one well were trypsinized and counted to calculate the MOI, and the virus stock was thawed and diluted in cell culture medium to achieve the desired MOI. Culture medium was aspirated from each well to be infected, and 500 μL of KB103-containing medium (or control medium) was added to each well. The plates were incubated at 37°C with 5-7.5% CO2 for 1.5-2 h with intermittent shaking every 15-20 min. 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 h. After incubation, cells and supernatants were harvested and processed for analysis.

[0150] mRNA quantification Col7 transcripts were amplified from RNA isolated from primary RDEB keratinocytes post-infection using the SYBR PCR assay (Sybr Select Master Mix, Life Technologies) according to the manufacturer's protocol. Col7 transcript levels were normalized to β-actin transcript levels.

[0151] Western blot analysis Cell lysates were generated from cells 48 hours postinfection, and Western blots were performed according to standard techniques using the following antibodies: rabbit anti-human Col7 polyclonal antibody (Sigma, catalog no. HPA042420), mouse anti-human GAPDH antibody (Santa Cruz Biotechnology, catalog no. sc-365062), rabbit anti-LH3 antibody (Protein Tech, catalog no. 11027-1-AP), and mouse anti-TSP1 antibody (Santa Cruz Biotechnology, catalog no. sc-59887).

[0152] Immunofluorescence Cells were seeded onto glass coverslips before infection, fixed 48 hours postinfection, stained with a primary rabbit anti-human Col7 polyclonal antibody (Sigma, Cat. No. HPA042420), washed, and further stained with a fluorescently labeled anti-rabbit secondary antibody (Invitrogen, Cat. 3 A11012). Cell nuclei were stained with DAPI using standard techniques.

[0153] cell adhesion 96-well plates were coated 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 100 μL reaction volume, then washed with PBS and blocked with PBS + 0.1% BSA for 1 h at 37°C. Mock (control)-infected or KB103-infected RDEB keratinocytes (2.4 × 10 cells in 100 μL of DMEM / HamF12 + 0.1% BSA) were cultured in 100 μL of DMEM / HamF12 + 0.1% BSA. 4 Cells were added to the plate and incubated at 37°C for 40–90 minutes. The wells were washed three times with PBS to remove any unbound cells, and adherent cells were fixed with PFE for 20 minutes. Fixed cells were then treated with 70% ethanol, stained with crystal violet, dissolved in 100% ethanol, and quantified by measuring absorbance at 630 nm.

[0154] Skin equivalent (SE) organotypic cultures Skin equivalent organotypic cultures composed of RDEB fibroblasts and keratinocytes were used to assess Col7 expression in the basement membrane zone (BMZ). Briefly, RDEB fibroblasts (2 x 10 per well) were cultured in 10 wells. 5 RDEB keratinocytes (1 x 10 cells per well) were embedded in a fibrin gel matrix in a 6-well plate and incubated in DMEM + 10% serum containing ascorbic acid and aprotinin at 37°C and 5% CO for 24 hours. 6Cells (keratinocytes) were seeded onto the matrix and grown to confluence in DMEM / F-12 keratinocyte medium containing 50 mg / mL ascorbic acid, allowing them to rise at the air-liquid interface. Two days after rise, KB103 virus was added to the cultures (at an MOI of 3) and incubated for 1.5 hours. After incubation, the cultures were washed and incubated for 5–14 days to promote stratification and epithelial differentiation. Skin equivalents (SEs) were manually peeled from the plates, embedded in optimal cutting temperature compound, frozen in liquid nitrogen, and cut into 6 mm sections for immunofluorescence staining using a monoclonal anti-Col7 antibody.

[0155] result KB103 pharmacology in normal and RDEB cells Numerous ex vivo approaches have been used to deliver the human COL7A1 gene to primary cells isolated from RDEB patients to correct 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). While successful in achieving persistent acquisition of key disease features, ex vivo gene delivery strategies for treating epidermolysis bullosa have a number of major drawbacks, including high cost, poor engraftment, surgical debridement, complex dressings and wound care, and a high likelihood of postoperative infection. An attractive alternative route to gene therapy is the use of viral or nonviral vectors to deliver gene products. However, non-viral vectors using plasmid DNA suffer from very low gene transfer efficiency when injected or administered topically, while the viral vectors most widely used in human gene therapy trials (retroviral vectors) do not infect non-dividing cells. This poses a problem for gene delivery to the skin because retroviral gene therapy requires tissue manipulation (e.g., wounding) to generate a suitable population of dividing cells. While large-capacity adenoviral vectors can deliver genome-sized transcription units and survive in transduced cells for extended periods, the toxicity and immunogenicity of adenoviral particles, as well as the need for a helper virus during vector production, remain major hurdles to their use in human gene therapy strategies. While replication-deficient HSV vectors have been used as delivery vehicles in numerous preclinical studies, no preclinical evidence has been reported to support the use of HSV-based viral vectors for epidermolysis bullosa or other dermatological applications.

[0156] To this end, we developed a replication-deficient herpes simplex type 1 virus (HSV-1) encoding the human COL7A1 gene as a novel vector useful for gene therapy treatment of DEB patients. The HSV-1 virus was engineered to have a complete deletion of the viral ICP4, ICP27, and UL55 genes. The ICP4 deletion resulted in the removal of the upstream promoter sequence that drives transcription of the immediate-early viral genes ICP22 and ICP47. The virus was further engineered to contain a human cytomegalovirus (HCMV) immediate-early promoter-driven human COL7A1 expression cassette encoded within both copies of the deleted ICP4 locus, resulting in a replication-deficient HSV-1 vector, designated KB103, suitable for delivering human COL7A1 to target cells (Figure 3).

[0157] To test the ability of KB103 to deliver and express Col7 in human cells and to restore the Col7 deficiency in RDEB patients, patient-derived human skin fibroblasts and keratinocytes were isolated from healthy and RDEB-affected individuals, and these primary cells were infected with KB103 at various MOIs. 24 to 72 hours after infection, COL7A1 gene expression was measured by real-time PCR in transduced cells, while Col7 protein expression was analyzed in parallel by both Western blot and immunofluorescence analysis.

[0158] A dose-dependent increase in COL7A1 gene expression was observed in RDEB keratinocytes (Figure 4A) and fibroblasts (Figure 4B) infected with KB103. KB103 infection increased COL7A1 gene expression by approximately 7.5-fold, 12.5-fold, and 25-fold in RDEB keratinocytes infected at MOIs of 0.3, 1, and 3, respectively (Figure 4A). Surprisingly, even 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, an approximately 30-fold increase in COL7A1 gene expression was measured in RDEB fibroblasts infected at an MOI of 1, whereas a 60-fold increase was observed in this cell type infected at an MOI of 3. This data indicated that COL7A1 gene expression was significantly upregulated in RDEB primary cells after infection with KB103.

[0159] As a result, strong Col7 protein expression was also observed in cells infected with KB103. Col7 protein expression was detected in both normal and RDEB keratinocytes (Figure 5A) and fibroblasts (Figure 5B) 48 hours after infection with KB103 at MOIs of 0.3, 1, and 3, and a clear dose-dependent increase in Col7 protein expression was observed with higher virus titers. Col7 expression was observed in both the supernatant and cell lysates from infected cells. Surprisingly, RDEB fibroblasts infected at an MOI of 0.3 exhibited higher levels of Col7 than those observed in uninfected normal fibroblasts (Figure 5B), suggesting complete restoration of Col7 expression in RDEB fibroblasts using KB103, even at low virus titers. No obvious effect on cell morphology was observed using high virus doses (MOI of 3). Furthermore, these experiments did not demonstrate any negative effect on fibroblast or keratinocyte cell proliferation using high doses of KB103, as determined by GAPDH expression.

[0160] Consistent with the above experiments, a strong and dose-dependent increase in Col7 protein expression was confirmed in normal and RDEB cells infected with KB103, as demonstrated by immunofluorescence detection of Col7 protein expression (Figure 6). As expected, no Col7 protein was detected in uninfected RDEB human skin fibroblasts or keratinocytes, and limited Col7 protein was detected in uninfected normal keratinocytes and fibroblasts. However, infection with KB103 was able to restore Col7 protein expression in RDEB fibroblasts and keratinocytes to levels above those observed in uninfected normal cells. Furthermore, the infection efficiency of KB103 (at an MOI of 3) was calculated to be ≥95% based on evaluation of three or more independent panels of each infection replicate, indicating that KB103 efficiently delivered and expressed the COL7A1 expression cassette. Taken together, this data suggested that KB103 was able to deliver and express COL7A1 in normal and RDEB primary cells, and furthermore, KB103 was well tolerated by both human dermal fibroblasts and keratinocytes.

[0161] Functional evaluation of KB103 in RDEB cells Next, we investigated the functionality of human Col7 protein expressed from KB103 in human skin fibroblasts and keratinocytes. First, we examined the effect of Col7 expression on the levels of lysyl hydroxylase 3 in KB103-infected cells. LH3 is required for extracellular matrix deposition and organization, and LH3 levels have been reported to be reduced in RDEB skin (Watt, SA et al. (2015) PLoS One 10(9):p.e0137639). Consistent with previous studies, little or no LH3 was observed in uninfected RDEB keratinocytes compared with normal keratinocytes (Figure 7, lanes 1 vs. 5). However, unexpectedly, we observed a dose-dependent increase in LH3 levels in KB103-infected RDEB keratinocytes, concomitant with increased Col7 protein expression (Figure 7), suggesting that KB103 was able to restore not only Col7 protein expression but also LH3 expression in RDEB cells.

[0162] Next, we examined the effect of Col7 expression on TSP-1 levels. TSP-1 is a negative regulator of angiogenesis, which 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 human dermal fibroblasts compared to normal human dermal fibroblasts (Figure 8, lanes 1 and 4). Surprisingly, TSP-1 protein expression was strongly inhibited upon infection of either normal or RDEB fibroblasts with KB103 (Figure 8). These data suggest that KB103 may promote angiogenesis not only by increasing Col7 and LH3 levels in infected cells but also by inhibiting the negative regulator TSP-1.

[0163] Finally, we tested the ability of KB103 to increase cell adhesion of RDEB keratinocytes to either collagen 1 or fibronectin. A dose-dependent increase in cell adhesion to both collagen 1 and fibronectin was observed in RDEB keratinocytes infected with KB103 at various MOIs (Figures 9A and 9B). Infection of RDEB keratinocytes at all MOIs tested demonstrated greater adhesion to wells treated with both substrates at all concentrations compared to uninfected (control) cells. Taken together, this data demonstrated that the human Col7 protein expressed from KB103 was functional in transduced cells. The functionality of this protein was demonstrated by its ability to increase LH3 protein levels, decrease TSP-1 protein levels, and improve cell adhesion to both collagen 1 and fibronectin compared to mock-infected samples.

[0164] KB103 pharmacology and toxicity in RDEB organotypic cultures Skin equivalent (SE) organotypic cultures composed of RDEB fibroblasts and keratinocytes were used to evaluate the expression of Col7 protein expressed by KB103 in the basement membrane zone (BMZ). RDEB fibroblasts and keratinocytes were mock-infected or infected with KB103 at an MOI of 3 and incubated for 5 days to promote stratification and epithelial differentiation. The resulting SEs were isolated, sectioned, and stained for immunofluorescence to detect Col7 protein expression. Col7 expression was detected in these organotypic cultures from KB103-infected cells, and the initiation of Col7 protein deposition in the BMZ was observed compared to mock-infected controls (Figure 10). These data suggested that KB103 not only delivered COL7A1 and efficiently expressed Col7 protein, but also that Col7 protein began to organize in the organotypic cultures in a pattern similar to that predicted for Col7 protein in vivo.

[0165] Collectively, these experiments demonstrated for the first time that a replication-deficient HSV-1 vector could be used as a vehicle to deliver a COL7A1 expression cassette into primary cells isolated from patients with epidermolysis bullosa. Furthermore, these data demonstrated that the Col7 protein could be expressed at high levels from this expression cassette in two different human cell types, one from healthy individuals and one from individuals with dermatological disorders. Finally, the Col7 protein was found to be functional, as it was able to increase LH3 expression, decrease TSP-1 expression, increase cell adhesion to collagen 1 and fibronectin, and organize in organotypic cultures in a pattern similar to the in vivo organization of Col7. Without wishing to be bound by theory, the data presented herein suggest that KB103 and other HSV-1 vectors may be useful as novel in vivo treatment strategies for epidermolysis bullosa and / or other dermatological applications.

[0166] Example 3: In vivo Col7 expression using replication-deficient HSV-1 The following example describes the use of a replication-deficient herpes simplex type 1 virus (modified to contain a human COL7A1 transgene) as a delivery vehicle for expressing the human Col7 protein in vivo.

[0167] method Construction and purification of KB103 KB103 virus was constructed and purified as described in Example 2 above.

[0168] Viral infection KB103 virus was delivered to wild-type Balb / c or skh1 elite mice by intradermal injection as follows: each animal received 1 × 10 of virus / site. 8 Plaque-forming units (PFU) were injected once at 2–4 sites in adjacent areas of the animal in a volume of 50 μL. Animals were sacrificed 48 h after KB103 administration, and the injection sites were harvested and processed for either real-time qPCR or immunofluorescence analysis.

[0169] For qPCR analysis, skin tissue was dissected down to the fascia using a 6 mm punch biopsy instrument. The biopsy was bisected into two specimens, and each specimen was flash-frozen using liquid nitrogen. Total RNA and DNA were isolated from one half of the biopsy using a Qiagen AllPrep DNA / RNA kit.

[0170] For immunofluorescence analysis, a circular area approximately 1 cm in diameter was excised from the skin at the injection site, cut in half, and mounted on OCT with the center of the circle facing upward. The prepared samples were flash-frozen in liquid nitrogen-cooled isopentane and stored at -80°C.

[0171] mRNA quantification Col7 transcripts were amplified from RNA isolated from mouse skin tissue after KB103 injection using a two-step protocol: 1) cDNA synthesis was performed using the Superscript III First Strand Synthesis Kit (Thermofisher, catalog no. 18-080-051), and 2) qPCR amplification was performed using the Quantitect Probe PCR Kit (Qiagen, catalog no. 204345) according to the manufacturer's protocol. 100 ng of cDNA was used for each reaction. Col7 transcript levels were normalized to GAPDH transcript levels.

[0172] Genome copy quantification The copy number of the KB103 viral genome in KB103-injected mice was quantified by qPCR amplification using the Quantitect Probe PCR kit (Qiagen catalog number 204345). 100 ng of mouse genomic DNA was used for each reaction, and mouse genomic GAPDH was used as a control.

[0173] Immunofluorescence Tissue sections from KB103-injected mice were fixed and subsequently stained with a primary rabbit anti-human Col7 polyclonal antibody (Sigma, Cat. No. HPA042420), washed, and further stained with a fluorescently labeled anti-rabbit secondary antibody (Invitrogen, Cat. 3 A11012). Cell nuclei were stained with DAPI using standard techniques.

[0174] result To test the ability of KB103 to successfully deliver and express human Col7 protein in vivo, mice were intradermally administered with KB103 virus. Viral genome copy numbers in infected mouse tissues were assessed, and high levels (>1,000,000 viral genome copies / 100 ng of mouse DNA) of KB103 viral genome delivery were observed in mice (Figure 11). Next, the ability of the virus to express human Col7 in vivo was examined. Quantitation of human Col7 transcripts in KB103-infected mice was measured and evaluated relative to the expression of a control mouse housekeeping gene. High levels of human Col7 transcripts were observed in infected mouse tissues (Figure 11), suggesting that the delivered viral genomes were able to successfully express their human gene cargo. Finally, the ability of KB103 to express Col7 protein was tested in infected mice. Mouse skin tissues were excised from mice after infection, and Col7 protein expression was assessed by immunohistochemical staining of mouse tissues. High levels of human Col7 protein were detected after histological staining (Figure 12). Surprisingly, not only was human Col7 protein expressed from the KB103 virus in the mouse dermis, but we also observed the initiation of human Col7 deposition in the basement membrane region of KB103-infected mice (Figure 12). Without wishing to be bound by theory, this data suggests the following: 1) the KB103 virus can successfully infect relevant tissues in vivo and deliver multiple genome copies to these tissues; 2) delivery of the KB103 virus to relevant tissues results in significant expression of the human genes encoded on this virus; and 3) KB103 not only successfully expresses human Col7 protein in vivo, but this protein can also initiate organization (e.g., in the basement membrane region), suggesting its ability to, in a sense, restore endogenous Col7 deficiency in affected individuals.

Claims

1. a) Pharmaceutical compositions comprising (i) and (ii) below: (i) Herpes simplex virus comprising a recombinant herpes simplex virus (HSV) genome, wherein the recombinant HSV genome comprises one or more polynucleotides encoding a transgene, and (ii) Pharmaceutically acceptable carriers; and, b) An accompanying document containing instructions for administering the pharmaceutical composition; A kit that includes, The recombinant HSV genome contains an inactivating mutation in one or both copies of the ICP4 herpes simplex virus gene. The aforementioned pharmaceutical composition is administered topically or transdermally to the target, and The kit comprising the pharmaceutical composition being administered to one or more affected areas of the subject due to a wound, injury, or disease of the skin.

2. The kit according to claim 1, wherein the pharmaceutical composition comprises an ointment, paste, cream, suspension, emulsion, fatty ointment, gel, powder, lotion, solution, spray, patch, or microneedle array.

3. The kit according to claim 1 or 2, wherein the HSV is suitable for delivering and expressing the one or more polynucleotides encoding the transgene into one or more target cells of the subject.

4. The kit according to any one of claims 1 to 3, wherein the recombinant HSV genome further comprises inactivating mutations in one or both copies of the ICP0 herpes simplex virus gene, the ICP22 herpes simplex virus gene, the ICP27 herpes simplex virus gene, the ICP47 herpes simplex virus gene, the thymidine kinase (tk) herpes simplex virus gene, the long unique region (UL) 41 herpes simplex virus gene, or the UL55 herpes simplex virus gene.

5. The kit according to any one of claims 1 to 4, wherein the inactivating mutation in one or both copies of the ICP4 herpes simplex virus gene is a deletion of at least a portion of the coding sequence of the ICP4 herpes simplex virus gene.

6. The kit according to any one of claims 1 to 5, wherein the one or more polynucleotides encoding the transgene are located within one or both of the ICP4 viral loci.

7. The kit according to any one of claims 1 to 6, wherein the one or more polynucleotides encoding the introduced gene do not contain a miRNA binding site.

8. The kit according to any one of claims 1 to 7, wherein the recombinant HSV genome is a recombinant HSV-1 genome.

9. The kit according to any one of claims 1 to 8, wherein the HSV containing the recombinant HSV genome is replication-deficient.

10. The kit according to any one of claims 1 to 9, wherein the pharmaceutically acceptable carrier comprises glycerol.

11. a) Pharmaceutical compositions comprising (i) and (ii) below: (i) Herpes simplex virus comprising a recombinant herpes simplex virus (HSV) genome, wherein the recombinant HSV genome comprises one or more polynucleotides encoding a transgene, and (ii) Pharmaceutically acceptable carriers; and, b) An accompanying document containing instructions for administering the pharmaceutical composition; A kit that includes, The recombinant HSV genome contains an inactivating mutation in one or both copies of the ICP4 herpes simplex virus gene. The pharmaceutical composition comprises an ointment, paste, cream, suspension, emulsion, fatty ointment, gel, powder, lotion, solution, spray, patch, or microneedle array, and The kit comprising the pharmaceutical composition, which is administered to one or more affected areas of a target due to a wound, injury, or disease of the skin.

12. The kit according to claim 11, wherein the pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, or intradermally.

13. The kit according to claim 11 or 12, wherein the HSV is suitable for delivering and expressing the one or more polynucleotides encoding the transgene into one or more target cells of the subject.

14. The kit according to any one of claims 11 to 13, wherein the recombinant HSV genome further comprises inactivating mutations in the ICP0 herpes simplex virus gene, the ICP22 herpes simplex virus gene, the ICP27 herpes simplex virus gene, the ICP47 herpes simplex virus gene, the thymidine kinase (tk) herpes simplex virus gene, the long unique region (UL) 41 herpes simplex virus gene, or the UL55 herpes simplex virus gene.

15. The kit according to claim 14, wherein the inactivating mutation in the ICP22 herpes simplex virus gene is a deletion of at least a portion of the coding sequence of the ICP22 herpes simplex virus gene.

16. The kit according to any one of claims 11 to 15, wherein the inactivating mutation in one or both copies of the ICP4 herpes simplex virus gene is a deletion of at least a portion of the coding sequence of the ICP4 herpes simplex virus gene.

17. The kit according to any one of claims 11 to 16, wherein the one or more polynucleotides encoding the transgene are located within one or both of the ICP4 viral loci.

18. The kit according to any one of claims 11 to 17, wherein the one or more polynucleotides encoding the introduced gene do not contain a miRNA binding site.

19. The kit according to any one of claims 11 to 18, wherein the recombinant HSV genome is a recombinant HSV-1 genome.

20. The kit according to any one of claims 11 to 19, wherein the HSV containing the recombinant HSV genome is replication-deficient.

21. The kit according to any one of claims 11 to 20, wherein the pharmaceutically acceptable carrier comprises glycerol.

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