Recombinant simplexvirus vector and uses thereof

Abstract

Provided herein, inter alia, is a recombinant herpesvirus vector genome including a heterologous herpesvirus Infected Cell Polypeptide 0 (ICP0) gene, a polynucleotide comprising the recombinant herpesvirus vector genome, and a host cell including the recombinant herpesvirus vector genome. The recombinant herpesvirus vector genome provided herein including embodiments thereof is contemplated to be useful for expressing a transgene in the host cell. In embodiments, the recombinant herpesvirus vector genome provided herein including embodiments thereof causes no detectable cellular toxicity to the host cell.

Description

RECOMBINANT SIMPLEXVIRUS VECTOR AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 688,821, filed August 29, 2024 and US. Provisional Application No. 63 / 786,032, filed April 9, 2025, which are hereby incorporated by reference in their entirety and for all purposes.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (061404-511001WO_SequenceListing_ST26.xml; Size: 75,420 bytes; and Date of Creation: August 27, 2025) are hereby incorporated by reference in their entirety.BACKGROUND

[0003] Herpes simplex virus (HSV)-based vectors have been developed for therapeutic gene delivery, particularly to neuronal cells, due to the virus’s natural neurotropism. The wild type HSV vector is a highly cytotoxic human pathogen including at least 80 gene products. For development and suitability for gene therapy, HSV vectors have been modified to be replication defective, for example, by deleting or mutating genes required for viral replication. As such, the modified HSV vectors are unable to replicate but retain features of wild-type HSV vector, for example, the ability to express transgenes after having established latent infections. However, deletion of viral genes can repress transgene expression, thereby lowering the ability of the HSV-based vector to express a transgene long term or at high expression levels. Further, even low expression levels of specific HSV genes associated with viral gene expression and / or replication cause cytotoxicity in host cells, thereby rendering the HSV-based vector unsuitable for therapeutic purposes.

[0004] Provided herein, inter alia, are solutions to these and other problems in the art.BRIEF SUMMARY

[0005] In an aspect is provided a recombinant herpesvirus vector genome, wherein the recombinant herpesvirus vector genome includes a heterologous herpesvirus Infected Cell Polypeptide 0 (ICP0) gene.

[0006] In an aspect is provided a polynucleotide including the recombinant herpesvirus vector genome provided herein including embodiments thereof. In embodiments, the polynucleotide is a bacterial artificial chromosome (BAC).

[0007] In an aspect is provided a host cell including the polynucleotide provided herein including embodiments thereof. In embodiments, the host cell includes one or more polynucleotides encoding genes sufficient to package a simplexvirus viral particle.

[0008] In an aspect is provided a method of making a viral particle, the method including culturing a host cell provided herein including embodiments thereof under conditions that the host cell produces the viral particle.

[0009] In an aspect is provided a recombinant alphaherpesvirus vector, including the recombinant herpesvirus vector genome provided herein including embodiments thereof, a viral capsid, a viral envelope, and a viral tegument.

[0010] In an aspect is provided a method of transducing a population of cells, the method including contacting the population of cells with a recombinant alphaherpesvirus vector provided herein including embodiments thereof.

[0011] In an aspect is provided a pharmaceutical composition including the recombinant alphaherpesvirus vector provided herein including embodiments thereof.

[0012] In an aspect is provided a method of treating dystrophic epidermolysis bullosa (DEB) in a subject in need thereof, including administering an effective amount of the recombinant alphaherpesvirus vector provided herein including embodiments thereof or the pharmaceutical provided herein including embodiments thereof to the subject.

[0013] In an aspect is provided a method of expressing a protein in a subject in need thereof, including administering an effective amount of the recombinant alphaherpesvirus vector provided herein including embodiments thereof or the pharmaceutical composition vector provided herein including embodiments thereof to the subject by systemic, subcutaneous, topical, or intradermal injection.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1. Schematic of JDNI8 HSV vectors with various iterations of Nova designs inserted in the LAT locus. Nova vectors vary in the promoter designs for homolog genes where EFl A ensures constitutive expression and ICPO is the natural ICPO promoter from WT HSV. The exemplary Nova vectors contain a constitutive CAG promoter driving a reporter gene, ZsGreen. For generation of a gene therapy product, ZsGreen reporter gene can be replaced with a cDNA expressing a missing or mutated protein.

[0015] FIG.s 2A-2B. Reporter gene expression in fibroblasts. FIG. 2A. Nova-GFP HSV vectors highly transduce human dermal fibroblasts at an MOI of 10 and show durability across 30 days. Nova vectors increased expression compared to an ICPO containing HSV in two different WT human primary fibroblast cells (Lonza and ATCC nHDF). FIG. 2B. Nova-GFP HSV vectors highly transduce human dermal fibroblasts at an MOI of 10 and show durability across 24 days. Nova vectors increased expression compared to an ICPO containing HSV in two different WT human primary fibroblast cells (Lonza and ATCC nHDF).

[0016] FIG. 3. Nova-GFP HSV vectors show reduced toxicity compared to an ICPO containing HSV vector in both WT and RDEB fibroblasts at an MOI of 20.

[0017] FIG. 4. Nova-GFP HSV vectors show high expression of reporter gene ZsGreen compared to an ICPO containing HSV vector in WT human Keratinocyte.

[0018] FIG. 5. Nova-GFP HSV vectors show reduced toxicity compared to an ICPO containing HSV vector in human Keratinocytes at an MOI of 20.

[0019] FIG. 6. Nova addition to HSV results in increased viral titer compared to HSV without ICPO when produced in a U2OS-4-27 cell line.

[0020] FIG. 7. Nova HSV Vectors Show Enhanced Expression and Durability Compared to ICP0- HSV. Top panel shows representative fluorescence images showing transgene (green fluorescent protein) expression from Nova HSV vectors including heterologous ICPO gene or ICPO gene. The middle panel includes description of the vectors used, including HSV vectors with elCPO linked to an EFl promoter (Nova 1) or a promoter endogenous to the VR-733 HSV vector (Nova 3), and withbICPO linked to the promoter endogenous to the VR-733 HSV vector (Nova 2). Bottom panel illustrates level (left) and durablitity (right) of transgene expression. Results show that HSV vectors including heterologous ICPO gene have higher level and more durable transgene expression relative to a control HSV including endogenous ICPO.

[0021] FIG.s 8A-8B. Assessment of wound area reduction following topical administration of recombinant HSV including bovine ICPO (Nova HSV vector) or a control (vehicle control). FIG. 8A) Representative images of wounds in mice administered the Nova HSV vector or a control. FIG. 8B) Bar graph showing wound size of mice administered Nova HSV vector or a control. Results show that Nova HSV vector has superior wound healing relative to the control, as indicated by a smaller wound size.

[0022] FIG.s 9A-9C. Quantification of junctional Col7Al expression in virus-treated vs. gel- treated wounds. FIG. 9A) Schematic showing treatment schedule and timepoint for assessing Col7Al expression at the dermal-epidermal junction (DEJ) post-treatment. FIG. 9B) Representative immunofluorescence images taken at Day 28 after Nova HSV vector administration showing Col7A protein expression. FIG. 9C) Bar graph showing quantification of Col7A protein expression at DEJ. Results show that the mice administered the Nova HSV vector have higher Col7A protein expression relative to mice administered a control.

[0023] FIG.s 10A-10E. Nova-Col7A infected cells express Col7Al transgene. FIG. 10A) Schematic of recombinant Nova HSV vector including bovine ICPO gene and Col7A transgene. FIG. 10B) Representative images of a Western blot gel illustration that WT fibroblasts, RDEB fibroblasts, and RDEB keratinocytes infected with Nova-ColA HSV vector express and secrete Col7Al protein in a dose-dependent manner. Cells infected with the control Nova-ZsG vector do not express Col7Al protein. FIG. 10C) Representative fluorescent images showing dose-dependent Col7A expression in Nova-Col7A infected RDEB fibroblasts. FIG. 10D) ELISA analysis was performed to quantify Col7A expression in cells infected with Nova-Col7A at an MOI of 0.1 on Days 3, 7, 14, 21, and 28 post-infection. Results show that Col7A is durably expressed at least 28 days post Nova-Col7A infection even at a lower MOI of 0.1. FIG. 10E) Bar graph depicting RT- qPCR results showing RDEB fibroblast Col7A expression on Day 3 post Nova-Col7A infection.The results show that Col7A protein levels showed a dose-dependent increase with higher MOIs of Nova-Col7 HSV.DETAILED DESCRIPTION

[0024] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0025] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0026] Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other components.

[0027] The term “about” refers to any minimal alteration in the concentration or amount of an agent that does not change the efficacy of the agent in preparation of a formulation and in treatment of a disease or disorder. The term “about” with respect to concentration range of the agents (e. ., therapeutic / active agents) of the current disclosure also refers to any variation of a stated amount or range which would be an effective amount or range. In embodiments, the term "about" means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about means the specified value.

[0028] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. It is also understood that throughout the application, data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0029] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0030] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0031] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar(ribose or deoxyribose). Non-limiting examples, of nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered stmctures such as dendrimers and the like.

[0032] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. For example, the nucleic acid provided herein may be part of a vector. For example, the nucleic acid provided herein may be part of a herpesvirus vector genome, which may be transduced into a cell. Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.

[0033] Nucleic acids, including e.g., nucleic acids with a phosphothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amio acid on a protein or polypeptide through a covalent, non-covalent or other interaction.

[0034] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodi thioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0035] Nucleic acids can include nonspecific sequences. As used herein, the term "nonspecific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0036] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.

[0037] The term “complement,” as used herein, in reference to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides refers to base pairing with a complementary nucleotide or sequence of nucleotides. As described herein and commonly known in the art the complementary (matching) nucleotide of adenosine is thymidine and the complementary (matching) nucleotide of guanosine is cytosine. Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence.

[0038] As described herein the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other, may have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region).

[0039] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O- phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0040] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0041] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acidpolymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0042] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0043] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue. One skilled in the art will immediately recognize the identity and location of residues corresponding to a specific position in a protein (e.g., COL7A1) in other proteins with different numbering systems. For example, by performing a simple sequence alignment with a protein (e.g., COL7A1) the identity and location of residues corresponding to specific positions of the protein are identified in other protein sequences aligning to the protein. For example, a selected residue in a selected protein corresponds to glutamic acid at position 138 when the selected residue occupies the same essential spatial or other structural relationship as a glutamic acid at position 138. In some embodiments, where a selected protein is aligned for maximum homology with a protein, the position in the aligned selected protein aligning with glutamic acid 138 is to correspond to glutamic acid 138. Instead of a primary sequence alignment, a three-dimensional structural alignment can also be used, e.g., where the structure of the selected protein is aligned for maximumcorrespondence with the glutamic acid at position 138, and the overall structures compared. In this case, an amino acid that occupies the same essential position as glutamic acid 138 in the structural model is to correspond to the glutamic acid 138 residue.

[0044] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations.Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0045] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.

[0046] The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)(see, e.g., Creighton, Proteins (1984)).

[0047] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http: / / www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50- 100 amino acids or nucleotides in length.

[0048] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0049] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Set. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0050] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) AMC. Acids Res. 25:3389-3402, and Altschul et al. (1990) Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from itsmaximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0051] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0052] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0053] The term "COL7A1 protein" or “COL7A1" as used herein includes any of the recombinant or naturally-occurring forms of collagen alpha-1 (VII) (COL7A1) protein, also known as Long- chain collagen (LC collagen), Collagen alpha-l(VII) chain, Col7A, or variants or homologs thereofthat maintain C0L7A1 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to COL7A1). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring COL7A1 protein. In embodiments, the COL7A1 protein is substantially identical to the protein identified by the UniProt reference number Q02388 or a variant or homolog having substantial identity thereto. In embodiments, the COL7A1 protein is encoded by a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID N0: 19.

[0054] The term “SERPINA1 protein" or “SERPINA1" as used herein includes any of the recombinant or naturally-occurring forms of SERPINA 1, also known as Alpha- 1 Antitrypsin, or variants or homologs thereof that maintain SERPINA 1 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to SERPINA1). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring SERPINA1 protein. In embodiments, the SERPINA1 protein is substantially identical to the protein identified by the UniProt reference number P01009 or a variant or homolog having substantial identity thereto.

[0055] The term “PKD1 protein" or “PKD1" as used herein includes any of the recombinant or naturally-occurring forms of PKD1, also known as Polycystin-1, or variants or homologs thereof that maintain PKD1 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to PKD1). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PKD1 protein. In embodiments, the PKD1 protein is substantially identical to the protein identified by the UniProt reference number P98161 or a variant or homolog having substantial identity thereto.

[0056] The term “ABCB 11 protein" or “ABCB 11" as used herein includes any of the recombinant or naturally-occurring forms of ABCB 11, also known as Bile salt export pump, or variants orhomologs thereof that maintain ABCB11 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to ABCB11). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring ABCB11 protein. In embodiments, the ABCB11 protein is substantially identical to the protein identified by the UniProt reference number 095342 or a variant or homolog having substantial identity thereto.

[0057] The term “RPE65 protein" or “RPE65" as used herein includes any of the recombinant or naturally-occurring forms of RPE65, also known as Retinal Pigment Epithelium-Specific 65 kDa Protein, or variants or homologs thereof that maintain RPE65 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to RPE65). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring RPE65 protein. In embodiments, the RPE65 protein is substantially identical to the protein identified by the UniProt reference number QI 6518 or a variant or homolog having substantial identity thereto.

[0058] The term “DMD protein" or “DMD" as used herein includes any of the recombinant or naturally-occurring forms of DMD, also known as Dystrophin, or variants or homologs thereof that maintain DMD activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to DMD). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring DMD protein. In embodiments, the DMD protein is substantially identical to the protein identified by the UniProt reference number Pl 1532 or a variant or homolog having substantial identity thereto.

[0059] The term “HBB protein" or “HBB" as used herein includes any of the recombinant or naturally-occurring forms of HBB, also known as Hemoglobin Subunit Beta, or variants or homologs thereof that maintain HBB activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HBB). In some aspects, the variants or homologs have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HBB protein. In embodiments, the HBB protein is substantially identical to the protein identified by the UniProt reference number P68871 or a variant or homolog having substantial identity thereto.

[0060] The term “CFH protein" or “CFH" as used herein includes any of the recombinant or naturally-occurring forms of CFH, also known as Complement Factor H, or variants or homologs thereof that maintain CFH activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CFH). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CFH protein. In embodiments, the CFH protein is substantially identical to the protein identified by the UniProt reference number P08603 or a variant or homolog having substantial identity thereto.

[0061] The term "HSV-1 ICP0 protein" or " HSV-1 ICP0" as used herein includes any of the recombinant or naturally-occurring forms of HSV-1 ICP0 protein, also known as HSV-1 E3 ubiquitin-protein ligase ICP0, or variants or homologs thereof that maintain HSV-1 ICP0 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HSV-1 ICP0). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HSV-1 ICP0 protein. In embodiments, the HSV-1 ICP0 protein is substantially identical to the protein identified by the UniProt reference number P08393 or a variant or homolog having substantial identity thereto.

[0062] The term "HSV-2 ICP0 protein" or " HSV-2 ICP0" as used herein includes any of the recombinant or naturally-occurring forms of HSV-2 ICP0 protein, also known as HSV-2 E3 ubiquitin-protein ligase 1CP0, HSV-2 RING-type E3 ubiquitin transferase ICP0 or variants or homologs thereof that maintain HSV-2 ICP0 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HSV-2 ICP0). In some aspects, the variants orhomologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HSV-2 ICP0 protein. In embodiments, the HSV-2 ICP0 protein is substantially identical to the protein identified by the UniProt reference number P28284 or a variant or homolog having substantial identity thereto.

[0063] The term "bICPO protein" or "bICPO" as used herein includes any of the recombinant or naturally-occurring forms of bICPO protein, also known as varicellovirus bovinealphal ICP0, varicellovirus bovinealphal RING-type E3 ubiquitin transferase ICP0, IER 2.9 / ER2.6, or variants or homologs thereof that maintain bICPO activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to bICPO). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring bICPO protein. In embodiments, the bICPO protein is substantially identical to the protein identified by the UniProt reference number P29836 or a variant or homolog having substantial identity thereto. In embodiments, the bICPO protein is substantially identical to the bICPO protein having at least 80% identity to SEQ ID NO:7 or a variant or homolog having substantial identity thereto. In embodiments, the bICPO protein includes the sequence of SEQ ID NO:7. In embodiments, the bICPO protein is the sequence of SEQ ID NO:7.

[0064] The term "elCPO protein" or "elCPO" as used herein includes any of the recombinant or naturally-occurring forms of elCPO protein, also known as varicellovirus equidalphal ICP0, varicellovirus equidalphal E3 ubiquitin-protein ligase ICP0, varicellovirus equidalphal RING-type E3 ubiquitin transferase ICP0, or variants or homologs thereof that maintain elCPO activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to elCPO). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring elCPO protein. In embodiments, the elCPO protein is substantially identical to the protein identified by the UniProt reference number P28990 or a variant or homolog having substantial identity thereto. In embodiments, the elCPO protein is substantially identical to the elCPO protein having at least 80% identity to SEQ ID NO:9 or a variant or homolog having substantial identity thereto. Inembodiments, the elCPO protein includes the sequence of SEQ ID NO:9. In embodiments, the elCPO protein is the sequence of SEQ ID NO:9.

[0065] The term "HSV-1 ICP4 protein" or "HSV-1 ICP4" as used herein includes any of the recombinant or naturally-occurring forms of HSV-1 ICP4 protein, also known as HSV-1 mRNA export factor, HSV-1 Immediate-early protein IE63, or variants or homologs thereof that maintain HSV-1 ICP4 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HSV-1 ICP4). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HSV-1 ICP4 protein. In embodiments, the HSV-1 ICP4 protein is substantially identical to the protein identified by the UniProt reference number P10238 or a variant or homolog having substantial identity thereto.

[0066] The term "HSV-2 ICP4 protein" or "HSV-2 ICP4" as used herein includes any of the recombinant or naturally-occurring forms of HSV-2 ICP4 protein, also known as HSV-2 mRNA export factor, HSV-2 Immediate-early protein IE63, or variants or homologs thereof that maintain HSV-2 ICP4 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HSV-2 ICP4). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HSV-2 ICP4 protein. In embodiments, the HSV-2 ICP4 protein is substantially identical to the protein identified by the UniProt reference number P28276 or a variant or homolog having substantial identity thereto.

[0067] The term "HSV-1 ICP27 protein" or "HSV-1 ICP27" as used herein includes any of the recombinant or naturally-occurring forms of HSV-1 ICP27 protein, HSV-1 Infected cell protein 27 (ICP27), or variants or homologs thereof that maintain HSV-1 ICP27 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HSV-1 ICP27). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HSV-1 ICP27 protein.In embodiments, the HSV-1 ICP27 protein is substantially identical to the protein identified by the UniProt reference number Pl 0238 or a variant or homolog having substantial identity thereto.

[0068] The term "HSV-2 ICP27 protein" or "HSV-2 ICP27" as used herein includes any of the recombinant or naturally-occurring forms of HSV-2 ICP27 protein, HSV-2 Infected cell protein 27 (ICP27), or variants or homologs thereof that maintain HSV-2 ICP27 activity (e g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HSV-2 ICP27). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HSV-2 ICP27 protein. In embodiments, the HSV-2 ICP27 protein is substantially identical to the protein identified by the UniProt reference number P28276 or a variant or homolog having substantial identity thereto.

[0069] The term "HSV-1 UL41 protein" or "HSV-1 UL41" as used herein includes any of the recombinant or naturally-occurring forms of HSV-1 UL41 protein, also known as HSV-1 Virion host shutoff (vhs) protein, or variants or homologs thereof that maintain HSV-1 UL41 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HSV-1 UL41). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HSV-1 UL41 protein. In embodiments, the HSV-1 UL41 protein is substantially identical to the protein identified by the UniProt reference number Q82171 or a variant or homolog having substantial identity thereto.

[0070] The term "HSV-2 UL41 protein" or "HSV-2 UL41" as used herein includes any of the recombinant or naturally-occurring forms of HSV-2 UL41 protein, also known as HSV-2 Virion host shutoff (vhs) protein, or variants or homologs thereof that maintain HSV-2 UL41 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HSV-2 UL41). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HSV-2 UL41 protein. In embodiments, the HSV-2 UL41 protein is substantially identical to the protein identifiedby the UniProt reference number 039988 or a variant or homolog having substantial identity thereto.

[0071] The term "HSV-1 ICP47 protein" or "HSV-1 ICP47 " as used herein includes any of the recombinant or naturally-occurring forms of HSV-1 ICP47 protein, also known as HSV-1 Immediate-early protein IE12, HSV-1 Infected cell protein 47, HSV-1 Immediate-early-5 or variants or homologs thereof that maintain HSV-1 ICP47 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HSV-1 ICP47). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HSV-1 ICP47 protein. In embodiments, the HSV-1 ICP47 protein is substantially identical to the protein identified by the UniProt reference number P03170 or a variant or homolog having substantial identity thereto.

[0072] The term "HSV-2 ICP47 protein" or "HSV-2 ICP47 " as used herein includes any of the recombinant or naturally-occurring forms of HSV-2 ICP47 protein, also known as HSV-2 Immediate-early protein IE12, HSV-2 Infected cell protein 47, HSV-2 Immediate-early-5 or variants or homologs thereof that maintain HSV-2 ICP47 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HSV-2 ICP47). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HSV-2 ICP47 protein. In embodiments, the HSV-2 ICP47 protein is substantially identical to the protein identified by the UniProt reference number P14345 or a variant or homolog having substantial identity thereto.

[0073] The term "varicellovirus bovinealpha5 ICP0 protein " or "bovine alphaherpesvirus 5 ICP0" as used herein includes any of the recombinant or naturally-occurring forms of bovine alphaherpesvirus 5 ICP0 protein, also known as bovine alphaherpesvirus 5 ICP0, BoHV-5 ICP0, or variants or homologs thereof that maintain BoHV-5 ICP0 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to BoHV-5 1CP0). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring BoHV-5 ICPO protein. In embodiments, the BoHV-5 ICPO protein is substantially identical to the protein identified by the UniProt reference number Q6X208 or a variant or homolog having substantial identity thereto.

[0074] The term "varicellovirus bubalinealphal ICPO protein" or "varicellovirus bubalinealphal ICPO" as used herein includes any of the recombinant or naturally-occurring forms of varicellovirus bubalinealphal ICPO protein, also known as bubaline alphaherpesvirus 1 ICPO, BuHV-1 ICPO, or variants or homologs thereof that maintain BuHV-1 ICPO activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to BuHV-1 ICPO). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring BuHV-1 ICPO protein. In embodiments, the BuHV-1 ICPO protein is substantially identical to the protein identified by the UniProt reference number A0A1L5JKJ2 or a variant or homolog having substantial identity thereto.

[0075] The term "varicellovirus canidalphal ICPO protein" or “varicellovirus canidalphal ICPO" as used herein includes any of the recombinant or naturally-occurring forms of varicellovirus canidalphal ICPO protein, also known as Canid alphaherpesvirus 1 ICPO, Canine herpesvirus ICPO, CHV ICPO, or variants or homologs thereof that maintain CHV ICPO activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CHV ICPO). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CHV ICPO protein. In embodiments, the CHV ICPO protein is substantially identical to the protein identified by the UniProt reference number UPI0007C5E32F or a variant or homolog having substantial identity thereto.

[0076] The term "varicellovirus equidalpha3 ICPO protein" or “varicellovirus equidalpha3 ICPO" as used herein includes any of the recombinant or naturally-occurring forms of varicellovirus equidalpha3 ICPO protein, also known as Equid alphaherpesvirus 3 ICPO, Equine herpesvirus 3 ICPO, EHV-3 ICPO, or variants or homologs thereof that maintain EHV-3 ICPO activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to EHV-3 ICPO).In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring EHV-3 ICP0 protein. In embodiments, the EHV-3 ICP0 protein is substantially identical to the protein identified by the UniProt reference number A0A077B626 or a variant or homolog having substantial identity thereto.

[0077] The term "varicellovirus equidalpha4 ICP0 protein" or “varicellovirus equidalpha4 ICP0" as used herein includes any of the recombinant or naturally-occurring forms of varicellovirus equidalpha4 ICP0 protein, also known as Equid alphaherpesvirus 4 ICP0, Equine herpesvirus 4 ICP0, EHV-4 ICP0, or variants or homologs thereof that maintain EHV-4 ICP0 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to EHV-4 ICP0). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring EHV-4 ICP0 protein. In embodiments, the EHV-4 ICP0 protein is substantially identical to the protein identified by the UniProt reference number A0A120HUE4 or a variant or homolog having substantial identity thereto.

[0078] The term "varicellovirus equidalpha8 ICP0 protein" or “varicellovirus equidalpha8 ICP0" as used herein includes any of the recombinant or naturally-occurring forms of varicellovirus equidalpha8 ICP0 protein, also known as Equid alphaherpesvirus 8 ICP0, Equine herpesvirus 8 ICP0, EHV-8 ICP0, or variants or homologs thereof that maintain EHV-8 ICP0 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to EHV-8 ICP0). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring EHV-8 ICP0 protein. In embodiments, the EHV-8 ICP0 protein is substantially identical to the protein identified by the UniProt reference number A0A2K9QPL7 or a variant or homolog having substantial identity thereto.

[0079] The term "varicellovirus equidalpha9 ICP0 protein" or “varicellovirus equidalpha9 ICP0 as used herein includes any of the recombinant or naturally-occurring forms of varicellovirusequidalpha9 ICPO protein, also known as Equid alphaherpesvirus 9 ICPO, Equine herpesvirus 9 ICPO, EHV-9 ICPO, or variants or homologs thereof that maintain EHV-9 ICPO activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to EHV-9 ICPO). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring EHV-9 ICPO protein. In embodiments, the EHV-9 ICPO protein is substantially identical to the protein identified by the UniProt reference number B7FEK5 or a variant or homolog having substantial identity thereto.

[0080] The term "varicellovirus felidalphal ICPO protein" or “varicellovirus felidalphal ICPO" as used herein includes any of the recombinant or naturally-occurring forms of varicellovirus felidalphal ICPO protein, also known as Feline herpesvirus 1 ICPO, FeHV-1 ICPO, or variants or homologs thereof that maintain FeHV-1 ICPO activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to FeHV-1 ICPO). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring FeHV-1 ICPO protein. In embodiments, the FeHV-1 ICPO protein is substantially identical to the protein identified by the UniProt reference number 037928 or a variant or homolog having substantial identity thereto.

[0081] The term " varicellovirus humanalpha3 ICPO protein" or “varicellovirus humanalpha3 ICPO" as used herein includes any of the recombinant or naturally-occurring forms of varicellovirus humanalpha3 ICPO protein, also known as Varicella zoster virus ICPO, human herpesvirus 3 ICPO, HHV-3 ICPO, or variants or homologs thereof that maintain HHV-3 ICPO activity (e g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HHV-3 ICPO). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HHV-3 ICPO protein. In embodiments, the HHV-3 ICPO protein is substantially identical to the protein identified by the UniProt reference number 037928 or a variant or homolog having substantial identity thereto

[0082] The terms “bICPO gene”, “bICPO gene”, or the like, as used herein refer to the any of the recombinant or naturally-occurring forms of the bICPO gene or variants or homologs thereof that code for a bICPO polypeptide capable of maintaining the activity of the bICPO polypeptide (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to bICPO). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous nucleic acid portion) compared to a naturally occurring bICPO. In embodiments, the bICPO gene includes the nucleic acid sequence of SEQ ID NO:3. In embodiments, the bICPO gene is the nucleic acid sequence of SEQ ID NO:3.

[0083] The terms “elCPO gene”, “elCPO gene”, or the like, as used herein refer to the any of the recombinant or naturally-occurring forms of the elCPO gene or variants or homologs thereof that code for a elCPO polypeptide capable of maintaining the activity of the elCPO polypeptide (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to elCPO). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous nucleic acid portion) compared to a naturally occurring elCPO. In embodiments, the elCPO gene includes the nucleic acid sequence of SEQ ID NO: 5. In embodiments, the elCPO gene is the nucleic acid sequence of SEQ ID NO:5.

[0084] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence (e.g. a promoter 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. Operably linked means that the nucleotide sequences being linked are typically contiguous. However, as enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome. Linking may be accomplished by ligation at convenient sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0085] The term "gene" means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a "protein gene product" is a protein expressed from a particular gene.

[0086] The terms "plasmid", "vector" or "expression vector" refer to a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, the gene and the regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids.

[0087] A “viral vector” refers to a recombinant viral genome and a viral capsid and / or envelope, where the viral vector is capable of delivering genomic material into a host cell for expression of genetic material. For example, in embodiments, a viral vector (e.g. recombinant alphaherpesvirus vector) includes a recombinant viral genome having one or more transgenes (e.g. a gene encoding a therapeutic protein) capable of being expressed in a host cell. Thus, in embodiments, a viral vector may be a delivery vehicle for genetic material (transgene (e.g. gene encoding a therapeutic protein)) into a target host cell. In embodiments, the viral vector includes a recombinant viral genome, a viral capsid, and a viral envelope. In embodiments, the viral vector further includes a viral tegument.

[0088] The term "recombinant" when used with reference, e.g., to a virus, cell, nucleic acid, protein, or vector, indicates that the virus, cell, nucleic acid, protein or vector, has been modified by the introduction of an exogenous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. For example, a recombinant virus (herpes simplex virus (e.g. HSV-1, HSV-2)) is generated by combining portions of nucleic acids using recombinant nucleic acid technology. For example, a recombinant virus may be generated by replacing one or more viral genes with an exogenous gene. For example, a recombinant virus may be generated by replacing a gene endogenous to the virus with an exogenous gene (e.g. an exogenous ICP0 gene). In embodiments, a recombinant virus may be generated by inserting an exogenous gene (e.g. a nucleic acid encoding a transgene (e.g. Col7A, an exogenous ICP0)) into said virus. In embodiments, the exogenous gene is heterologous to the virus. In embodiments, therecombinant herpesvirus vector genome provided herein including embodiments thereof includes one or more heterologous nucleic acids (e g. a heterologous ICPO gene, a heterologous transgene (Col7A gene)). Thus, in embodiments, the virus provided herein including embodiments thereof is a recombinant virus. In embodiments, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0089] The term "exogenous" refers to a molecule or substance (e.g., a compound, nucleic acid or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that does not originate from the cell or organism it is expressed by. The heterologous ICPO gene (e.g. bICPO gene, elCPO gene) provided herein including embodiments thereof is exogenous to the recombinant HSV genome vector provided herein. Thus, the heterologous ICPO gene does not originate from the recombinant HSV genome vector. Conversely, the term "endogenous" refers to a molecule or substance that is native to, or originates within, a given cell or organism. For example, an endogenous gene may refer to a gene that originates from or is native to a given genome or vector. For example, an HSV-1 vector may include an endogenous HSV-1 ICPO gene. In embodiments, an HSV-2 vector may include an endogenous HSV-2 ICPO gene. Thus, in embodiments, an ICPO gene that originates from or is native to a viral vector or a viral genome is referred to as an endogenous ICPO gene. For example, an ICPO gene that is native to or originates from the recombinant herpesvirus vector genome provided herein including embodiments thereof is an endogenous ICPO gene.

[0090] The term "isolated", when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A nucleic acid that is the predominant species present in a preparation is substantially purified.

[0091] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as a process of introducing a nucleic acid molecule or a protein to acell. Nucleic acids are introduced to a cell using non-viral or viral -based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Non- viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. In some embodiments, the nucleic acid molecules are introduced into a cell using electroporation following standard procedures well known in the art. For viral -based methods of transfection any useful viral vector (e.g. HSV vector) may be used in the methods described herein. Examples for viral vectors include, but are not limited to herpes simplex viral, retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some embodiments, the nucleic acid molecules are introduced into a cell using an HSV vector following standard procedures well known in the art. The terms "transfection" or "transduction" also refer to introducing proteins into a cell from the external environment. In embodiments, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8:1-4 and Prochi antz (2007) Nat. Methods 4: 119-20.

[0092] “ Transduce” or “transduction” are used according to their plain ordinary meanings and refer to the process by which one or more foreign nucleic acids (i.e. DNA not naturally found in the cell) are introduced into a cell. Typically, transduction occurs by introduction of a virus or viral vector (e.g. HSV vector) into the cell. For example, an HSV vector including a heterologous ICP0 gene may be transduced into a cell, thereby allowing expression of the heterologous ICP0 gene.

[0093] The word "expression" or "expressed" as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell. The level of expression of non-coding nucleic acid molecules (e.g., siRNA) may be detected by standard PCR or Northern blot methods well known in the art. See, Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88. Expression of a gene (e.g. heterologous ICP0 gene, transgene (e.g. gene encoding a therapeutic protein)) can be detected and / or measured by a variety of methods known in the art. Expression can be detected and / or measured using at the transcription level or atthe protein level. In embodiments, methods for detecting and / or measuring gene expression include RT-PCR, RT-qPCR, Northern blot, Fluorescence In Situ Hybridization (FISH), Western Blot, ELISA, immunohistochemistry, immunofluorescence, and mass spectroscopy. Thus, “detectable expression” and the like refer to gene expression that can be detected by a method known in the art.

[0094] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0095] When the label or detectable moiety is a radioactive metal or paramagnetic ion, the agent may be reacted with another long-tailed reagent having a long tail with one or more chelating groups attached to the long tail for binding to these ions. The long tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which the metals or ions may be added for binding. Examples of chelating groups that may be used according to the disclosure include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), DOTA, NOTA, NET A, TETA, porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups. The chelate is normally linked to the PSMA antibody or functional antibody fragment by a group, which enables the formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antibodies and carriers described herein. Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals including, but not limited to, radionuclides of gallium, yttrium and copper, respectively. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as223Ra for RAIT may be used. In certain embodiments, chelating moieties may be used to attach a PET imaging agent, such as an A1-18F complex, to a targeting molecule for use in PET analysis.

[0096] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. In embodiments, contacting refers to allowing a polynucleotide provided herein including embodiments thereof to interact with a cell so that the polynucleotide is delivered into the cell. In embodiments, contacting a cell with a polynucleotide refers to delivering the polynucleotide into the cell. In embodiments, contacting refers to allowing a recombinant viral vector (e.g. recombinant alphaherpesvirus vector) to interact with a cell (e.g. host cell) so that the virus enters the cell.

[0097] The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, a recombinant viral vector as described herein and a cell. In embodiments contacting includes, for example, allowing a virus as described herein to physically touch a cell.

[0098] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. DEB) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). In embodiments, a standard control may be an HSV vector that does not include the heterologous ICP0 gene provided herein In embodiments, a stardard control may be an HSV vector that includes an endogenous ICP0 gene. A standard control may be a composition (e.g. vehicle control) used for comparison to a test sample (e.g. HSV vector including an ICP0 gene). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e g. from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if valuesfor a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, etc).

[0099] One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values.Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.

[0100] “Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. In embodiments, a biological sample may be skin biopsy. In embodiments, a biological sample may be a tissue sample obtained from a subject (e.g. an RDEB or DEB patient).

[0101] A “cell” as used herein, refers to a cell carrying out metabolic or other functions sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaroytic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. In embodiments, the cell is a human cell. Cells may be useful when they are naturally nonadherentor have been treated not to adhere to surfaces, for example by trypsinization. In embodiments, the cell is a keratinocyte or a dermal fibroblast.

[0102] The terms “virus” or “virus particle” are used according to its plain ordinary meaning within Virology and refers to a virion including the viral genome (e.g. DNA, RNA, single strand, double strand), viral capsid and associated proteins, and in the case of enveloped viruses (e g. herpesvirus, poxvirus), an envelope including lipids and optionally components of host cell membranes, and / or viral proteins.

[0103] The term “replicate” is used in accordance with its plain ordinary meaning and refers to the ability of a cell or virus to produce progeny. A person of ordinary skill in the art will immediately understand that the term replicate when used in connection with DNA, refers to the biological process of producing two identical replicas of DNA from one original DNA molecule. Thus, the term “replicate” includes passaging and re-infecting progeny cells. In the context of a virus, the term “replicate” includes the ability of a virus to replicate (duplicate the viral genome and packaging said genome into viral particles) in a host cell and subsequently release progeny viruses from the host cell, which results in the lysis of the host cell.

[0104] The term “plaque forming units” is used according to its plain ordinary meaning in Virology and refers to the amount of plaques in a cell monolayer that can be formed per volume of viral particles. In some embodiments the units are based on the number of plaques that could form when infecting a monolayer of susceptible cells. For example, in embodiments 1,000 PFU / pl indicates that 1 pl of a solution including viral particles contains enough virus particles to produce 1000 infectious plaques in a cell monolayer. In embodiments, plaque forming units are abbreviated “PFU”.

[0105] The terms “multiplicity of infection” or “MOI” are used according to its plain ordinary meaning in Virology and refers to the ratio of infectious agent (e.g., herpesvirus) to the target (e.g., cell) in a given area or volume. In embodiments, the area or volume is assumed to be homogenous.

[0106] As defined herein, the term "inhibition", "inhibit", "inhibiting" and the like in reference to cell proliferation means negatively affecting (e.g., decreasing proliferation) or killing the cell. In some embodiments, inhibition refers to reduction of a disease or symptoms of disease (e.g. DEB,RDEB). In embodiments, "inhibitor" is a compound or protein that inhibits a receptor or another protein, e.g,, by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating activity (e.g., a receptor activity or a protein activity).

[0107] The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function.

[0108] The term “aberrant” as used herein refers to different from normal. When used to describe enzymatic activity, aberrant refers to activity that is greater or less than a normal control or the average of normal non-diseased control samples. Aberrant activity may refer to an amount of activity that results in a disease, wherein returning the aberrant activity to a normal or non-disease- associated amount (e.g. by using a method as described herein), results in reduction of the disease or one or more disease symptoms.

[0109] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including the range of normal doses for a given therapy, frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; and the route of administration. One of skill will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical or pharmaceutical composition, and depends on the route of administration. For example, a dosage form can be in a liquid form for nebulization, e.g., for inhalants, in a tablet or liquid, e.g., for oral delivery, or a saline solution, e.g., for injection.

[0110] By “effective dose or amount” or “therapeutically effective dose or amount” as used herein is meant a dose that produces the intended effects for which it is administered (e.g. expressing a transgene, expressing a protein, treating a condition or disease (e.g. DEB, RDEB)). The exact dose and formulation will depend on the purpose of the effect, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), andPickar, Dosage Calculations (1999)). For example, for the given parameter of transgene expression,an effective amount will show an increase of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100% in the level protein expression. In another example, for the given parameter of transgene expression, an effective amount will show an increase of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100% in the duration of detectable protein expression. Efficacy can also be expressed as “-fold” increase or decrease. For example, an effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect (e.g. protein expression level, duration of protein expression) over a standard control.[oni] As used herein, the term "administering" is used in accordance with its plain and ordinary meaning and includes oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intrapleural, intramuscular, intralesional, intratumoral, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini- osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal {e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By "co-administer" it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies. The compounds of the invention can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation). The compositions of the present invention can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0112] The terms "treat," "treating" or "treatment," and other grammatical equivalents as used herein, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relievinga condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include prophylaxis. The terms further include achieving a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. In embodiments, treating includes treating DEB (Dystrophic Epidermolysis Bullosa) or RDEB (Recessive Dystrophic Epidermolysis Bullosa).

[0113] The terms "prevent," "preventing," or "prevention," and other grammatical equivalents as used herein, include to keep from developing, occur, hinder or avert a disease or condition symptoms as well as to decrease the occurrence of symptoms. The prevention may be complete (i.e., no detectable symptoms) or partial, so that fewer symptoms are observed than would likely occur absent treatment. The terms further include a prophylactic benefit. For a disease or condition to be prevented, the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0114] The term “inhibiting” also means reducing an effect (disease state or expression level of a gene / protein / mRNA) relative to the state in the absence of a compound or composition of the present disclosure.

[0115] As used herein, the term “pharmaceutically acceptable” is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.

[0116] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (suchas Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0117] The term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration. In embodiments, the preparation is a topical formulation.

[0118] The pharmaceutical preparation is optionally in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The unit dosage form can be of a frozen dispersion.RECOMBINANT HERPESVIRUS VECTOR COMPOSITIONS

[0119] Provided herein, inter alia, is a recombinant herpesvirus vector genome including a heterologous herpesvirus Infected Cell Polypeptide 0 (ICPO) gene. As demonstrated herein, in embodiments, a heterologous ICPO gene (e.g. bICPO, elCPO) complements an inactivated endogenous ICPO gene (e.g. HSV-1 ICPO, HSV-2 ICPO) in a recombinant herpesvirus vector genome. In embodiments, a heterologous ICPO protein has substantially similar activity or functionality (e.g. initiating viral gene expression, promoter viral replication) relative to an ICPO protein endogenous to the herpesvirus (e.g. expressed from an endogenous ICPO gene). Thus, in embodiments, the recombinant herpesvirus vector genome provided including a heterologous ICPO gene herein including embodiments thereof is capable of activating herpesvirus gene expression andviral replication. Applicant has discovered that the recombinant herpesvirus vector genome including a heterologous ICPO gene is capable of infecting a host cell and producing viral particles without causing substantial cytotoxicity or detectable cytoxicity to the host cell. In embodiments, a host cell including the recombinant herpesvirus vector genome provided herein including embodiments thereof is capable of expressing a transgene for at least the duration and at least the same expression level as a host cell including a recombinant herpesvirus vector genome lacking the exogenous ICPO gene. In embodiments, the heterologous ICPO gene is inserted into the LAT region of the recombinant herpesvirus vector genome. In embodiments, the transgene is inserted into the LAT region of the recombinant herpesvirus vector genome. In embodiments, the host cell including the recombinant herpesvirus vector genome provided herein is capable of detectably expressing the transgene in vitro and / or in vivo. In an aspect is provided a recombinant herpesvirus vector genome, wherein the recombinant herpesvirus vector genome includes a heterologous herpesvirus Infected Cell Polypeptide 0 (ICPO) gene.

[0120] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, the heterologous ICPO gene provided herein including embodiments therof is not found in the same relationship to the herpesvirus vector genome provided herein including embodiments thereof in nature. For example, the heterologous ICPO gene originaates from a different species than the herpesvirus vector genome. Thus, in embodiments, “heterologous” when used in reference to a transgene (e.g. ICPO gene, gene encoding a therapeutic protein) indicates that the transgene is derived from one source (e.g. varicellovirus bovinealphal, varicellovirus equidalphal, etc.) and is inserted into a vector from a second source (e.g. Simplexvirus humanalphal (HSV-1), Simplexvirus humanalpha2 (HSV-2), etc.), where the second source is different from the first source. In embodiments, a heterologous gene may be a gene (e.g. bICPO gene, elCPO gene, etc.) derived from one source (e.g. varicellovirus bovinealphal, varicellovirus equidalphal, etc.) that is inserted into a vector from a second source (e.g. an HSV-1 vector, HSV-2 vector, etc.). In embodiments, the heterologous nucleic acid is recombinantly produced. In embodiments, a heterologous nucleic acid is a nucleic acid sequence from one species that is combined with a nucleic sequence from another species. For example, in embodiments, the heterologous nucleic acid is an ICPO gene from one species inserted into a herpesvirus vectorgenome from another species. Thus, a “heterologous ICPO gene” as used herein refers to an ICPO gene that is not native to the recombinant herpesvirus vector genome provided herein including embodiments thereof. In embodiments, the heterologous ICPO gene is not an HSV-1 ICPO gene or an HSV-2 ICPO gene. In embodiments, a heterologous nucleic acid includes two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. In embodiments, a heterologous nucleic acid includes a sequence from one source combined with a nucleic acid from another souce. In embodiments, a heterologous protein indicates that the protein is produced from a vector where the gene encoding the protein is not found in the vector genome in nature. In embodiments, a heterologous protein may indicate that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0121] For the recombinant herpesvirus vector genome provided herein, in embodiments, the recombinant herpesvirus vector genome is a recombinant alphaherpesvirus vector genome. “Alphaherpesvirus” or “Alphaherpesvirinae” is used in accordance with its plain and ordinary meaning and refers to the largest subfamily of the Herpesviridae family, which include doublestranded DNA viruses. The subfamily Alphaherpesvirinae includes five genera (Iltovirus, Mardivirus, Scutavirus, Simplexvirus, and Varicellovirus), which include related members capable of infecting humans and other animals, including herpes simplex virus, varicella-zoster virus, pseudorabies virus, bovine herpesvirus 1, among others. “Simplexvirus” is used in accordance with its plain and ordinary meaning and refers are double-stranded enveloped DNA viruses which typically include an envelope, a nucleocapsid, a DNA containing core, and a tegument. The simplexvirus genus includes at least 17 species including Simplexvirus atelinealphal, Simplexvirus bovinealpha2, Simplexvirus cercopithecinealpha2, Simplexvirus humanalpha 1 (HSV-1), Simplexvirus humanalpha2 (HSV-2), Simplexvirus leporidalpha4, Simplexvirus macacinealphal, Simplexvirus macacinealpha2, Simplexvirus macacinealpha3, Simplexvirus macropodidalphal, Simplexvirus macropodidalpha2, Simplexvirus macropodidalpha4, Simplexvirus paninealpha3, Simplexvirus papiinealpha2, Simplexvirus pteropodidalphal, Simplexvirus pteropodidalpha2, and Simplexvirus saimiriinealphal.

[0122] In embodiments, the recombinant alphaherpesvirus vector genome is a recombinant simplexvirus vector genome. In embodiments, the simplexvirus is Herpes simplex virus type 1(HSV-1) or Herpes simplex virus type 2 (HSV-2). Thus, in embodiments, the recombinant alphaherpesvirus vector genome is a recombinant HSV-1 vector genome or a recombinant HSV-2 vector genome. In embodiments, the recombinant alphaherpesvirus vector genome is a recombinant HSV-1 vector genome. In embodiments, the recombinant alphaherpesvirus vector genome is a recombinant HSV-2 vector genome.

[0123] The term “Herpes simplex virus type 1 (HSV-1)", also referred to as human herpesvirus- 1 , is used according to its common, ordinary meaning and refers to virus strains of the same or similar names and functional fragments and homologs thereof. The term includes recombinant or naturally occurring forms of HSV-1 or variants thereof that maintain HSV-1 activity (e.g. within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The term includes recombinant or naturally occurring forms of HSV-1 or variants thereof whose genome has sequence identity to the HSV-1 genome (e.g. about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the HSV-1 genome). HSV-1 may refer to variants having mutated amino acid residues that modulate (e.g. increase or decrease when compared to HSV-1) HSV-1 activity, expression, cellular targeting, or infectivity. HSV-1 may be modified as described herein. In embodiments, the HSV-1 refers to the virus strain identified by Genbank accession number IN555585, variants or homologs thereof.

[0124] The term “Herpes simplex virus type 2 (HSV-2)", also referred to as human herpesvirus-2, is used according to its common, ordinary meaning and refers to virus strains of the same or similar names and functional fragments and homologs thereof. The term includes recombinant or naturally occurring forms of HSV-2 or variants thereof that maintain HSV-2 activity (e.g. within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The term includes recombinant or naturally occurring forms of HSV-2 or variants thereof whose genome has sequence identity to the HSV-2 genome (e.g. about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the HSV-2 genome). HSV-2 may refer to variants having mutated amino acid residues that modulate (e.g. increase or decrease when compared to HSV-2) HSV-2 activity, expression, cellular targeting, or infectivity. HSV-2 may be modified as described herein. In embodiments, the HSV-2 refers to the virus strain identified by Genbank accession number IN561323, variants or homologs thereof.

[0125] As described herein, Applicant has discovered that, in embodiments, a heterologous ICP0 gene within a recombinant herpesvirus vector genome allows efficient delivery of the recombinantherpesvirus vector genome into a host cell and detectable expression of recombinant herpesvirus viral genes. In embodiments, the heterologous ICPO gene complements a recombinant herpesvirus vector genome including a non-functional endogenous ICPO, thereby allowing transduction of the host cell with the recombinant herpesvirus vector genome and expression of genes encoded by the recombinant herpesvirus vector genome. In embodiments, the heterologous herpesvirus ICPO gene is a heterologous alphaherpesvirus ICPO gene. In embodiments, the heterologous alphaherpesvirus ICPO gene is a heterologous varicellovirus ICPO gene.

[0126] As described above, Varicellovirus is a genus of viruses in the Alphaherpesvirinae subfamily. Natural hosts of Varicelloviruses are humans and other mammals serve as natural hosts. The Varicellovirus genus includes at least 19 species of virus including Varicellovirus bovinealphal, Varicellovirus bovinealpha5, Varicellovirus bubalinealphal, Varicellovirus canidalphal, Varicellovirus caprinealphal, Varicellovirus cercopithecinealpha9, Varicellovirus cervidalphal, Varicellovirus cervidalpha2, Varicellovirus cervidalpha3, Varicellovirus equidalphal, Varicellovirus equidalpha3, Varicellovirus equidalpha4, Varicellovirus equidalpha6, Varicellovirus equidalpha8, Varicellovirus equidalpha9, Varicellovirus felidalphal, Varicellovirus humanalpha3, Varicellovirus monodontidalphal, Varicellovirus phocidalphal, and Varicellovirus suidalphal. In embodiments, varicellovirus bovinealphal is referred to as bovine alphaherpesvirus 1 or BoHV-1. In embodiments, varicellovirus equidalphal ICPO is referred to as equine herpesvirus type 1 (EHV-1). In embodiments, varicellovirus humanalpha3 is referred to as Varicella-zoster virus (VZV). In embodiments, varicellovirus suidalphal is referred to as PICPO in Pseudorabies virus (PRV).

[0127] In embodiments, the heterologous varicellovirus ICPO gene is a varicellovirus bovinealphal ICPO gene, a varicellovirus bovinealpha5 ICPO gene, a varicellovirus bubalinealphal ICPO gene, a varicellovirus canidalphal ICPO gene, a varicellovirus caprinealphal ICPO gene, a varicellovirus cercopithecinealpha9 ICPO gene, a varicellovirus cervidalphal ICPO gene, a varicellovirus cervidalpha ICPO gene, a varicellovirus cervidalpha3 ICPO gene, a varicellovirus equidalphal ICPO (elCPOl) gene, a varicellovirus equidalpha3 ICPO gene, a varicellovirus equidalpha4 ICPO gene, a varicellovirus equidalpha6 ICPO gene, a varicellovirus equidalpha8 ICPO gene, a varicellovirus equidalpha9 ICPO gene, a varicellovirus felidalphal ICPO gene, a varicellovirus humanalpha3 ICPO gene, a varicellovirus monodontidalphal ICPO gene, a varicellovirus phocidalphal ICPO gene, or a varicellovirus suidalphal ICPO gene. In embodiments,the varicellovirus ICPO is a varicellovirus bovinealphal ICPO (bICPO) gene. In embodiments, the varicellovirus ICPO is a varicellovirus bovinealpha5 ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus bubalinealphal ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus canidalphal ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus caprinealphal ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus cercopithecinealpha9 ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus cervidalphal ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus cervidalpha2 ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus cervidalpha3 ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus equidalphal ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus equidalpha3 ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus equidalpha4 ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus equidalpha6 ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus equidalpha8 ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus equidalpha9 ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus felidalphal ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus humanalpha3 ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus monodontidalphal ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus phocidalphal ICPO gene. In embodiments, the varicellovirus ICPO is a varicellovirus suidalphal ICPO gene.

[0128] In embodiments, the heterologous ICPO gene is a functional homolog of an ICPO gene. The term “functional homolog” refers to a gene or protein that has substantially identical activity or function to a reference gene or protein. Thus, a functional homolog of an ICPO gene encodes a protein that has substantially identical activity or function to an ICPO protein. Similarly, a functional homolog of an ICPO protein is a protein that has substantially identical activity or function to an ICPO protein. In embodiments, the functional homolog of an ICPO protein has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity or function of an ICPO protein. In embodiments, the functional homolog of an ICPO protein has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity or function of an ICPO protein. In embodiments, the ICPO protein activity or function is activation of herpesvirus gene expression. In embodiments, the ICPO protein activity or function is activation of viral replication. In embodiments, the ICPO protein activity or function is ubiquitination of a host cell protein. Inembodiments, the ICPO protein activity or function is degradation of a host cell protein. In embodiments, the host cell protein is p53. In embodiments, the host cell protein is an antiviral host cell protein. In embodiments, the host cell antiviral gene or protein is promyelocytic leukemia protein (PML), SplOO, interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), or RNaseL. Thus, in embodiments, the ICPO protein activity or function is degradation of a host cell antiviral gene or protein. In embodiments, the ICPO protein activity or function is inhibition of the antivirus activity of a host cell protein. In embodiments, the ICPO protein activity or function is inhibition of the host immune response. For example, in embodiments, the ICPO protein activity or function is inhibition of the toll-like receptor mediated inflammatory response. In embodiments, the ICPO protein activity or function is inhibition of the interferon (INF) signaling pathway.

[0129] In embodiments, the functional homolog of an ICPO gene is a varicellovirus humanalpha3 ORF 61 gene.

[0130] In embodiments, the functional homolog of an ICPO gene is an adenovirus E1B-55K gene, an adenovirus E4orf6 gene, or a combination thereof. In embodiments, the functional homolog of an ICPO gene is an adenovirus E1B-55K gene and an adenovirus E4orf6 gene. In embodiments, the functional homolog of an ICPO gene is an adenovirus E1A gene. In embodiments, the functional homolog of an ICPO gene is an adenovirus E3-19K gene.

[0131] In embodiments, the heterologous ICPO gene is not a human herpesvirus-1 (HSV-1) ICPO gene. In embodiments, the heterologous ICPO gene does not include a functional fragment of a human HSV-1 ICPO gene. In embodiments, the heterologous ICPO gene is not a human herpesvirus- 2 (HSV-2) ICPO gene. In embodiments, the heterologous ICPO gene does not include a functional fragment of a human HSV-2 ICPO gene.

[0132] In embodiments, the varicellovirus ICPO is a varicellovirus bovinealphal ICPO (bICPO) gene. In embodiments, the bICPO gene includes a polynucleotide sequence according to SEQ ID NO:2 or SEQ ID NO:3 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:2 or SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence according to SEQ ID NO:2 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:2. In embodiments, the bICPO gene includes a polynucleotide sequence according to SEQ ID NO: 3 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:3. In embodiments, the bICPO geneincludes a polynucleotide sequence according to SEQ ID NO:2. In embodiments, the bICPO gene includes a polynucleotide sequence according to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:2. In embodiments, the bICPO gene includes a polynucleotide sequence at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:3.

[0133] In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 80%, wherein the polynucleotide sequence having a sequence identity of at least 80% may be contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 80%, and the polynucleotide sequence having at least 80% sequence identity is a non-contiguous sequence. In embodiments, the "non-contiguous sequence" is a sequence including one or more sequence fragments having no sequence identity to SEQ ID NO:3. In embodiments, the non-contiguous sequence is a sequence including a first sequence fragment having at least 80% sequence identity to SEQ ID NO:3 connected to a second sequence fragment having at least 80% sequence identity to SEQ ID NO:3 through a sequence fragment having no sequence identity to SEQ ID NO:3. In embodiments, the non-contiguous sequence is a sequence including a plurality of sequence fragments having at least 80% sequence identity to SEQ ID NO: 3 connected through a plurality of sequence fragments having no sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene further includes a nucleotide insertion, deletion or mutation.

[0134] In embodiments, the bICPO gene includes a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 81% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 82% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 83% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 84% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 86% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 87% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 88% sequence identity to SEQ ID NO:3. In embodiments, the bICPO geneincludes a polynucleotide sequence having at least 89% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 91% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 92% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 93% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 94% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 96% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 97% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 98% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes the polynucleotide sequence of SEQ ID NO:3. In embodiments, the bICPO gene is the polynucleotide sequence of SEQ ID NO:3.

[0135] In embodiments, the bICPO gene includes a polynucleotide sequence having about 80% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 81% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 82% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 83% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 84% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 85% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 86% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 87% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 88% sequence identity to SEQ ID NO: 3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 89% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 90% sequenceidentity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 91% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 92% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 93% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 94% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 95% sequence identity to SEQ ID NO: 3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 96% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 97% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 98% sequence identity to SEQ ID NO:3. In embodiments, the bICPO gene includes a polynucleotide sequence having about 99% sequence identity to SEQ ID NO:3.

[0136] In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 80% to SEQ ID NO:3, and the polynucleotide sequence having at least 80% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 81% to SEQ ID NO:3, and the polynucleotide sequence having at least 81% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 82% to SEQ ID NO:3, and the polynucleotide sequence having at least 82% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 83% to SEQ ID NO:3, and the polynucleotide sequence having at least 83% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 84% to SEQ ID NO:3, and the polynucleotide sequence having at least 84% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 85% to SEQ ID NO:3, and the polynucleotide sequence having at least 85% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 86% to SEQ ID NO:3, and the polynucleotide sequence having at least 86% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 87% to SEQ ID NO:3, and the polynucleotide sequence having at least 87% sequence identity is contiguous. In embodiments,the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 88% to SEQ ID NO:3, and the polynucleotide sequence having at least 88% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 89% to SEQ ID NO:3, and the polynucleotide sequence having at least 89% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 90% to SEQ ID NO:3, and the polynucleotide sequence having at least 90% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 91% to SEQ ID NO:3, and the polynucleotide sequence having at least 91% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 92% to SEQ ID NO:3, and the polynucleotide sequence having at least 92% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 93% to SEQ ID NO:3, and the polynucleotide sequence having at least 93% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 94% to SEQ ID NO:3, and the polynucleotide sequence having at least 94% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 95% to SEQ ID NO:3, and the polynucleotide sequence having at least 95% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 96% to SEQ ID NO:3, and the polynucleotide sequence having at least 96% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 97% to SEQ ID NO:3, and the polynucleotide sequence having at least 97% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 98% to SEQ ID NO:3, and the polynucleotide sequence having at least 98% sequence identity is contiguous. In embodiments, the bICPO gene includes a polynucleotide sequence having a sequence identity of at least 99% to SEQ ID NO:3, and the polynucleotide sequence having at least 99% sequence identity is contiguous.

[0137] In embodiments, the bICPO gene encodes a bICPO protein according to SEQ ID NO:6 or SEQ ID NO:7 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. In embodiments, the bICPO gene encodes a bICPO protein according to SEQ ID NO:6 or atleast 80%, at least 90%, or at least 95% identical to SEQ ID NO:6. In embodiments, the bICPO gene encodes a bICPO protein according to SEQ ID NO:7 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:7. In embodiments, the bICPO gene encodes a bICPO protein according to SEQ ID NO:6. In embodiments, the bICPO gene encodes a bICPO protein at least 80% identical to SEQ ID NO:6. In embodiments, the bICPO gene encodes a bICPO protein at least 90% identical to SEQ ID NO:6. In embodiments, the bICPO gene encodes a bICPO protein at least 95% identical to SEQ ID NO:6. In embodiments, the bICPO gene encodes a bICPO protein according to SEQ ID NO:7. In embodiments, the bICPO gene encodes a bICPO protein at least 80% identical to SEQ IDNO:7. In embodiments, the bICPO gene encodes a bICPO protein at least 90% identical to SEQ IDNO:7. In embodiments, the bICPO gene encodes a bICPO protein at least 95% identical to SEQ IDNO:7.

[0138] In embodiments, the bICPO protein includes a sequence having at least 80% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 81% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 82% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 83% sequence identity to SEQ ID NO: 7. In embodiments, the bICPO protein includes a sequence having at least 84% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 85% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 86% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 87% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 88% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 89% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 90% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 91% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 92% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 93% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 94% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 95% sequence identity to SEQ ID NO:7. In embodiments, the bICPOprotein includes a sequence having at least 96% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 97% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having at least 98% sequence identity to SEQ ID NO: 7. In embodiments, the bICPO protein includes a sequence having at least 99% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes the sequence of SEQ ID NO:7. In embodiments, the bICPO protein is the sequence of SEQ ID NO:7.

[0139] In embodiments, the bICPO protein includes a sequence having about 70% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 71% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 72% sequence identity to SEQ ID NO: 7. In embodiments, the bICPO protein includes a sequence having about 73% sequence identity to SEQ ID NO: 7. In embodiments, the bICPO protein includes a sequence having about 74% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 75% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 76% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 77% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 78% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 79% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 80% sequence identity to SEQ ID NO: 7. In embodiments, the bICPO protein includes a sequence having about 81% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 82% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 83% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 84% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 85% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 86% sequence identity to SEQ ID NO: 7. In embodiments, the bICPO protein includes a sequence having about 87% sequence identity to SEQ ID NO: 7. In embodiments, the bICPO protein includes a sequence having about 88% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 89% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 90% sequence identity to SEQID NO:7. In embodiments, the bICPO protein includes a sequence having about 91% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 92% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 93% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 94% sequence identity to SEQ ID NO: 7. In embodiments, the bICPO protein includes a sequence having about 95% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 96% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 97% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 98% sequence identity to SEQ ID NO:7. In embodiments, the bICPO protein includes a sequence having about 99% sequence identity to SEQ ID NO:7.

[0140] In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 80% to SEQ ID NO:7, and the sequence having at least 80% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 81% to SEQ ID NO:7, and the sequence having at least 81% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 82% to SEQ ID NO:7, and the sequence having at least 82% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 83% to SEQ ID NO:7, and the sequence having at least 83% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 84% to SEQ ID NO:7, and the sequence having at least 84% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 85% to SEQ ID NO:7, and the sequence having at least 85% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 86% to SEQ ID NO:7, and the sequence having at least 86% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 87% to SEQ ID NO:7, and the sequence having at least 87% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 88% to SEQ ID NO:7, and the sequence having at least 88% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 89% toSEQ ID N0:7, and the sequence having at least 89% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 90% to SEQ ID NO:7, and the sequence having at least 90% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 91% to SEQ ID NO:7, and the sequence having at least 91% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 92% to SEQ ID NO:7, and the sequence having at least 92% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 93% to SEQ ID NO:7, and the sequence having at least 93% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 94% to SEQ ID NO:7, and the sequence having at least 94% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 95% to SEQ ID NO:7, and the sequence having at least 95% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 96% to SEQ ID NO:7, and the sequence having at least 96% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 97% to SEQ ID NO:7, and the sequence having at least 97% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 98% to SEQ ID NO:7, and the sequence having at least 98% sequence identity is contiguous. In embodiments, the bICPO protein includes a sequence having a sequence identity of at least 99% to SEQ ID NO:7, and the sequence having at least 99% sequence identity is contiguous.

[0141] For the recombinant herpes vector genome provided herein, in embodiments, the heterologous ICPO gene encodes a ICPO protein including a “nuclear localization signal or sequence” (NLS). The term “nuclear localization signal or sequence” (NLS) is used in accordance to its plain ordinary meaning in the art and refers to an amino acid sequence that mediates transport of a protein into the cell nucleus. The NLS may facilitate delivery of a protein through the nuclear envelope by facilitating association of proteins in the nuclear pore complex. In embodiments, the NLS includes one or more short sequences of positively charged lysine residues or arginine residues. In embodiments, the NLS includes the sequence KRRR (SEQ ID NO: 10). In embodiments, the NLS is the sequence of SEQ ID NO: 10.

[0142] In embodiments, the bICPO gene encodes a bICPO protein including a nuclear localization sequence (NLS). In embodiments, the NLS includes the sequence KRRR (SEQ ID NO: 10). In embodiments, the NLS is the sequence of SEQ ID NO: 10.

[0143] For the recombinant herpes vector genome provided herein, in embodiments, the varicellovirus ICPO gene is a varicellovirus equidalphal ICPO (elCPO) gene. In embodiments, the elCPO gene includes a polynucleotide sequence according to SEQ ID NO:4 or SEQ ID NO:5 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:4 or SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence according to SEQ ID NO:4. In embodiments, the elCPO gene includes a polynucleotide sequence at least 80% identical to SEQ ID NO:4. In embodiments, the elCPO gene includes a polynucleotide sequence at least 90% identical to SEQ ID NO:4. In embodiments, the elCPO gene includes a polynucleotide sequence at least 95% identical to SEQ ID NO:4. In embodiments, the elCPO gene includes a polynucleotide sequence according to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence at least 80% identical to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence at least 90% identical to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence at least 95% identical to SEQ ID NO:5.

[0144] In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 80%, wherein the polynucleotide sequence having a sequence identity of at least 80% may be contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 80%, and the polynucleotide sequence having at least 80% sequence identity is a non-contiguous sequence. In embodiments, the "non-contiguous sequence" is a sequence including one or more sequence fragments having no sequence identity to SEQ ID NO: 5. In embodiments, the non-contiguous sequence is a sequence including a first sequence fragment having at least 80% sequence identity to SEQ ID NO:5 connected to a second sequence fragment having at least 80% sequence identity to SEQ ID NO: 5 through a sequence fragment having no sequence identity to SEQ ID NO:5. In embodiments, the non-contiguous sequence is a sequence including a plurality of sequence fragments having at least 80% sequence identity to SEQ ID NO: 5 connected through a plurality of sequence fragments having no sequence identity to SEQ ID NO: 5. In embodiments, the elCPO gene further includes a nucleotide insertion, deletion or mutation.

[0145] In embodiments, the elCPO gene includes a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 81% sequence identity to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 82% sequence identity to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 83% sequence identity to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 84% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 86% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 87% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 88% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 89% sequence identity to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 91% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 92% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 93% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 94% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 96% sequence identity to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 97% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 98% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes the polynucleotide sequence of SEQ ID NO: 5. In embodiments, the elCPO gene is the polynucleotide sequence of SEQ ID NO:5.

[0146] In embodiments, the elCPO gene includes a polynucleotide sequence having about 80% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotidesequence having about 81% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 82% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 83% sequence identity to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 84% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 85% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 86% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 87% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 88% sequence identity to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 89% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 90% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 91% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 92% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 93% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 94% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 95% sequence identity to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 96% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 97% sequence identity to SEQ ID NO: 5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 98% sequence identity to SEQ ID NO:5. In embodiments, the elCPO gene includes a polynucleotide sequence having about 99% sequence identity to SEQ ID NO:5.

[0147] In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 80% to SEQ ID NO:5, and the polynucleotide sequence having at least 80% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 81% to SEQ ID NO:5, and the polynucleotide sequence having at least 81% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 82% to SEQ ID NO: 5, andthe polynucleotide sequence having at least 82% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 83% to SEQ ID NO:5, and the polynucleotide sequence having at least 83% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 84% to SEQ ID NO:5, and the polynucleotide sequence having at least 84% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 85% to SEQ ID NO:5, and the polynucleotide sequence having at least 85% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 86% to SEQ ID NO:5, and the polynucleotide sequence having at least 86% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 87% to SEQ ID NO: 5, and the polynucleotide sequence having at least 87% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 88% to SEQ ID NO:5, and the polynucleotide sequence having at least 88% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 89% to SEQ ID NO:5, and the polynucleotide sequence having at least 89% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 90% to SEQ ID NO:5, and the polynucleotide sequence having at least 90% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 91% to SEQ ID NO:5, and the polynucleotide sequence having at least 91% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 92% to SEQ ID NO: 5, and the polynucleotide sequence having at least 92% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 93% to SEQ ID NO:5, and the polynucleotide sequence having at least 93% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 94% to SEQ ID NO:5, and the polynucleotide sequence having at least 94% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 95% to SEQ ID NO:5, and the polynucleotide sequence having at least 95% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotidesequence having a sequence identity of at least 96% to SEQ ID NO:5, and the polynucleotide sequence having at least 96% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 97% to SEQ ID NO: 5, and the polynucleotide sequence having at least 97% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 98% to SEQ ID NO:5, and the polynucleotide sequence having at least 98% sequence identity is contiguous. In embodiments, the elCPO gene includes a polynucleotide sequence having a sequence identity of at least 99% to SEQ ID NO:5, and the polynucleotide sequence having at least 99% sequence identity is contiguous.

[0148] In embodiments, the elCPO gene encodes an elCPO protein at least 90% or at least 95% identical to SEQ ID NO:8 or SEQ ID NON. In embodiments, the elCPO gene encodes an elCPO protein according to SEQ ID NO:8. In embodiments, the elCPO gene encodes an elCPO protein at least 90% identical to SEQ ID NO: 8. In embodiments, the elCPO gene encodes an elCPO protein at least 95% identical to SEQ ID NO: 8. In embodiments, the elCPO gene encodes an elCPO protein according to SEQ ID NON. In embodiments, the elCPO gene encodes an elCPO protein at least 90% identical to SEQ ID NON. In embodiments, the elCPO gene encodes an elCPO protein at least 95% identical to SEQ ID NON.

[0149] In embodiments, the elCPO protein includes a sequence having at least 80% sequence identity to SEQ ID NON. In embodiments, the elCPO protein includes a sequence having at least 81% sequence identity to SEQ ID NON. In embodiments, the elCPO protein includes a sequence having at least 82% sequence identity to SEQ ID NON. In embodiments, the elCPO protein includes a sequence having at least 83% sequence identity to SEQ ID NON. In embodiments, the elCPO protein includes a sequence having at least 84% sequence identity to SEQ ID NON. In embodiments, the elCPO protein includes a sequence having at least 85% sequence identity to SEQ ID NON. In embodiments, the elCPO protein includes a sequence having at least 86% sequence identity to SEQ ID NON. In embodiments, the elCPO protein includes a sequence having at least 87% sequence identity to SEQ ID NON. In embodiments, the elCPO protein includes a sequence having at least 88% sequence identity to SEQ ID NON. In embodiments, the elCPO protein includes a sequence having at least 89% sequence identity to SEQ ID NON. In embodiments, the elCPO protein includes a sequence having at least 90% sequence identity to SEQ ID NON. Inembodiments, the elCPO protein includes a sequence having at least 91% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having at least 92% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having at least 93% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having at least 94% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having at least 95% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having at least 96% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having at least 97% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having at least 98% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having at least 99% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes the sequence of SEQ ID NO:9. In embodiments, the elCPO protein is the sequence of SEQ ID NO:9.

[0150] In embodiments, the elCPO protein includes a sequence having about 80% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 81% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 82% sequence identity to SEQ ID NO: 9. In embodiments, the elCPO protein includes a sequence having about 83% sequence identity to SEQ ID NO: 9. In embodiments, the elCPO protein includes a sequence having about 84% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 85% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 86% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 87% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 88% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 89% sequence identity to SEQ ID NO: 9. In embodiments, the elCPO protein includes a sequence having about 90% sequence identity to SEQ ID NO: 9. In embodiments, the elCPO protein includes a sequence having about 91% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 92% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 93% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 94% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 95%sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 96% sequence identity to SEQ ID NO: 9. In embodiments, the elCPO protein includes a sequence having about 97% sequence identity to SEQ ID NO: 9. In embodiments, the elCPO protein includes a sequence having about 98% sequence identity to SEQ ID NO:9. In embodiments, the elCPO protein includes a sequence having about 99% sequence identity to SEQ ID NO:9.

[0151] In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 80% to SEQ ID NO:9, and the sequence having at least 80% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 81% to SEQ ID NO:9, and the sequence having at least 81% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 82% to SEQ ID NO:9, and the sequence having at least 82% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 83% to SEQ ID NO:9, and the sequence having at least 83% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 84% to SEQ ID NO:9, and the sequence having at least 84% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 85% to SEQ ID NO:9, and the sequence having at least 85% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 86% to SEQ ID NO:9, and the sequence having at least 86% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 87% to SEQ ID NO:9, and the sequence having at least 87% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 88% to SEQ ID NO:9, and the sequence having at least 88% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 89% to SEQ ID NO:9, and the sequence having at least 89% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 90% to SEQ ID NO:9, and the sequence having at least 90% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 91% to SEQ ID NO:9, and the sequence having at least 91% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 92% toSEQ ID N0:9, and the sequence having at least 92% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 93% to SEQ ID NO:9, and the sequence having at least 93% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 94% to SEQ ID NO:9, and the sequence having at least 94% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 95% to SEQ ID NO:9, and the sequence having at least 95% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 96% to SEQ ID NO:9, and the sequence having at least 96% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 97% to SEQ ID NO:9, and the sequence having at least 97% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 98% to SEQ ID NO:9, and the sequence having at least 98% sequence identity is contiguous. In embodiments, the elCPO protein includes a sequence having a sequence identity of at least 99% to SEQ ID NO:9, and the sequence having at least 99% sequence identity is contiguous.

[0152] In embodiments, the elCPO protein includes a nuclear localization sequence (NLS), preferably KRRR (SEQ ID NO: 10). In embodiments, the NLS includes SEQ ID NO: 10. In embodiments, the NLS is SEQ ID NO: 10.

[0153] For the recombinant herpesvirus vector genome provided herein, in embodiments, the varicellovirus ICP0 gene includes a polynucleotide sequence encoding a varicellovirus ICP0 ring domain. As used herein, the term “ring domain” refers to a protein structural domain including a zinc finger domain typically 20 to 80 amino acid residues in length. In embodiments, the zinc finger domain includes a Cys3HisCys4 amino acid motif which binds two zinc cations. In embodiments, the zinc finger domain includes seven cysteines and one histidine arranged non-consecutively. In embodiments, a ring domain includes ubiquitin ligase activity. Thus, in embodiments, a varicellovirus ICP0 protein including a ring domain is capable of targeting cellular proteins for degradation via the ubiquitin-proteosome pathway.

[0154] In embodiments, the variellovirus ICP0 ring domain is a varicellovirus bovinealphal ICP0 (bICPO) ring domain, a varicellovirus bovinealpha5 ICP0 ring domain, a varicellovirusbubalinealphal ICPO ring domain, a varicellovirus canidalphal ICPO ring domain, a varicellovirus caprinealphal ICPO ring domain, a varicellovirus cercopithecinealpha9 ICPO ring domain, a varicellovirus cervidalphal ICPO ring domain, a varicellovirus cervidalpha2 ICPO ring domain, a varicellovirus cervidalpha3 ICPO ring domain, a varicellovirus equidalphal ICPO (elCPO) ring domain, a varicellovirus equidalpha3 ICPO ring domain, a varicellovirus equidalpha4 ICPO ring domain, a varicellovirus equidalpha6 ICPO ring domain, a varicellovirus equidalpha8 ICPO ring domain, a varicellovirus equidalpha9 ICPO ring domain, a varicellovirus felidalphal ICPO ring domain, a varicellovirus humanalpha3 ICPO ring domain, a varicellovirus monodontidalphal ICPO ring domain, a varicellovirus phocidalphal ICPO ring domain, or a varicellovirus suidalphal ICPO ring domain.

[0155] In embodiments, the variellovirus ICPO ring domain is a varicellovirus bovinealphal ICPO (bICPO) ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus bovinealpha5 ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus bubalinealphal ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus canidalphal ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus caprinealphal ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus cercopithecinealpha9 ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus cervidalphal ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus cervidalpha ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus cervidalpha3 ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus equidalphal ICPO (elCPO) ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus equidalpha3 ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus equidalpha4 ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus equidalpha6 ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus equidalpha8 ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus equidalpha9 ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus felidalphal ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus humanalpha3 ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus monodontidalphal ICPO ring domain. Inembodiments, the variellovirus ICPO ring domain is a varicellovirus phocidalphal ICPO ring domain. In embodiments, the variellovirus ICPO ring domain is a varicellovirus suidalphal ICPO ring domain. In embodiments, the varicellovirus ICPO ring domain does not include a human HSV-1 ICPO ring domain or a functional fragment thereof.

[0156] In embodiments, the bICPO ring domain has at least 80% sequence identity to a sequence corresponding to positions 13 to 52 of SEQ ID NO:7. In embodiments, the elCPO ring domain has at least 80% sequence identity a sequence corresponding to residues 8-47 of SEQ ID NO:9.

[0157] In embodiments, the heterologous ICPO gene further includes a polynucleotide sequence encoding a purification tag or a detectable moiety. In embodiments, the detectable moiety is a myc epitope tag. In embodiment, the myc epitope tag is encoded by a polynucleotide of SEQ ID NO: 16. In embodiments, the myc epitope tag is the sequence of SEQ ID NO: 18. In embodiments, the purification tag or the detectable moiety is attached to the heterologous ICPO by a linker.

[0158] For the recombinant herpesvirus vector genome provided herein, in embodiments, the heterologous ICPO gene further includes a polynucleotide encoding a linker. In embodiments, the linker is encoded by the polynucleotide of SEQ ID NO: 17. In embodiments, the linker sequence includes the sequence of GASG. In embodiments, the linker sequence is the sequence of GASG.

[0159] Expression of the heterologous ICPO gene provided herein may be controlled by a promoter. Therefore, in embodiments, the heterologous ICPO gene is operably linked to a promoter. In embodiments, the promoter is endogenous to the recombinant herpesvirus vector genome. In embodiments, the promoter is an endogenous ICPO gene promoter. In embodiments, the promoter is endogenous to the recombinant herpesvirus vector genome provided herein including embodiments thereof and is moved to an ectopic location within the genome. In embodiments, the promoter is a heterologous promoter. Thus, in embodiments, the promoter is not typically found in a herpesvirus genome in nature.

[0160] In embodiments, the promoter is a constitutive promoter. In embodiments, the promoter is a CbH promoter, a CBA promoter, a SFFV promoter, a MSCV promoter, a SV40 promoter, a hPGK promoter, a RSV promoter, a CMV promoter, an EFla promoter, a mitochondrial heavy-strand promoter, a UBC promoter, a CAG promoter, a GAPDH promoter, an ACTB promoter. Inembodiments, the promoter is a CbH promoter. The CbH is a hybrid promoter derived from the CBA promoter, wherein the CbH promoter includes a CMV promoter, a truncated chicken 0-actin intron, and an MVM (minute virus of mice) intron. In embodiments, the promoter is a CBA promoter. The CBA promoter includes a sequence derived from a chicken-actin promoter and an CMV immediate-early enhancer sequence. In embodiments, the promoter is a SFFV promoter. The SFFV promoter is a constitutive promoter derived from a Spleen Focus-Forming Virus (SFFV) promoter sequence. In embodiments, the promoter is a MSCV promoter. The MSCV promoter is derived from the Long Terminal Repeat (LTR) of the murine stem cell virus. In embodiments, the promoter is a SV40 promoter. The SV40 promoter is derived from a DNA region from Simian Virus 40 (SV40). In embodiments, the SV40 promoter includes TATA box sequences, 21 -bp repeats including GC rich regions having multiple GC poxes, and 72-bp repeats including transcriptional enhancers. In embodiments, the promoter is a hPGK promoter. The hPGK promoter is derived from the promoter sequence for the human phosphoglycerate kinase 1 gene. In embodiments, the promoter is an RSV promoter. The RSV promoter is derived from a region of DNA located at the 5' end of the RSV genome functional for regulating transcription of viral genes. In embodiments, the promoter is a CMV promoter. The CMV promoter is derived from the immediate early promoter of the human cytomegalovirus. In embodiments, the promoter is an EFla promoter. The EFla promoter is a constitutive promoter derived from the human EEF1A1 gene, which encodes the alpha subunit of the eukaryotic elongation factor 1. In embodiments, the promoter is a mitochondrial heavy-strand promoter. The mitochondrial heavy-strand promoter (HSP) is derived from a region of the human mitochondrial DNA (mtDNA). In embodiments, the region from the human mtDNA includes the starting point for transcription of genes encoded on the heavy strand of mtDNA. In embodiments, the promoter is a UBC promoter. The UBC promoter is derived from the promoter of the polyubiquitin C gene (UBC). In embodiments, the UBC promoter includes heat shock elements (HSEs). In embodiments, the promoter is a CAG promoter. The CAG promoter is a hybrid promoter including the CMV early enhancer, the chicken beta-actin promoter and its first intron, and the rabbit beta-globin splice acceptor site. In embodiments, the promoter is a GAPDH promoter. In embodiments, the GAPDH promoter includes Hypoxia Responsive Elements (HREs), Spl binding sites, and myeloid zinc finger- 1 (MZF-1) transcription factor binding sites. In embodiments, thepromoter is an ACTB promoter. The ACTB promoter is derived from the ACTB (beta-actin) promoter sequence.

[0161] In embodiments, the promoter is a synthetic promoter. The term “synthetic promoter” refers to a man-made sequence typically produced by recombinant nucleic acid technology for regulating gene expression. For example, a synthetic promoter may include sequences not typically found in nature, or combinations of regulatory polynucleotide sequences not typically found together in nature. In embodiments, the synthetic promoter is an inducible promoter. The term “inducible promoter” refers to a regulatory sequence that can be activated or inactivated in response to a specific stimulus to regulate gene expression. Examples of stimuli include chemical, light, or temperature stimuli. Thus, an inducible promoter is capable of activating or inactivating expression of the operably linked gene based on presence or absence of the specific stimulus.

[0162] In embodiments, the promoter is a hybrid promoter. The term “hybrid promoter” refers to a polynucleotide sequence including sequences derived from at least two different regulatory elements (e.g. two different promoters). In embodiments, a hybrid promoter may include sequences from at least two different promoters. Thus, in embodiments, a synthetic promoter may be a hybrid promoter.

[0163] In embodiments, the constitutive promoter is a human elongation factor-1 alpha (EF-1 alpha) promoter, wherein the EF-1 alpha promoter includes SEQ ID NO: 14 or is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter is at least 80% identical to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter is at least 90% identical to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter is at least 95% identical to SEQ ID NO: 14.

[0164] In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 81% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 82% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 83% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 84% sequence identity to SEQ ID NO: 14. In embodiments, theEF-1 alpha promoter includes a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 86% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 87% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 88% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 89% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 91% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 92% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 93% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 94% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes the nucleic acid sequence of SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter is the nucleic acid sequence of SEQ ID NO: 14.

[0165] In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 80% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 81% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 82% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 83% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 84% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 85% sequence identity to SEQID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 86% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 87% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 88% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 89% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 90% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 91% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 92% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 93% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 94% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 95% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 96% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 97% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 98% sequence identity to SEQ ID NO: 14. In embodiments, the EF-1 alpha promoter includes a nucleic acid sequence having about 99% sequence identity to SEQ ID NO: 14.

[0166] In embodiments, the promoter is an ICPO promoter, optionally an endogenous ICPO promoter of the recombinant herpesvirus vector genome or the native promoter of the heterologous ICPO gene. In embodiments, the promoter is an endogenous ICPO promoter of the recombinant herpesvirus vector genome. In embodiments, the promoter is the native promoter of the heterologous ICPO gene. Methods for making and using herpesvirus vector promoters are described in more detail in U.S. Patent No. 5,849,572, which is incorporated herein in its entirety and for all purposes.

[0167] In embodiments, the promoter is an inducible promoter, optionally a chemically inducible promoter, a temperature inducible promoter, or a light induced promoter. In embodiments, the promoter is an inducible promoter. In embodiments, the promoter is a chemically inducible promoter. In embodiments, the promoter is a temperature inducible promoter. In embodiments, the promoter is a light induced promoter. In embodiments, the chemical promoter is Tet-on system. Inembodiments, the chemical promoter is Tet-off system. In embodiments, the temperature inducible promoter is a Hsp70 promoter. In embodiments, the temperature inducible promoter is a Hsp90 promoter. In embodiments, the temperature inducible promoter is derived from a Hsp70 promoter. In embodiments, the temperature inducible promoter is derived from a Hsp90 promoter. In embodiments, the light induced promoter is a YFI system. In embodiments, the light induced promoter is derived from a YFI system.

[0168] In embodiments, the recombinant herpesvirus vector genome includes an inactivating mutation in an endogenous ICPO gene. The term “inactivating mutation” in reference to a gene refers to a mutation that causes a gene or a protein expressed from said gene to lose its activity. For example, an inactivating mutation in a gene may cause a protein expressed from the gene to have less than 50%, 40, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% activity than the naturally occurring protein. In embodiments, an inactivating mutation in a gene may cause a protein expressed from the gene to have no detectable activity. In embodiments, an inactivating mutation in a gene may decrease or inhibit transcription of the gene. In embodiments, the inactivating mutation may be in a regulatory element of the gene. In embodiments, the inactivating mutation may be in the promoter region of the gene. In embodiments, the inactivating mutation may be in the coding region of the gene. In embodiments, the inactivating mutation may be deletion of a start codon or a stop codon. In embodiments, the inactivating mutation may be deletion of a start codon and a stop codon. In embodiments, the inactivating mutation may decrease the stability of the mRNA transcript produced from the gene. In embodiments, the inactivating mutation may cause the protein expressed from the gene to have decreased stability or to be misfolded.

[0169] In embodiments, the inactivating mutation produces a non-functional endogenous ICPO protein. A non-functional endogenous ICPO refers to a mutated endogenous ICPO protein that is not capable of maintaining the activity or function of the wild type protein. For example, in embodiments, a non-functional endogenous ICPO protein is unable to substantially regulate viral gene transcription, reactivate latent HSV, activate viral replication, inhibit host cell antiviral protein expression (e g. interferons) and / or regulate host cell protein expression. In embodiments, viral gene expression and replication can be assessed by detection of viral mRNA or protein expression. In embodiments, regulation of host cell protein and anti-viral protein expression can be assessed by detection host cell mRNA or protein expression. In embodiments, a non-functional endogenousICPO gene does not cause substantial cytotoxicity in a host cell including the recombinant herpesvirus vector genome provided herein including embodiments thereof. In embodiments, the inactivating mutation produces a mutated endogenous ICPO protein. In embodiments, the inactivating protein produces a fragment of an endogenous ICPO protein. In embodiments, the inactivating mutation does not produce an endogenous ICPO protein or a fragment of the endogenous ICPO protein.

[0170] In embodiments, the inactivating mutation is a deletion, insertion, point mutation or rearrangement. In embodiments, the inactivating mutation is an inactivating mutation in the endogenous ICPO gene. In embodiments, the inactivating mutation is an inactivating insertion in the endogenous ICPO gene. In embodiments, the inactivating mutation is a partial or complete deletion of the endogenous ICPO gene. In embodiments, the inactivating mutation is a deletion in the coding sequence of the endogenous ICPO gene (e.g. one or both copies of the endogenous ICPO gene). In embodiments, the inactivating mutation is a deletion in the promoter region of the ICPO gene. In embodiments, the inactivating mutation is a mutation in the promoter region of the ICPO gene. In embodiments, both copies of the endogenous human HSV1 ICPO in the recombinant herpesvirus vector are deleted. In embodiments, the inactivating mutation is an insertion of the varicellovirus ICPO gene into endogenous ICPO gene. For example, in embodiments, the inactivating mutation is a deletion of the endogenous ICPO gene or a portion of the endogenous ICPO gene and insertion of the varicellovirus ICPO gene into the endogenous ICPO gene.

[0171] In embodiments, the heterologous ICPO gene is inserted into the LAT locus of the recombinant herpesvirus vector genome. The “latency associated transcripts locus” or “LAT locus” is used in accordance to its plain ordinary meaning in the art and refers to a region within the HSV-1 genome which encodes transcripts collectively known as latency associated transcripts (LATs). The HSV genome typically has the structure of TRL-UL-IRL-IRS-US- RS, where the UL and Us are regions flanked by inverted repeats TRL / IRL and TRs / IRs, respectively. The LAT locus is located in TRL / TKL and therefore is present in two copies in the genome. The LAT locus produces multiple transcripts including an 8.3 kb IncRNA, as well as various miRNAs, and overlaps with the genes encoding regulatory proteins, including ICPO and ICP4. Thus, in embodiments, the heterologous ICPO gene is inserted into the LAT locus. In embodiments, upon insertion of the heterologous ICPO gene into the LAT locus, the LAT locus or portions thereof may be deleted. In embodiments, theL AT locus includes the region corresponding to 118,802: 121,417 in HSV-1 strain 17syn+ genome (GenBank accession JN555585.1).

[0172] In embodiments, the recombinant herpesvirus vector genome includes an inactivating mutation in one or more genes. In embodiments, the inactivating mutation decreases or inhibits cytotoxicity of the recombinant herpesvirus vector genome relative to a recombinant herpesvirus vector genome that does not include the inactivating mutation. In embodiments, the recombinant herpesvirus vector genome includes an inactivating mutation in an ICP4 gene, an ICP27 gene, a UL41 gene, a UL47 gene, or a combination thereof. In embodiments, the recombinant herpesvirus vector genome includes an inactivating mutation in an ICP4 gene, an ICP27 gene, a UL41 gene, or a combination thereof. In embodiments, the recombinant herpesvirus vector genome includes an inactivating mutation in an ICP4 gene and / or an ICP27 gene. In embodiments, both copies of the ICP4 in the recombinant herpesvirus vector genome are deleted. In embodiments, both copies of the ICP27 in the recombinant herpesvirus vector genome are deleted. Methods for making inactivating mutations in ICP4 and ICP27 are described in more detail in U.S. Patent No. 5,658,724, U.S. Patent No. 5,879,934, and U.S. Patent No. 7,531,167, which are incorporated herein in their entirety and for all purposes.

[0173] In embodiments, the recombinant herpesvirus vector genome includes an inactivating mutation in an ICP4 gene. In embodiments, the inactivating mutation produces a non-functional ICP4 protein. A non-functional ICP4 protein refers to a mutated ICP4 protein that is not capable of maintaining the activity or function of the wild type ICP4 protein. For example, in embodiments, a non-functional ICP4 protein is unable to regulate host cell gene expression or regulates host cell gene expression at substantially undetectable levels. In embodiments, the inactivating mutation produces a mutated ICP4 protein. In embodiments, the inactivating protein produces a fragment of an 1CP4 protein. In embodiments, the inactivating mutation does not produce an ICP4 protein.

[0174] In embodiments, the inactivating mutation is a deletion, insertion, point mutation or rearrangement in the ICP4 gene. In embodiments, the inactivating mutation is an inactivating mutation in the 1CP4 gene. In embodiments, the inactivating mutation is an inactivating insertion in the ICP4 gene. In embodiments, the inactivating mutation is a partial or complete deletion of the ICP4 gene. In embodiments, the inactivating mutation is a deletion in the coding sequence of theICP4 gene (e.g. one or both copies of the ICP4 gene). In embodiments, the inactivating mutation is a deletion in the promoter region of the ICP4 gene. In embodiments, the inactivating mutation is a mutation in the promoter region of the ICP4 gene. In embodiments, both copies of the ICP4 gene in the recombinant herpesvirus vector are deleted. In embodiments, the inactivating mutation is an insertion of the heterologous ICPO gene into ICP4 gene.

[0175] In embodiments, the recombinant herpesvirus vector genome includes an inactivating mutation in an ICP27 gene. In embodiments, the inactivating mutation produces a non-functional ICP27 protein. A non-functional ICP27 protein refers to a mutated ICP27 protein that is not capable of maintaining the activity or function of the wild type ICP27 protein. For example, in embodiments, a non-functional ICP27 protein is unable to regulate host cell gene expression or regulates host cell gene expression at substantially undetectable levels. In embodiments, the inactivating mutation produces a mutated ICP27 protein. In embodiments, the inactivating protein produces a fragment of an ICP27 protein. In embodiments, the inactivating mutation does not produce an ICP27 protein.

[0176] In embodiments, the inactivating mutation is a deletion, insertion, point mutation or rearrangement in the ICP27 gene. In embodiments, the inactivating mutation is an inactivating mutation in the ICP27 gene. In embodiments, the inactivating mutation is an inactivating insertion in the ICP27 gene. In embodiments, the inactivating mutation is a partial or complete deletion of the ICP27 gene. In embodiments, the inactivating mutation is a deletion in the coding sequence of the ICP27 gene (e.g. one or both copies of the ICP27 gene). In embodiments, the inactivating mutation is a deletion in the promoter region of the ICP27 gene. In embodiments, the inactivating mutation is a mutation in the promoter region of the ICP27 gene. In embodiments, both copies of the ICP27 gene in the recombinant herpesvirus vector are deleted. In embodiments, the inactivating mutation is an insertion of the heterologous ICP27 gene into ICP27 gene.

[0177] In embodiments, the recombinant herpesvirus vector genome includes an inactivating mutation in a virion host shutoff (vhs) gene (UL41). In embodiments, the inactivating mutation produces a non-functional UL41 protein. A non-functional UL41 protein refers to a mutated UL41 protein that is not capable of maintaining the activity or function of the wild type UL41 protein. For example, in embodiments, a non-functional UL41 protein is unable to regulate host cell proteinsynthesis or degrade host cell mRNA or regulates host cell protein synthesis or degrades host cell mRNA at substantially undetectable levels. In embodiments, the inactivating mutation produces a mutated UL41 protein. In embodiments, the inactivating protein produces a fragment of an UL41 protein. In embodiments, the inactivating mutation does not produce an UL41 protein.

[0178] In embodiments, the inactivating mutation is a deletion, insertion, point mutation or rearrangement in the UL41 gene. In embodiments, the inactivating mutation is an inactivating mutation in the UL41 gene. In embodiments, the inactivating mutation is an inactivating insertion in the UL41 gene. In embodiments, the inactivating mutation is a partial or complete deletion of the UL41 gene. In embodiments, the inactivating mutation is a deletion in the coding sequence of the UL41 gene (e.g. one or both copies of the UL41 gene). In embodiments, the inactivating mutation is a deletion in the promoter region of the UL41 gene. In embodiments, the inactivating mutation is a mutation in the promoter region of the UL41 gene. In embodiments, both copies of the UL41 gene in the recombinant herpesvirus vector are deleted. In embodiments, the inactivating mutation is an insertion of the heterologous ICPO gene into UL41 gene.

[0179] In embodiments, embodiments, the recombinant herpesvirus vector genome includes an inactivating mutation in an ICP47 gene. In embodiments, the inactivating mutation produces a nonfunctional ICP47 protein. A non-functional ICP47 protein refers to a mutated ICP47 protein that is not capable of maintaining the activity or function of the wild type ICP47 protein. For example, in embodiments, a non-functional ICP47 protein is unable to regulate mRNA degradation or regulates mRNA degradation at substantially undetectable levels. In embodiments, the inactivating mutation produces a mutated ICP47 protein. In embodiments, the inactivating protein produces a fragment of an ICP47 protein. In embodiments, the inactivating mutation does not produce an ICP47 protein.

[0180] In embodiments, the inactivating mutation is a deletion, insertion, point mutation or rearrangement in the ICP47 gene. In embodiments, the inactivating mutation is an inactivating mutation in the ICP47 gene. In embodiments, the inactivating mutation is an inactivating insertion in the ICP47 gene. In embodiments, the inactivating mutation is a partial or complete deletion of the ICP47 gene. In embodiments, the inactivating mutation is a deletion in the coding sequence of the ICP47 gene (e.g. one or both copies of the ICP47 gene). In embodiments, the inactivating mutation is a deletion in the promoter region of the ICP47 gene. In embodiments, the inactivating mutation isa mutation in the promoter region of the ICP47 gene. In embodiments, both copies of the ICP47 gene in the recombinant herpesvirus vector are deleted. In embodiments, the inactivating mutation is an insertion of the heterologous ICPO gene into ICP47 gene.

[0181] For the recombinant herpesvirus vector genome provided herein, in embodiments, the recombinant herpesvirus vector genome includes an inactivating mutation in a joint region. As used herein, “joint” or “joint region” in reference to a herpesvirus genome refers to the region that separates the unique long (UL) and unique short (Us) portions of the genome, including the internal repeats (IRL and IRs) and the terminal repeats (TRL and TRs). The joint region includes copies of regulatory genes, including the ICPO gene and the ICP4 gene. The LAT locus, which plays a crucial role in latency, is located in the inverted repeat regions associated with the long repeat (IRL and TRL), antisense to the ICPO gene.

[0182] In embodiments, the inactivating mutation in the joint region is a deletion, insertion, point mutation or rearrangement of a gene within the joint region. For example, in embodiments, the inactivating mutation is a deletion, insertion, point mutation or rearrangement in the LAT locus, endogenous ICPO gene, ICP4 gene, or a combination thereof. In embodiments, the inactivating mutation in the joint region is a complete or partial deletion of the joint region. In embodiments, the complete or partial deletion of the joint region increases the stability of the recombinant herpesvirus vector genome relative to a recombinant herpesvirus vector genome that does not include the complete or partial deletion of the joint region. In embodiments, deletion of the joint region includes deletion of one copy of an endogenous ICPO gene within the recombinant herpesvirus vector genome. In embodiments, deletion of the joint region includes deletion of one copy of an ICP4 gene. In embodiments, the recombinant herpesvirus vector genome includes an inactivating mutation in a UL / US (unique long / unique short) joint region. In embodiments, the inactivating mutation includes an inactivating mutation in the joint region separating the UL and the Us genome elements of the recombinant herpesvirus vector genome.

[0183] In embodiments, the recombinant herpesvirus vector genome includes a joint region. In embodiments, the recombinant herpesvirus vector genome includes a deletion of the joint region.

[0184] In embodiments, the recombinant herpesvirus vector genome includes a transgene. As used herein, a “transgene” refers to a nucleic acid sequence that originates from outside a given cell,organism, or virus. A transgene as provided herein is therefore not native to, or does not originate from within the herpesvirus vector genome. A transgene as provided herein may encode a protein or may be a non-coding nucleic acid sequence. Transgenes provided herein may a polynucleotide sequence useful for treating a disease (e.g., polynucleotide sequences that encode for therapeutic proteins) or detectable moiety-encoding polynucleotide sequences.

[0185] In embodiments, the recombinant herpesvirus vector genome includes a plurality of transgenes. In embodiments, the recombinant herpesvirus vector genome includes a first transgene and a second transgene. In embodiments, the first transgene encodes a first therapeutic protein and the second transgene encodes a second therapeutic protein. In embodiments, the first transgene encodes a therapeutic protein and the second transgene encodes is a detectable moiety-encoding polynucleotide sequence.

[0186] A “therapeutic protein” as used herein refers to a recombinant protein for treating or preventing a disease (e.g. RDEB, DEB) or a condition. In embodiments, a therapeutic protein replaces a dysfunctional or deficient protein (e.g. Col7A protein) in a disease pathway. In embodiments, a therapeutic protein performs an enzymatic reaction. In embodiments, the therapeutic protein is a regulatory protein.

[0187] A “detectable moiety-encoding polynucleotide sequence” as used herein refers to a polynucleotide sequence that encodes a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Detectable moiety-encoding polynucleotide sequences may encode a fluorescent moiety. Non-limiting examples of fluorescent moieties are zsGreen, mCherry, Emerald, and firefly luciferase.

[0188] In embodiments, the transgene is operably linked to one or more insulator sequences within the HSV vector genome. Without wishing to be bound by scientific theory, the one or more insulator sequences may inhibit formation of heterochromatin at the sites of the insulator sequences and within from about 1 kb to about 5 kb from the insulator sequences. Thus, in embodiments, the one or more insulator sequences may inhibit binding of a protein that induces formation of heterochromatin, or formation of heterochromatin, which would decrease or inhibit expression of the transgene. Non-limiting examples of suitable insulator sequences include HSV chromatin boundary (CTRL / CTCF-binding / insulator) elements CTRL I and CTRL2, chicken hypersensitive site 4insulator (cHS4), human HNRP A2B1— CBX3 ubiquitous chromatin opening element (UCOE), and the scaffold / matrix attachment region (S / MAR) from the human interferon beta gene (IFNB1). In embodiments, the insulator sequences are CTRL1 and CTRL2. In embodiments, the CTRL1 and CTRL2 insulator sequences are endogenous to the recombinant herpesvirus vector genome provided herein including embodiments thereof. In embodiments, the CTRL1 and CTRL2 insulator sequences are native to (e.g. endogenous) to the LAT locus. In embodiments, the CTRL1 and CTRL2 insulator sequences are endogenous to the recombinant herpesvirus vector genome provided herein including embodiments thereof and are moved to an ectopic location within the genome. Insulator sequences and methods for enhancing transgene expression are described in more detail in Emery, Hum. Gene Ther. 22,761- 74 (2011) and Antoniou et aL, Hum. Gene Ther. 24, 363-74 (2013), which are incorporated herein in their entirety and for all purposes.

[0189] In embodiments, the transgene encodes a therapeutic protein. In embodiments, the therapeutic protein is COL7A1 (collagen alpha-1 (VII) chain), SERPINA1 (Alpha-1 Antitrypsin), PKD1 (Polycystin 1), ABCB11 (ATP Binding Cassette Subfamily B Member 11), PKD2 (Polycystin 2), ATP7B (ATPase Copper Transporting Beta), SERPINC1 (Antithrombin III), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), ATP8B1 (ATPase Phospholipid Transporting 8B1), ABCB4 (ATP Binding Cassette Subfamily B Member 4), CFH (Complement Factor H), F8 (Coagulation Factor VIII), APOB (Apolipoprotein B), LDLR (Low Density Lipoprotein Receptor), SMN1 (Survival Motor Neuron 1), RPE65 (Retinal Pigment Epithelium- Specific 65 kDa Protein), F9 (Factor IX), IL-2 (Interleukin-2), a CAR (Chimeric Antigen Receptor), ABCA4 (ATP Binding Cassette Subfamily A Member 4), GAA (Acid Alpha-Glucosidase), GBA (Glucocerebrosidase), SGSH (N-Sulfoglucosamine Sulfohydrolase), IDUA (Alpha-L-Iduronidase), ADA (Adenosine Deaminase), OTC (Ornithine Transcarbamylase), HEXA (Hexosaminidase A), FANCA (Fanconi Anemia Complementation Group A), MECP2 (Methyl CpG Binding Protein 2), ASPA (Aspartoacylase), BTK (Bruton Tyrosine Kinase), HBB (Hemoglobin Subunit Beta), DMD (Dystrophin), CNGB3 (Cyclic Nucleotide Gated Channel Beta 3), PRKAG2 (Protein Kinase AMP- Activated Non-Catalytic Subunit Gamma 2), CLN2 (Ceroid-Lipofuscinosis, Neuronal 2), ARSA (Aryl sulfatase A), GUSB (Beta-Glucuronidase), GALC (Galactosylceramidase), SGCB (Sarcoglycan Beta), MY07A (myosin VIIA), PCHD15 (protocadherin related 15), CDH23 (cadherin related 23), USH2A (usherin), or GPR98 (adhesion G protein-coupled receptor VI). Inembodiments, the therapeutic protein is is C0L7A1 (collagen alpha-1 (VII) chain). In embodiments, the therapeutic protein is SERPINA1 (Alpha-1 Antitrypsin). In embodiments, the therapeutic protein is PKD1 (Polycystin 1). In embodiments, the therapeutic protein is ABCB11 (ATP Binding Cassette Subfamily B Member 11), In embodiments, the therapeutic protein is PKD2 (Polycystin 2). In embodiments, the therapeutic protein is ATP7B (ATPase Copper Transporting Beta). In embodiments, the therapeutic protein is SERPINC1 (Antithrombin III). In embodiments, the therapeutic protein is CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). In embodiments, the therapeutic protein is ATP8B1 (ATPase Phospholipid Transporting 8B1). In embodiments, the therapeutic protein is ABCB4 (ATP Binding Cassette Subfamily B Member 4). In embodiments, the therapeutic protein is CFH (Complement Factor H). In embodiments, the therapeutic protein is F8 (Coagulation Factor VIII). In embodiments, the therapeutic protein is APOB (Apolipoprotein B). In embodiments, the therapeutic protein is LDLR (Low Density Lipoprotein Receptor). In embodiments, the therapeutic protein is SMN1 (Survival Motor Neuron 1). In embodiments, the therapeutic protein is RPE65 (Retinal Pigment Epithelium-Specific 65 kDa Protein). In embodiments, the therapeutic protein is F9 (Factor IX). In embodiments, the therapeutic protein is IL-2 (Interleukin-2). In embodiments, the therapeutic protein is a CAR (Chimeric Antigen Receptor). In embodiments, the therapeutic protein is ABCA4 (ATP Binding Cassette Subfamily A Member 4). In embodiments, the therapeutic protein is GAA (Acid Alpha-Glucosidase). In embodiments, the therapeutic protein is GBA (Glucocerebrosidase). In embodiments, the therapeutic protein is SGSH (N-Sulfoglucosamine Sulfohydrolase). In embodiments, the therapeutic protein is IDUA (Alpha-L-Iduronidase). In embodiments, the therapeutic protein is ADA (Adenosine Deaminase). In embodiments, the therapeutic protein is OTC (Ornithine Transcarbamylase). In embodiments, the therapeutic protein is HEXA (Hexosaminidase A). In embodiments, the therapeutic protein is FANCA (Fanconi Anemia Complementation Group A). In embodiments, the therapeutic protein is MECP2 (Methyl CpG Binding Protein 2). In embodiments, the therapeutic protein is ASPA (Aspartoacylase). In embodiments, the therapeutic protein is BTK (Bruton Tyrosine Kinase). In embodiments, the therapeutic protein is HBB (Hemoglobin Subunit Beta). In embodiments, the therapeutic protein is DMD (Dystrophin). In embodiments, the therapeutic protein is CNGB3 (Cyclic Nucleotide Gated Channel Beta 3). In embodiments, the therapeutic protein is PRKAG2 (Protein Kinase AMP -Activated Non-Catalytic Subunit Gamma 2). In embodiments, thetherapeutic protein is CLN2 (Ceroid-Lipofuscinosis, Neuronal 2). In embodiments, the therapeutic protein is ARSA (Arylsulfatase A). In embodiments, the therapeutic protein is GUSB (Beta- Glucuronidase). In embodiments, the therapeutic protein is GALC (Galactosylceramidase). In embodiments, the therapeutic protein is SGCB (Sarcoglycan Beta). In embodiments, the therapeutic protein is MY07A (myosin VIIA). In embodiments, the therapeutic protein is PCHD15 (protocadherin related 15). In embodiments, the therapeutic protein is CDH23 (cadherin related 23). In embodiments, the therapeutic protein is USH2A (usherin). In embodiments, the therapeutic protein is GPR98 (adhesion G protein-coupled receptor VI).

[0190] In embodiments, the recombinant herpesvirus vector genome includes genes essential for alpha herpesvirus replication.

[0191] In embodiments, the recombinant herpesvirus vector genome is a Herpes Simplex Virus 1 (HSV-1) vector genome. In embodiments, the recombinant herpesvirus vector genome is a Herpes Simplex Virus 2 (HSV-2) vector genome. Recombinant herpesvirus vector genomes and methods for making the same are described in more detail in WO 2015 / 009952; U.S. Patent No. 5,998,174; Miyagawa, Y., et al. Deletion of the Virion Host Shut-off Gene Enhances Neuronal -Selective Transgene Expression from an HSV Vector Lacking Functional IE Genes. Mol Ther Methods Clin Dev. 2017 Jun 16;6:79-90. doi: 10.1016 / j.omtm.2017.06.001. PMID: 28702475; PMCID: PMC5493822.; and Miyagawa, Y., et al. Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity. Proc Natl Acad Sci U S A. 2015 Mar 31;112(13):E1632-41. doi: 10.1073 / pnas.1423556112. Epub 2015 Mar 16. PMID: 25775541;PMCID: PMC4386379., which are incorporated by reference in its entirety and for all purposes.

[0192] In one embodiment, the recombinant herpesvirus vector genome is a recombinant HSV-1 vector genome, wherein the recombinant HSV-1 vector genome includes a heterologous ICP0 gene, wherein the heterologous ICP0 gene is a bICPO gene, wherein the recombinant HSV-1 vector genome includes an inactivating mutation in the endogenous ICP0 gene, an inactivating mutation in the ICP4 gene, an inactivating mutation in the ICP27 gene, and an inactivating mutation in the ICP47 gene, wherein the inactivating mutation in the endogenous ICP0 gene is a deletion in the coding sequence of the endogenous ICP0 gene, the inactivating mutation in the ICP4 gene is a deletion in the coding sequence of the ICP4 gene, the inactivating mutation in the ICP27 gene is adeletion in the coding sequence of the ICP27 gene, and the inactivating mutation in the ICP47 gene is a deletion in the promoter region, the start codon or the stop codon of the ICP47 gene, and wherein the recombinant HSV-1 vector genome further includes a transgene, and wherein the transgene is inserted into the LAT locus of the recombinant HSV-1 vector genome.

[0193] In one embodiment, the recombinant herpesvirus vector genome is a recombinant HSV-1 vector genome, wherein the recombinant HSV-1 vector genome includes a heterologous ICPO gene, wherein the heterologous ICPO gene is an elCPO gene, wherein the recombinant HSV-1 vector genome includes an inactivating mutation in the endogenous ICPO gene, an inactivating mutation in the ICP4 gene, an inactivating mutation in the ICP27 gene, and an inactivating mutation in the ICP47 gene, wherein the inactivating mutation in the endogenous ICPO gene is a deletion in the coding sequence of the endogenous ICPO gene, the inactivating mutation in the ICP4 gene is a deletion in the coding sequence of the ICP4 gene, the inactivating mutation in the ICP27 gene is a deletion in the coding sequence of the ICP27 gene, and the inactivating mutation in the ICP47 gene is a deletion in the promoter region, the start codon or the stop codon of the ICP47 gene, and wherein the recombinant HSV-1 vector genome further includes a transgene, and wherein the transgene is inserted into the LAT locus of the recombinant HSV-1 vector genome.

[0194] In one embodiment, the recombinant herpesvirus vector genome is a recombinant HSV-1 vector genome, wherein the recombinant HSV-1 vector genome includes a heterologous ICPO gene, wherein the heterologous ICPO gene is a bICPO gene, wherein the recombinant HSV-1 vector genome includes an inactivating mutation in the endogenous ICPO gene, an inactivating mutation in the ICP4 gene, an inactivating mutation in the ICP27 gene, an inactivating mutation in the ICP47 gene, and an inactivating mutation in the UL41 gene, wherein the inactivating mutation in the endogenous ICPO gene is a deletion in the coding sequence of the endogenous ICPO gene, the inactivating mutation in the ICP4 gene is a deletion in the coding sequence of the ICP4 gene, the inactivating mutation in the ICP27 gene is a deletion in the coding sequence of the ICP27 gene, the inactivating mutation in the ICP47 gene is a deletion in the promoter region, the start codon or the stop codon of the ICP47 gene, the inactivating mutation in the UL41 gene is a deletion in the coding sequence of the UL41 gene, and wherein the recombinant HSV-1 vector genome further includes a transgene, and wherein the transgene is inserted into the LAT locus of the recombinant HSV-1 vector genome.

[0195] In one embodiment, the recombinant herpesvirus vector genome is a recombinant HSV-1 vector genome, wherein the recombinant HSV-1 vector genome includes a heterologous ICPO gene, wherein the heterologous ICPO gene is an elCPO gene, wherein the recombinant HSV-1 vector genome includes an inactivating mutation in the endogenous ICPO gene, an inactivating mutation in the ICP4 gene, an inactivating mutation in the ICP27 gene, an inactivating mutation in the ICP47 gene, and an inactivating mutation in the UL41 gene, wherein the inactivating mutation in the endogenous ICPO gene is a deletion in the coding sequence of the endogenous ICPO gene, the inactivating mutation in the ICP4 gene is a deletion in the coding sequence of the ICP4 gene, the inactivating mutation in the ICP27 gene is a deletion in the coding sequence of the ICP27 gene, and the inactivating mutation in the ICP47 gene is a deletion in the promoter region, the start codon or the stop codon of the ICP47 gene, and the inactivating mutation in the UL41 gene is a deletion in the coding sequence of the UL41 gene, and wherein the recombinant HSV-1 vector genome further includes a transgene, and wherein the transgene is inserted into the LAT locus of the recombinant HSV-1 vector genome.POLYNUCLEOTIDE COMPOSITIONS

[0196] The compositions provided herein include polynucleotides including the recombinant herpesvirus vector genome provided herein including embodiments thereof. Thus, in an aspect is provided a polynucleotide including the recombinant herpesvirus vector genome provided herein including embodiments thereof.

[0197] In embodiments, the polynucleotide includes one or more sequences that facilitate propagation of the recombinant herpesvirus vector genome provided herein including embodiments thereof in a bacteria cell. For example, in embodiments, the polynucleotide may include one or more sequences necessary for host cell infection and viral replication. Thus, in embodiments, the polynucleotide includes a bacterial artificial chromosome (BAC). The term “bacterial artificial chromosome” is used in accordance to its plain ordinary meaning in the art and refers to a circular DNA construct capable of carrying large DNA inserts, for example, DNA inserts from about 100 kb to about 350 kb in length. In embodiments, the BAC includes one or more sequences derived from a bacteria. In embodiments, the BAC includes a selectable gene, for example, a gene that confers bacterial resistance to an antibiotic or toxin (e.g., chloramphenicol, tetracycline, ampicillin, zeocin,etc.). The BAC can further include a detectable-moiety encoding polynucleotide. In embodiments, the detectable moiety-encoding polynucleotide is a reporter gene (e.g., LacZ (encoding betagalactosidase), or a fluorescent protein-encoding gene (e.g., gfp (encoding green fluorescent protein), yfp (encoding yellow fluorescent protein), rfp (encoding red fluorescent protein), and analogues thereof (e.g., encoding iRFP, EGFP, and the like)) under the control of a eukaryotic promoter, such as a constitutive mammalian promoter (e.g., an SV40, RSV, CMV, ubiquitin C (UbC), CAG, or 0-actin promoter, etc.).

[0198] For the polynucleotide provided herein, in embodiments, the BAC is flanked by sequences facilitating removal of the BAC, for example, site-specific recombinase recognition sites. In embodiments, the BAC is flanked by consensus sequences recognized by enzymes including ere, dre, flp, KD, B2, B3, R, etc. In embodiments, the sequences facilitate excision of the BAC from the polynucleotide.

[0199] In embodiments, the polynucleotide provided herein including embodiments thereof is codon-optimized. As used herein, codon-optimization refers to genetic engineering methods that use synonymous codon substitutions to increase protein production in host expression system. In embodiments, codon-optimization refers to the substitution of less frequent codons with more frequent codons according to genomic codon usage in an organism that serves as the host for protein expression. In embodiments, codon-optimization including substitution of codons with codons that are more efficiently recognized by the host’s tRNA pool. For example, codons may be selected which correspond to tRNAs that are abundunt in the host expression system. In embodiments, the polynucleotide is codon-optimized for human cells. In embodiments, the polynucleotide is codon- optimized for mouse cells. In embodiments, the polynucleotide is codon-optimized for rat cells. In embodiments, the polynucleotide is codon-optimized for hamster cells. In embodiments, the polynucleotide is codon-optimized for yeast cells. In embodiments, the polynucleotide is codon- optimized for bacterial cells. In embodiments, the polynucleotide is codon-optimized for A. coli.

[0200] In embodiments, the polynucleotide provided herein including embodiments thereof is a DNA polynucleotide, RNA polynucleotide, or a combination thereof.

[0201] The polynucleotide provided herein may form part of a vector. In embodiments, the polynucleotide may form part of a vector DNA including regulatory sequences that facilitateexpression of a gene in a transfected cell. In embodiments, the vector directs the cell's machinery to make RNA and protein(s). In an aspect is provided a recombinant alphaherpesvirus vector, including the recombinant herpesvirus vector genome provided herein including embodiments thereof, a viral capsid, a viral envelope, and a viral tegument.

[0202] “ Viral capsid” is used in accordance to its plain ordinary meaning in the art, and refers to a layer of protein that surrounds the viral genome. The viral capsid may prevent or inhibit viral genome degradation. In embodiments, the viral capsid facilitates attachment and entry into a host cell for the purpose of delivering the viral genome into the host for replication.

[0203] “Viral envelope” is used in accordance to its plain ordinary meaning in the art, and refers to an outer layer of a virus. The viral envelope may function to prevent or inhibit viral genome degradation. The viral envelope facilitates the entry of a virus into the host cell and the exit of a virus from the host cell.

[0204] “Viral tegument” is used in accordance to its plain ordinary meaning in the art, and refers to a layer of protein that surrounds the viral capsid. Typically, the viral tegument includes a variety of viral proteins. In embodiments, viral tegument proteins function to facilitate efficient viral replciation, mediate viral envelopment, and faclilate viral capside transport. In embodiments, the viral tegument includes proteins associated with suppressing the host’s immune system.

[0205] In embodiments, the recombinant alphaherpesvirus vector is a recombinant simplexvirus vector.

[0206] In embodiments, the recombinant alphaherpesvirus vector is a recombinant Herpes Simplex Virus 1 (HSV-1) particle. In embodiments, the recombinant alphaherpesvirus vector is a recombinant Herpes Simplex Virus 2 (HSV-2) particle.

[0207] In embodiments, the recombinant alphaherpesvirus vector is capable of transducing a cell. In embodiments, the recombinant alphaherpesvirus vector is non-toxic to transduced cells. Toxicity of the recombinant alphaherpesvirus vector in a transduced cell can be assessed by cytotoxicity assays, for example to detect cell apoptosis (e.g. degradation of cell proteins, genome fratgmentation, detection of cellular membrane, detection of cellular cytotoxicity biomarkers (e.g.LDH or G6PD). In embodiments, a cell transduced with the recombinant alphaherpesvirus vector provided herein does not have substantially decreased viability relative to a non-transduced cell.

[0208] In embodiments, the recombinant alphaherpesvirus vector is not oncolytic. For example, in embodiments, the recombinant alphaherpesvirus vector viruses does not specifically infect and replicate in cancer cells. In embodiments, the recombinant alphaherpesvirus vector does not selectivity replicate and kill dividing cells as compared to non-dividing cells.

[0209] In embodiments, the recombinant herpesvirus vector genome includes a transgene. In embodiments, the recombinant herpesvirus vector genome includes a single transgene. In embodiments, the recombinant herpesvirus vector genome includes a plurality of transgenes. In embodiments, each of the plurality of transgenes are the same transgene. In embodiments, each of the plurality of transgenes is a different transgene. In embodiments, the transgene encodes a therapeutic protein. In embodiments, the therapeutic protein is COL7A1 (collagen alpha- 1 (VII) chain), SERPINAl (Alpha-1 Antitrypsin), PKD1 (Polycystin 1), ABCB11 (ATP Binding Cassette Subfamily B Member 11), PKD2 (Poly cystin 2), ATP7B (ATPase Copper Transporting Beta), SERPINC1 (Antithrombin III), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), ATP8B1 (ATPase Phospholipid Transporting 8B1), ABCB4 (ATP Binding Cassette Subfamily B Member 4), CFH (Complement Factor H), F8 (Coagulation Factor VIII), APOB (Apolipoprotein B), LDLR (Low Density Lipoprotein Receptor), SMN1 (Survival Motor Neuron 1), RPE65 (Retinal Pigment Epithelium-Specific 65 kDa Protein), F9 (Factor IX), IL-2 (Interleukin-2), a CAR (Chimeric Antigen Receptor), ABCA4 (ATP Binding Cassette Subfamily A Member 4), GAA (Acid Alpha-Glucosidase), GBA (Glucocerebrosidase), SGSH (N-Sulfoglucosamine Sulfohydrolase), IDUA (Alpha-L-Iduronidase), ADA (Adenosine Deaminase), OTC (Ornithine Transcarbamylase), HEXA (Hexosaminidase A), FANCA (Fanconi Anemia Complementation Group A), MECP2 (Methyl CpG Binding Protein 2), ASPA (Aspartoacylase), BTK (Bruton Tyrosine Kinase), HBB (Hemoglobin Subunit Beta), DMD (Dystrophin), CNGB3 (Cyclic Nucleotide Gated Channel Beta 3), PRKAG2 (Protein Kinase AMP -Activated Non-Catalytic Subunit Gamma 2), CLN2 (Ceroid- Lipofuscinosis, Neuronal 2), ARSA (Aryl sulfatase A), GUSB (Beta-Glucuronidase), GALC (Galactosylceramidase), SGCB (Sarcoglycan Beta), MY07A (myosin VIIA), PCHD15 (protocadherin related 15), CDH23 (cadherin related 23), USH2A (usherin), or GPR98 (adhesion G protein-coupled receptor VI). In embodiments, the therapeutic protein is COL7A1.

[0210] In embodiments, the transgene is codon-optimized.

[0211] In embodiments, the recombinant herpesvirus vector genome provided herein including embodiments thereof include a plurality of transgenes encoding COL7A1 protein. In embodiments, each of the plurality of transgenes encoding COL7A1 protein include codon variations. In embodiments, the codon variations in each of the transgenes encoding COL7A1 protein reduce sequence homology, thereby preventing recombination between the transgenes.

[0212] In embodiments, the recombinant alphaherpesvirus vector causes cells transduced with the recombinant alphaherpesvirus vector to express a therapeutic protein provided herein including embodiments thereof at a level and / or for a duration greater than a reference vector including a vector genome lacking the heterologous varicellovirus ICPO. A “reference vector” refers to a recombinant herpes virus vector to which a test recombinant herpes virus vector is compared. In embodiments, the reference vector is identical to the test recombinant herpes virus vector with the exception of the heterologous ICPO gene included in the test vector. Thus, in embodiments, the reference vector does not include the heterologous IPCO gene provided herein including embodiments thereof. The reference vector may produce a value (e.g. reference value) to which a measured quantity is compared (e.g. therapeutic protein expression level, duration of therapeutic protein expression, cellular toxicity, etc.). Thus, in embodiments, the reference vector produces a reference value (or control value) which is measured in parallel with a test vector.

[0213] In embodiments, the recombinant alphaherpesvirus vector causes cells transduced with the recombinant alphaherpesvirus vector to express a therapeutic protein provided herein including embodiments thereof at a level at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than a reference vector including a vector genome lacking the heterologous varicellovirus ICPO provided herein including embodiments thereof. In embodiments, the recombinant alphaherpesvirus vector causes cells transduced with the recombinant alphaherpesvirus vector to express a therapeutic protein provided herein including embodiments for a duration at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times longer than a reference vector including a vector genome lacking the heterologous varicellovirus ICPO provided herein including embodiments thereof. In embodiments, the recombinant alphaherpesvirus vector provided herein causes cellstransduced with the recombinant alphaherpesvirus vector to detectably express a therapeutic protein provided herein including embodiments thereof for at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32 days, 34 days, 36 days, 38 days, or 40 days.CELL COMPOSITIONS

[0214] The polynucleotide compositions provided herein including the recombinant herpesvirus vector genome provided herein including embodiments thereof are capable of transducing a cell. In embodiments, delivery (e.g. transduction) of the polynucleotide thereof cause no or substantially no cellular toxicity to the transduced cell. Thus, in an aspect is provided a host cell including the polynucleotide provided herein including embodiments thereof.

[0215] In embodiments, the host cell is a complementing cell. For example, in embodiments, the complementing cell expresses a gene that has been inactivated (e.g. by an inactivating mutation) in the recombinant herpesvirus vector genome provided herein including embodiments thereof. In embodiments, the host cell includes one or more polynucleotides encoding Human Herpes Simplex Virus 1 ICP4, Human Herpes Simplex Virus 1 ICP0, ICP27, or a combination thereof. Methods for generating a complementing cell are described in more detail in U.S. Patent No. 5,804,413, which is incorporated herein in its entirety and for all purposes.

[0216] In embodiments, the host cell includes one or more polynucleotides encoding genes sufficient to package a simplexvirus viral particle. In embodiments, the gene sufficient to package a simplexvirus viral particle includes Human Herpes Simplex Virus 1 ICP4, Human Herpes Simplex Virus 1 ICP0, Human Herpes Simplex Virus 1 ICP27 or a combination thereof. In embodiments, the gene sufficient to package a simplexvirus viral particle includes Human Herpes Simplex Virus 1 ICP4. In embodiments, the gene sufficient to package a simplexvirus viral particle includes Human Herpes Simplex Virus 1 ICP0. In embodiments, the gene sufficient to package a simplexvirus viral particle includes Human Herpes Simplex Virus 1 ICP27. In embodiments, the gene sufficient to package a simplexvims viral particle includes Human Herpes Simplex Virus 1 ICP4 and Human Herpes Simplex Virus 1 ICP0.METHODS OF USE

[0217] Provided herein are methods for making a viral particle, wherein the viral particle includes a heterologous ICPO gene as provided herein including embodiments thereof. In embodiments, the method includes contacting a host cell with recombinant herpesvirus vector genome provided herein including embodiments thereof or a polynucleotide provided herein including embodiments thereof. In embodiments, subsequent to the contacting, the host cell expresses the heterologous ICPO gene and the transgene provided herein including embodiments thereof. As described throughout the specification, in embodiments, the host cell expresses increased levels of the transgene relative to a host cell including a virus particle without the heterologous ICPO gene. Thus, in an aspect is provided a method of making a viral particle including culturing the host cell provided herein including embodiments thereof under conditions that the host cell produces the viral particle.

[0218] In embodiments, the host cell is a keratinocyte or a dermal fibroblast. In embodiments, the host cell is a keratinocyte. In embodiments, the host cell is a dermal fibroblast.

[0219] In embodiments, the host cell includes one or more genes sufficient to package a simplexvirus viral particle. In embodiments, the one or more genes sufficient to package a simplexvirus viral particle is an essential viral gene. In embodiments, the host cell expresses an essential viral gene. The term “essential viral gene” as used herein in relation to the virus provided herein, refers to a gene required for one or more of viral replication, viral amplification or viral spread (e.g. transmission of the virus from one cell to another cell). Thus, in embodiments, an essential viral gene is a gene required for viral propagation. In embodiments, the essential viral gene is a gene essential for viral replication. In embodiments, the essential viral gene is a gene essential for viral amplification. In embodiments, the essential viral gene is a gene essential for viral spread. For example, the essential viral gene may be essential for virus entry into a host cell. In another example, the essential viral gene may be a gene required for viral DNA replication.

[0220] In embodiments, the essential viral gene is UL9, UL29 (ICP8), UL5, UL52, UL8, UL30, UL23, UL19 (Major capsid protein), UL6, UL25, UL27 (gB), UL28, UL15, UL33, or a combination thereof. In embodiments, the essential viral gene is UL9. In embodiments, the essential viral gene is UL29 (ICP8) . In embodiments, the essential viral gene is UL5. In embodiments, the essential viral gene is UL52. In embodiments, the essential viral gene is UL8. In embodiments, the essential viral gene is UL30. In embodiments, the essential viral gene is UL23. In embodiments, the essential viralgene is UL19 (Major capsid protein) . In embodiments, the essential viral gene is UL6. In embodiments, the essential viral gene is UL25. In embodiments, the essential viral gene is UL27 (gB) . In embodiments, the essential viral gene is UL28. In embodiments, the essential viral gene is UL15. In embodiments, the essential viral gene is UL33.

[0221] In embodiments, the host cell includes a Human Herpes Simplex Virus 1 ICP4 gene, a Human Herpes Simplex Virus 1 ICPO gene, a Human Herpes Simplex Virus 1 ICP27 gene, or a combination thereof. In embodiments, the host cell includes a Human Herpes Simplex Virus 1 ICP4 gene and a Human Herpes Simplex Virus 1 ICPO gene. In embodiments, the host cell includes a Human Herpes Simplex Virus 1 ICP4 gene. In embodiments, the host cell includes a Human Herpes Simplex Virus 1 ICPO gene. In embodiments, the host cell includes a Human Herpes Simplex Virus 27 ICPO gene.

[0222] In embodiments, the host cell is a U2OS cell. In embodiments, the U2OS cell includes a Human Herpes Simplex Virus 1 ICP4 gene, a Human Herpes Simplex Virus 1 ICPO gene, a Human Herpes Simplex Virus 1 ICP27 gene or a combination thereof. In embodiments, the U2OS cell includes a Human Herpes Simplex Virus 1 ICP4 gene, a Human Herpes Simplex Virus 1 ICPO gene, or a combination thereof.

[0223] In embodiments, the method includes the contacting the host cell with a polynucleotide including the recombinant herpesvirus vector genome provided herein including embodiments thereof. In embodiments, the method includes delivering into the host cell a polynucleotide including the recombinant herpesvirus vector genome provided herein including embodiments thereof. In embodiments, the delivering includes electroporating the host cell with the polynucleotide including the recombinant herpesvirus vector genome provided herein including embodiments thereof. In embodiments, the polynucleotide includes a bacterial artificial chromosome (BAC).

[0224] In an aspect is provided a method of transducing a population of cells, the method including contacting the population of cells with the recombinant alphaherpesvirus vector genome provided herein including embodiments thereof. In embodiments, the contacting occurs in a subject. In embodiments, the occurs in subject who has DEB. In embodiments, the contacting is in vitro. Inembodiments, contacting the population of cells includes electroporating the cells in the presence of the recombinant alphaherpesvirus vector genome provided herein including embodiments thereof.

[0225] The compositions provided herein, including polynucleotides and pharmaceutical compositions, are contemplated to be useful for expressing Collagen alpha- 1 (COL7) in a subject having dystrophic epidermolysis bullosa (DEB). In embodiments, the recombinant alphaherpesvirus genome provided herein including embodiments thereof, or a vector including the same increases the level of COL7 protein expressed in a cell in a subject having DEB. In an aspect is provided a method of treating dystrophic epidermolysis bullosa (DEB) in a subject in need thereof, including administering an effective amount of the recombinant alphaherpesvirus genome provided herein including embodiments thereof or a pharmaceutical composition including the same to the subject. In embodiments, the recombinant alphaherpesvirus genome is provided in a recombinant alphaherpesvirus vector including a viral capsid, a viral envelope, and a viral tegument.

[0226] In an aspect is provided a method of treating dystrophic epidermolysis bullosa (DEB) in a subject in need thereof, including administering an effective amount of the recombinant alphaherpesvirus vector provided herein including embodiments thereof or the pharmaceutical provided herein including embodiments thereof to the subject. Dystrophic epidermolysis bullosa (DEB) refers to a genetic disorder that typically causes the skin and mucous membranes of an affected individual to blister and erode in response to minor injury or friction. Symptoms of this condition vary widely among affected individuals, ranging from mild (blistering may only affect the hands, feet, knees, and elbows) to severe (widespread blistering and scarring, possibly leading to vision loss, disfigurement, and other serious, and sometimes fatal, medical conditions). In embodiments, treating DEB is a wound-healing effect. In embodiments, treating can be assessed by the decrease in the size of a wound in a subject having DEB. In embodiments, treating can be assessed by expression of COL7A (e.g. at a wound, at the dermal-epidermal junction). In embodiments, treating is assessed by a decrease or reduction in the number of new blisters over a defined period. In embodiments, treating is assessed by an increase in anchoring fibril density at the dermal-epidermal junction.

[0227] In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 10% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 20% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 30% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 40% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 50% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 60% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 70% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 80% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 85% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirusvector is increased by at least about 90% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 91% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 92% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 93% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 94% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 95% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 96% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 97% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 98% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 99% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirusvector is increased by at least about 100% relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector.

[0228] In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 150-fold, or 200-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 0.1 -fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 0.2-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 0.4-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 0.5-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 0.6-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 0.7-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 0.8-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirusvector is increased by at least about 0.9-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 1-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 2-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 3-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 4-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 4-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 5-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 6-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 7-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 8-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirusvector is increased by at least about 9-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 10-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 15-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 20-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 25-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 30-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 35-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 40-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 45-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 50-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirusvector is increased by at least about 100-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 150-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the wound healing effect of the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector is increased by at least about 200-fold relative to the effect in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector.

[0229] In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 10% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 20% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 30% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 40% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 50% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 60% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 70% relative to the size in the absence of therecombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 80% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 85% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 90% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 91% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 92% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 93% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 94% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 95% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 96% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 97% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 98% relative to the sizein the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 99% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 100% relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector.

[0230] In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 150-fold, or 200-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 0.1 -fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 0.2-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 0.4-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 0.5-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 0.6-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 0.7-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or therecombinant alphaherpesvirus vector decreases wound size by at least about 0.8-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 0.9-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 1-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 2-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 3 -fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 4-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 4-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 5-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 6-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 7-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 8-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinantalphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 9-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 10-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 15-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 20-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 25-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 30-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 35-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 40-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 45-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 50-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 100-fold relative to the size in the absence of the recombinant alphaherpesvirus genomeor recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 150-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector decreases wound size by at least about 200-fold relative to the size in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector.

[0231] In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression (e.g. at the dermal-epidermal junction) by least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 10% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 20% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 30% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 40% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 50% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 60% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 70% relative tothe expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 80% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 85% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 90% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 91% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 92% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 93% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 94% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 95% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 96% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 97% relative tothe expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 98% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 99% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 100% relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector.

[0232] In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 150-fold, or 200-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 0.1 -fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 0.2-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 0.4-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 0.5-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 0.6-foldrelative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 0.7-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 0.8-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 0.9-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 1-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 2-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 3-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 4-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 4-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 5-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 6-fold relative tothe expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 7-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 8-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 9-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 10-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 15-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 20-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 25-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 30-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 35-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 40-fold relative tothe expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 45-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 50-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 100-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 150-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector. In embodiments, the recombinant alphaherpesvirus genome or the recombinant alphaherpesvirus vector increases Col7A expression by at least about 200-fold relative to the expression in the absence of the recombinant alphaherpesvirus genome or recombinant alphaherpesvirus vector.

[0233] In embodiments, the administration is systemic administration, subcutaneous administration, topical administration, or intradermal administration. In embodiments, the administration is systemic administration. In embodiments, the administration is subcutaneous administration. In embodiments, the administration is topical administration. In embodiments, the administration is intradermal administration.

[0234] As described throughout the specification, in embodiments, a host cell including the polynucleotide provided herein including embodiments thereof is capable of expressing a transgene encoding a therapeutic protein provided herein including embodiments thereof. In embodiments, the therapeutic protein is expressed and is detectable after the contacting step. In embodiments, the therapeutic protein remains detectable for at least 10, 12, 14, 16, 18, 20, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 days after contacting the population of cells. Thus, in an aspect is provided a method of expressing a therapeutic protein in a subject in need thereof, the method including administering an effective amount of the recombinant alphaherpesvirus vector providedherein including embodiments thereof or the pharmaceutical composition provided herein including embodiments thereof to the subject by systemic administration, subcutaneous administration, topical administration, or intradermal administration. In embodiments, the administration is systemic administration. In embodiments, the administration is subcutaneous administration. In embodiments, the administration is topical administration. In embodiments, the administration is intradermal administration.

[0235] In embodiments, the vector is administered no more than once every two weeks. In embodiments, the vector is administered no more than once every three weeks. In embodiments, the vector is administered no more than once a month. In embodiments, the vector is administered no more than once every 5 weeks. In embodiments, the vector is administered no more than once every six weeks. In embodiments, the vector is administered no more than once every seven weeks. In embodiments, the vector is administered no more than once every two months.

[0236] In embodiments, the administering includes sequential administration of the vector on the same day, or not on the same day (e.g., occurs on consecutive days). For example, in embodiments, the administering includes sequential administration of the vector once a day for 2, 3, 4, 5, or 6 consecutive days. In embodiments, the sequential administering occurs no more than once every two weeks. In embodiments, the sequential administering occurs no more than once every three weeks. In embodiments, the sequential administering occurs no more than once a month. In embodiments, the sequential administering occurs no more than once every five weeks. In embodiments, the sequential administering occurs no more than once every six weeks. In embodiments, the sequential administering occurs no more than once every seven weeks. In embodiments, the sequential administering occurs no more than once every 2 months.

[0237] In embodiments, the administering includes intermittent administration wherein the vector is administered for a period of time (which can be considered a “first period of administration”), followed by a time during which the vector is not taken or is taken at a lower maintenance dose (which can be considered “off-period”) followed by a period during which the vector is administered again (which can be considered a “second period of administration”). Generally, during the second phase of administration, the dosage level of the vector will match that administered during the first period of administration but can be increased or decreased as medically necessary.

[0238] For the method provided herein, in embodiments, the therapeutic protein is expressed and is detectable after a first administering step. In embodiments, the therapeutic protein is detectable for at least 10 days after the administering. In embodiments, the therapeutic protein is detectable for at least 12 days after the administering. In embodiments, the therapeutic protein is detectable for at least 14 days after the administering. In embodiments, the therapeutic protein is detectable for at least 16 days after the administering. In embodiments, the therapeutic protein is detectable for at least 18 days after the administering. In embodiments, the therapeutic protein is detectable for at least 20 days after the administering. In embodiments, the therapeutic protein is detectable for at least 24 days after the administering. In embodiments, the therapeutic protein is detectable for at least 26 days after the administering. In embodiments, the therapeutic protein is detectable for at least 28 days after the administering. In embodiments, the therapeutic protein is detectable for at least 30 days after the administering. In embodiments, the therapeutic protein is detectable for at least 32 days after the administering. In embodiments, the therapeutic protein is detectable for at least 34 days after the administering. In embodiments, the therapeutic protein is detectable for at least 36 days after the administering. In embodiments, the therapeutic protein is detectable for at least 38 days after the administering. In embodiments, the therapeutic protein is detectable for at least 40 days after the administering. In embodiments, the therapeutic protein is detectable for at least 42 days after the administering. In embodiments, the therapeutic protein is detectable for at least 44 days after the administering. In embodiments, the therapeutic protein is detectable for at least 46 days after the administering. In embodiments, the therapeutic protein is detectable for at least 48 days after the administering. In embodiments, the therapeutic protein is detectable for at least 50 days after the administering.

[0239] For the method provided herein, in embodiments, the recombinant alphaherpesvirus vector causes transduced cells to express the therapeutic protein at a level and / or for a duration greater than a reference vector having a vector genome lacking the heterologous vari cellovirus ICP0 provided herein including embodiments thereof. In embodiments, the therapeutic protein is expressed at a level at least 10-fold greater for at least twice the duration of the expression level of the therapeutic protein relative to a reference vector having a vector genome lacking the heterologous varicellovirus ICP0 provided herein including embodiments thereof.

[0240] In embodiments, the therapeutic protein is expressed at a level at least 10-fold, 15-fold, 20- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater of the expression level of the therapeutic protein relative to a reference vector having a vector genome lacking the heterologous varicellovirus ICPO provided herein including embodiments thereof. In embodiments, the therapeutic protein is expressed for at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 30x, 40x, or 50x the duration of the expression of the therapeutic protein relative to a reference vector having a vector genome lacking the heterologous varicellovirus ICPO provided herein including embodiments thereof.

[0241] In embodiments, the method causes no or substantially no detectable cellular toxicity when administered in a therapeutically effective amount. In embodiments, substantially no detectable cellular toxicity refers to no detectable decrease in cell viability of transduced cells relative to nontransduced cells. In embodiments, no cellular toxicity refers to less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, or 1% decrease in cell viability of transduced cells relative to non-transduced cells. In embodiments, no cellular toxicity refers to no detectable increase in cell death of transduced cells relative to non-transduced cells. In embodiments, no cellular toxicity or no substantial cellular toxicity refers to no greater than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, or 1% increase in cell death in transduced cells relative to non-transduced cells. In embodiments, cellular toxicity is assessed by apoptosis of transduced cells. As described above, in embodiments, cellular toxicvity may be assessed by cytotoxicity assays, for example to detect cell apoptosis (e.g. degradation of cell proteins, genome fratgmentation, detection of cellular membrane, detection of cellular cytotoxicity biomarkers (e.g. LDH or G6PD). In embodiments, the methods and compositions provided herein do not cause substantial cellular toxicity relative to a cell in the absence of the method or composition.PHARMACEUTICAL COMPOSITIONS

[0242] In an aspect is provided a pharmaceutical composition including the recombinant alphaherpesvirus vector provided herein including embodiments thereof. In embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be formulated for an administration method as providedherein including embodiments thereof. For example, in embodiments, the pharmaceutically acceptable excipient is formulated for systemic administration, topical administration, transdermal administration, subcutaneous administration or intradermal administration.

[0243] In embodiments, the pharmaceutically acceptable excipient is suitable for topical administration or transdermal administration. Examples of excipients suitable for use in topical or transdermal administration include, but are not limited to, ointments, pastes, creams, suspensions, emulsions, fatty ointments, gels, powders, lotions, solutions, sprays, patches, microneedle arrays, and inhalants. In embodiments, the pharmaceutical carrier includes a patch (e.g. a patch that adheres to the skin). In embodiments, the pharmaceutically acceptable excipient includes a microneedle array.

[0244] 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 persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.EMBODIMENTS

[0245] Embodiment 1. A recombinant herpesvirus vector genome, wherein the recombinant herpesvirus vector genome comprises a heterologous herpesvirus Infected Cell Polypeptide 0 (ICPO) gene.

[0246] Embodiment 2. The recombinant herpesvirus vector genome of embodiment 1, wherein the recombinant herpesvirus vector genome is a recombinant alphaherpesvirus vector genome.

[0247] Embodiment 3. The recombinant herpesvirus vector genome of embodiment 2, wherein the recombinant alphaherpesvirus vector genome is a recombinant simplexvirus vector genome.

[0248] Embodiment 4. The recombinant herpesvirus vector genome of any one of embodiments 1 to 3, wherein the heterologous herpesvirus ICPO gene is a heterologous alphaherpesvirus ICPO gene.

[0249] Embodiment 5. The recombinant herpesvirus vector genome of embodiment 4, wherein the heterologous alphaherpesvirus ICPO gene is a heterologous vari cellovirus ICPO gene.

[0250] Embodiment 6. The recombinant herpesvirus vector genome of embodiment 5, wherein the varicellovirus ICPO is a varicellovirus bovinealphal ICPO gene, a varicellovirus bovinealpha5 ICPO gene, a varicellovirus bubalinealphal ICPO gene, a varicellovirus canidalphal ICPO gene, a varicellovirus caprinealphal ICPO gene, a varicellovirus cercopithecinealpha9 ICPO gene, a varicellovirus cervidalphal ICPO gene, a varicellovirus cervidalpha2 ICPO gene, a varicellovirus cervidalpha3 ICPO gene, a varicellovirus equidalphal ICPO gene, a varicellovirus equidalphaS ICPO gene, a varicellovirus equidalpha4 ICPO gene, a varicellovirus equidalpha6 ICPO gene, a varicellovirus equidalpha8 ICPO gene, a varicellovirus equidalpha9 ICPO gene, a varicellovirus felidalphal ICPO gene, a varicellovirus humanalpha3 ICPO gene, a varicellovirus monodontidalphal ICPO gene, a varicellovirus phocidalphal ICPO gene, or a varicellovirus suidalphal ICPO gene.

[0251] Embodiment 7. The recombinant herpesvirus vector genome of embodiment 5, wherein the varicellovirus ICPO gene is a varicellovirus bovinealphal ICPO (bICPO) gene.

[0252] Embodiment 8. The recombinant herpesvirus vector genome of embodiment 7, wherein the bICPO gene comprises a polynucleotide sequence according to SEQ ID NO:3 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:3.

[0253] Embodiment 9. The recombinant herpesvirus vector genome of embodiment 7 or 8, wherein the bICPO gene encodes a bICPO protein according to SEQ ID NO: 7 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:7.

[0254] Embodiment 10. The recombinant herpesvirus vector genome of embodiment 9, wherein the bICPO gene encodes a bICPO protein comprising a nuclear localization sequence (NLS), preferably KRRR.

[0255] Embodiment 11. The recombinant herpesvirus vector genome of embodiment 5, wherein the varicellovirus ICPO gene is a varicellovirus equidalphal ICPO (elCPO) gene.

[0256] Embodiment 12. The recombinant herpesvirus vector genome of embodiment 11, wherein the elCPO gene comprises a polynucleotide sequence according to SEQ ID NO:5 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:5.

[0257] Embodiment 13. The recombinant herpesvirus vector genome of embodiment 11, wherein the elCPO gene encodes a elCPO protein at least 80%, at least 90% or at least 95% identical to SEQ ID NO:9.

[0258] Embodiment 14. The recombinant herpesvirus vector genome of embodiment 13, wherein the elCPO protein comprises a nuclear localization sequence (NLS), preferably KRRR (SEQ ID NOTO).

[0259] Embodiment 15. The recombinant herpesvirus vector genome of any one of embodiments 1 to 14, wherein the ICP0 gene is operably linked to a promoter.

[0260] Embodiment 16. The recombinant herpesvirus vector genome of embodiment 15, wherein the promoter is a constitutive promoter, optionally a CbH promoter, a CBA promoter, a SFFV promoter, a MSCV promoter, a SV40 promoter, a hPGK promoter, a RSV promoter, a CMV promoter, an EF 1 a promoter, a mitochondrial heavy-strand promoter, a UBC promoter, a CAG promoter, a GAPDH promoter, an ACTB promoter.

[0261] Embodiment 17. The recombinant herpesvirus vector genome of embodiment 15, wherein the promoter is a synthetic promoter.

[0262] Embodiment 18. The recombinant herpesvirus vector genome of embodiment 15, wherein the promoter is a hybrid promoter.

[0263] Embodiment 19. The recombinant herpesvirus vector genome of embodiment 16, wherein the constitutive promoter is a human elongation factor- 1 alpha (EF-1 alpha) promoter, wherein the EF-1 alpha promoter comprises SEQ ID NO: 14 or is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 14.

[0264] Embodiment 20. The recombinant herpesvirus vector genome of embodiment 15, wherein the promoter is an ICP0 promoter, optionally an endogenous ICP0 promoter of the recombinant herpesvirus vector genome or a promoter of the heterologous ICP0 gene.

[0265] Embodiment 21. The recombinant herpesvirus vector genome of embodiment 15, wherein the promoter is an inducible promoter, optionally a chemically inducible promoter, a temperature inducible promoter, or a light induced promoter

[0266] Embodiment 22. The recombinant herpesvirus vector genome of any one of embodiments 1 to 21, wherein the recombinant herpesvirus vector genome comprises an inactivating mutation in an endogenous ICPO gene.

[0267] Embodiment 23. The recombinant herpesvirus vector genome of embodiment 22, wherein the inactivating mutation is a partial or complete deletion of the endogenous ICPO gene.

[0268] Embodiment 24. The recombinant herpesvirus vector genome of embodiment 22, wherein the inactivating mutation is an insertion of the varicellovirus ICPO gene into endogenous ICPO gene.

[0269] Embodiment 25. The recombinant herpesvirus vector genome of any one of embodiments 1 to 24, wherein the recombinant herpesvirus vector genome comprises inactivating mutations in an ICP4 gene and / or an ICP27 gene.

[0270] Embodiment 26. The recombinant herpesvirus vector genome of any one of embodiments 1 to 25, wherein the recombinant herpesvirus vector genome comprises a JOINT region.

[0271] Embodiment 27. The recombinant herpesvirus vector genome of any one of embodiments 1 to 26, wherein the vector genome comprises a deletion of the JOINT region.

[0272] Embodiment 28. The recombinant herpesvirus vector genome of any one of embodiments 1 to 27, wherein the recombinant herpesvirus vector genome comprises a transgene.

[0273] Embodiment 29. The recombinant herpesvirus vector genome of embodiment 28, wherein the transgene encodes a therapeutic protein.

[0274] Embodiment 30. The recombinant herpesvirus vector genome of embodiment 29, wherein the therapeutic protein is COL7A1 (collagen alpha- 1 (VII) chain), SERPINA1 (Alpha- 1 Antitrypsin), PKD1 (Polycystin 1), ABCB11 (ATP Binding Cassette Subfamily B Member 11), PKD2 (Poly cystin 2), ATP7B (ATPase Copper Transporting Beta), SERPINC1 (Antithrombin III),CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), ATP8B1 (ATPase Phospholipid Transporting 8B1), ABCB4 (ATP Binding Cassette Subfamily B Member 4), CFH (Complement Factor H), F8 (Coagulation Factor VIII), APOB (Apolipoprotein B), LDLR (Low Density Lipoprotein Receptor), SMN1 (Survival Motor Neuron 1), RPE65 (Retinal Pigment Epithelium- Specific 65 kDa Protein), F9 (Factor IX), IL-2 (Interleukin-2), a CAR (Chimeric Antigen Receptor), ABCA4 (ATP Binding Cassette Subfamily A Member 4), GAA (Acid Alpha-Glucosidase), GBA (Glucocerebrosidase), SGSH (N-Sulfoglucosamine Sulfohydrolase), IDUA (Alpha-L-Iduronidase), ADA (Adenosine Deaminase), OTC (Ornithine Transcarbamylase), HEXA (Hexosaminidase A), FANCA (Fanconi Anemia Complementation Group A), MECP2 (Methyl CpG Binding Protein 2), ASPA (Aspartoacylase), BTK (Bruton Tyrosine Kinase), HBB (Hemoglobin Subunit Beta), DMD (Dystrophin), CNGB3 (Cyclic Nucleotide Gated Channel Beta 3), PRKAG2 (Protein Kinase AMP- Activated Non-Catalytic Subunit Gamma 2), CLN2 (Ceroid-Lipofuscinosis, Neuronal 2), ARSA (Aryl sulfatase A), GUSB (Beta-Glucuronidase), GALC (Galactosylceramidase), SGCB (Sarcoglycan Beta), MY07A (myosin VIIA), PCHD15 (protocadherin related 15), CDH23 (cadherin related 23), USH2A (usherin), or GPR98 (adhesion G protein-coupled receptor VI).

[0275] Embodiment 31 . The recombinant herpesvirus vector genome of embodiment 29 or 30, wherein the therapeutic protein is COL7A1.

[0276] Embodiment 32. The recombinant herpesvirus vector genome of any one of embodiments 1 to 31, wherein the recombinant herpesvirus vector genome comprises one or more genes sufficient to package a simplexvirus viral particle.

[0277] Embodiment 33. The recombinant herpesvirus vector genome of any one of embodiments 1 to 32, wherein the recombinant herpesvirus vector genome is a Herpes Simplex Virus 1 (HSV-1) vector genome.

[0278] Embodiment 34. The recombinant herpesvirus vector genome of any one of embodiments 1 to 32, wherein the recombinant herpesvirus vector genome is a Herpes Simplex Virus 2 (HSV-2) vector genome.

[0279] Embodiment 35. A polynucleotide comprising the recombinant herpesvirus vector genome of any one of embodiments 1 to 34, wherein optionally the polynucleotide comprises a bacterial artificial chromosome (BAC).

[0280] Embodiment 36. A host cell comprising the polynucleotide of embodiment 35 and optionally one or more polynucleotides encoding genes sufficient to package a simplexvirus viral particle.

[0281] Embodiment 37. The host cell of embodiment 36, wherein gene sufficient to package a simplexvirus viral particle comprises Human Herpes Simplex Virus 1 ICP4, Human Herpes Simplex Virus 1 ICP0, Human Herpes Simplex Virus 1 ICP27, or a combination thereof.

[0282] Embodiment 38. A method of making a viral particle, comprising culturing the host cell of embodiment 36 under conditions that the host cell to produce the viral particle.

[0283] Embodiment 39. A recombinant alphaherpesvirus vector, comprising the recombinant herpesvirus vector genome of any one of embodiments 1 to 34, a viral capsid, a viral envelope, and a viral tegument.

[0284] Embodiment 40. The recombinant alphaherpesvirus vector of embodiment 39, wherein the recombinant alphaherpesvirus vector is a recombinant simplexvirus vector.

[0285] Embodiment 41. The recombinant alphaherpesvirus vector of embodiment 40, wherein the recombinant alphaherpesvirus vector is a recombinant Herpes Simplex Virus 1 (HSV-1) particle.

[0286] Embodiment 42. The recombinant alphaherpesvirus vector of embodiment 40, wherein the recombinant alphaherpesvirus vector is a recombinant Herpes Simplex Virus 2 (HSV-2) particle.

[0287] Embodiment 43. The recombinant alphaherpesvirus vector of any one of embodiments 39 to 42, wherein the recombinant alphaherpesvirus vector is capable of transducing a cell.

[0288] Embodiment 44. The recombinant alphaherpesvirus vector of any one of embodiments 39 to 43, wherein the recombinant alphaherpesvirus vector is non-toxic to transduced cells.

[0289] Embodiment 45. The recombinant alphaherpesvirus vector of any one of embodiments 39 to 44, wherein the recombinant alphaherpesvirus vector is not oncolytic.

[0290] Embodiment 46. The recombinant alphaherpesvirus vector of any one of embodiments 39 to 45, wherein the recombinant herpesvirus vector genome comprises a transgene.

[0291] Embodiment 47. The recombinant alphaherpesvirus herpesvirus vector of embodiment 46, wherein the transgene encodes a therapeutic protein.

[0292] Embodiment 48. The recombinant herpesvirus vector of embodiment 47, wherein the therapeutic protein is wherein the therapeutic protein is COL7A1 (collagen alpha-1 (VII) chain), SERPINA1 (Alpha-1 Antitrypsin), PKD1 (Polycystin 1), ABCB11 (ATP Binding Cassette Subfamily B Member 11), PKD2 (Polycystin 2), ATP7B (ATPase Copper Transporting Beta), SERPINC1 (Antithrombin III), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), ATP8B1 (ATPase Phospholipid Transporting 8B1), ABCB4 (ATP Binding Cassette Subfamily B Member 4), CFH (Complement Factor H), F8 (Coagulation Factor VIII), APOB (Apolipoprotein B), LDLR (Low Density Lipoprotein Receptor), SMN1 (Survival Motor Neuron 1), RPE65 (Retinal Pigment Epithelium-Specific 65 kDa Protein), F9 (Factor IX), IL-2 (Interleukin-2), a CAR (Chimeric Antigen Receptor), ABCA4 (ATP Binding Cassette Subfamily A Member 4), GAA (Acid Alpha-Glucosidase), GBA (Glucocerebrosidase), SGSH (N-Sulfoglucosamine Sulfohydrolase), IDUA (Alpha-L-Iduronidase), ADA (Adenosine Deaminase), OTC (Ornithine Transcarbamylase), HEXA (Hexosaminidase A), FANCA (Fanconi Anemia Complementation Group A), MECP2 (Methyl CpG Binding Protein 2), ASPA (Aspartoacylase), BTK (Bruton Tyrosine Kinase), HBB (Hemoglobin Subunit Beta), DMD (Dystrophin), CNGB3 (Cyclic Nucleotide Gated Channel Beta 3), PRKAG2 (Protein Kinase AMP -Activated Non-Catalytic Subunit Gamma 2), CLN2 (Ceroid- Lipofuscinosis, Neuronal 2), ARSA (Aryl sulfatase A), GUSB (Beta-Glucuronidase), GALC (Galactosylceramidase), SGCB (Sarcoglycan Beta), MY07A (myosin VIIA), PCHD15 (protocadherin related 15), CDH23 (cadherin related 23), USH2A (usherin), or GPR98 (adhesion G protein-coupled receptor VI).

[0293] Embodiment 49. The recombinant herpesvirus vector genome of embodiment 47 or 48, wherein the therapeutic protein is COL7A1.

[0294] Embodiment 50. The recombinant alphaherpesvirus vector of any one of embodiments 47 to 49, wherein the recombinant alphaherpesvirus vector causes cells transduced with said recombinant alphaherpesvirus vector to express the therapeutic protein at a level and / or for aI l lduration greater than a reference vector having a vector genome lacking the heterologous varicellovirus ICPO.

[0295] Embodiment 51. A method of transducing a population of cells, the method comprising contacting the population of cells with the recombinant alphaherpesvirus vector of any one of embodiments 39 to 50.

[0296] Embodiment 52. The method of embodiment 51, wherein the therapeutic protein is expressed and is detectable after the contacting step.

[0297] Embodiment 53. The method of embodiment 52, wherein the therapeutic protein remains detectable at least 10 days after contacting the population of cells.

[0298] Embodiment 54. The method of embodiment one of embodiments 51 to 53, wherein the recombinant alphaherpesvirus vector causes transduced cells to express the therapeutic protein at a level and / or for a duration greater than a reference vector having a vector genome lacking the heterologous varicellovirus ICPO, optionally at least 10-fold greater level and at least twice the duration.

[0299] Embodiment 55. A pharmaceutical composition comprising the recombinant alphaherpesvirus vector of any one of embodiments 39 to 50.

[0300] Embodiment 56. A method of treating dystrophic epidermolysis bullosa (DEB) in a subject in need thereof, comprising administering an effective amount of the recombinant alphaherpesvirus vector of any one of embodiments 39 to 50 or the pharmaceutical composition of embodiment 55 to the subject.

[0301] Embodiment 57. The method of embodiment 56, wherein the administering is systemic administration, subcutaneous administration, topical administration, or intradermal administration.

[0302] Embodiment 58. A method of expressing a therapeutic protein in a subject in need thereof, comprising administering an effective amount of the recombinant alphaherpesvirus vector of any one of embodiments 39 to 50 or the pharmaceutical composition of embodiment 55 to the subject by systemic administration, subcutaneous administration, topical administration, or intradermal administration.

[0303] Embodiment 59. The method of any one of embodiments 56 to 58, wherein the vector is administered no more than once every two weeks.

[0304] Embodiment 60. The method of any one of embodiments 56 to 58, wherein the vector is administered no more than once a month.

[0305] Embodiment 61. The method of any one of embodiments 56 to 60, wherein the therapeutic protein is expressed and is detectable after a first administering step.

[0306] Embodiment 62. The method of embodiment 61, wherein the therapeutic protein is detectable for at least 10 days after the administering.

[0307] Embodiment 63. The method of any one of embodiments 58 to 62, wherein the vector causes transduced cells to express the therapeutic protein at a level and / or for a duration greater than a reference vector having a vector genome lacking the heterologous varicellovirus ICP0, optionally at least 10-fold greater level and at least twice the duration.

[0308] Embodiment 64. The method of any one of embodiments 58 to 63, wherein the method causes no or substantially no detectable cellular toxicity when administered in a therapeutically effective amount.EXAMPLESExample 1: Exemplary Method for Making a Recombinant Herpesvirus Vector

[0309] A recombinant herpesvirus vector genome was generated from the recombinant herpesvirus vector genome JANI8. As previously described, JANI8 was derived from the HSV-1 KOS strain. In embodiments, HSV-1 KOS strain is identified by GenBank reference JQ673480. Briefly, the ICPO gene, ICP4 gene, ICP27 gene, virion host shutoff (vhs) genes (UL41), and the UL / US joint region were deleted from the HSV-1 KOS strain. Gateway recombinase sites were inserted into the LAT locus at 3’ of the LATP2 of the HSV-1 for insertion of one or more therapeutic genes. The LAT locus was designed to be flanked by insulator sequences CTRL1 and CTRL2 elements to prevent silencing of transgenes expression in the LAT locus. Heterologous elCPO or bICPO genes were inserted into the LAT locus and operably linked to either the natural (e.g. endogenous) ICPO promoter or an EF1A promoter, thereby generating recombinant herpesvirus vector genomes, referred to as Nova 1, Nova 2, Nova 3 and Nova 4 (FIG. 1) in Examples 1-4.

[0310] The recombinant herpesvirus was produced by electroporation of a BAC plasmid encoding the recombinant HSV vector genome (e.g. Nova 1, Nova 2, Nova 3, Nova 4) into U2OS-ICP4 / 27 cells expressing HSV ICP4 and ICP27 proteins. Specifically, 2 pL of BAC plasmid DNA in 23 pL of SE solution (Lonza) was electroporated into 5xl05U2OS-ICP4 / 27 cells.

[0311] The cells were recovered in VP-SFM media (Gibco), transferred to a 33 °C incubator and monitored for 85-90% cytopathic effect (CPE), which took approximately 8-10 days. Upon 85-90% CPE, 70% of supernatant was removed. Cells were packaged with the remaining 30% supernatant, and subsequently underwent three freeze-thaw cycles.

[0312] To produce larger scale of viruses, viruses were expanded by infection of sequentially larger cultures at an MOI of 0.001 PFU per cell in an 33 °C incubator.Example 2: Recombinant Herpesvirus Vector Transduced Human Fibroblasts Durably Express Transgene without Substantial Cytotoxic Effects

[0313] Primary normal human dermal fibroblasts (nHDF) from two different sources (ATCC and Lonza Bioscience) were transduced with recombinant herpesvirus vectors Nova 1, Nova 2, and Nova 3 at a MOI of 10. The Nova vectors used in this study all included a GFP encodingpolynucleotide, thereby allowing detection of the transgene. GFP detection by Incucyte Imaging was assessed from day 0 (day of transduction) to Day 30. As shown in FIG.s 2A and 2B, all of Nova 1 to Nova 3 increased GFP expression by relative to a reference HSV vector including an endogenous ICPO gene.

[0314] Dystrophic epidermolysis bullosa (RDEB) fibroblasts and nHDF cells were transduced with recombinant herpesvirus vectors Nova 1, Nova 2, and Nova 3 at a MOI of 20. Cell survival was assessed from the time of transduction to day 9 after transduction. As illustrated in FIG. 3, cells transduced with the Nova vectors show reduced toxicity relative to cells transduced with a reference HSV vector including an endogenous ICPO gene.Example 3: Recombinant Herpesvirus Vector Transduced Human Keratinocytes Durably Express Transgene without Substantial Cytotoxic Effects

[0315] Normal human epidermal keratinocytes (NHEK), the major cell type in the epidermis, were transduced with Nova 1, Nova 2, Nova 3, and Nova 4. Each of the Nova vectors included a transgene encoding the reporter protein ZsGreen.

[0316] Reporter gene expression was assessed from the time of transduction to 15 days post transduction. As illustrated in FIG. 4, the transgene was expressed at higher levels and for longer term relative to expression of the gene in an HSV vector including an endogenous ICPO gene.

[0317] The transduced NHEK cells were further assessed for cell viability. As illustrated in FIG. 5, the NHEK cells transduced with the Nova vectors show reduced toxicity relative to cells transduced with a reference HSV vector including an endogenous ICPO gene.Example 4: Viral Titer of Cells Transduced with Recombinant Herpesvirus Vector

[0318] U2OS cells including ICP4 and ICP27 genes (U2OS-4-27 cells) were transfected with Nova 1, Nova 2, and Nova 3 vectors. The U2OS-4-27 cells were cultured as described in Example 1, and viral titers were assessed at day. As illustrated in FIG. 6, U2OS-4-27 cells transduced with the Nova vectors produced increased viral titers relative to cells transduce with an HSV vector including an endogenous ICPO gene.Example 5: Recombinant Herpesvirus Vector Shows In Vivo Transgene Expression and Wound Healing

[0319] WT Balb / C mice were topically administered either a vehicle control or the recombinant herpesvirus including a bovine ICPO gene and Col7Al transgene (Nova-Col7A) at a dose of 1 x 108pfu / ml (100 pl per dose) on Day 0 and Day 2, corresponding to a total administered dose of approximately 2* 107pfu. The Nova-Col7A composition is provided in a topical gel formulation and the vehicle control is the identical formulation, except without the Nova-Col7A vector.

[0320] Wound healing and protein expression ...

Claims

WHAT IS CLAIMED IS:

1. A recombinant herpesvirus vector genome, wherein the recombinant herpesvirus vector genome comprises a heterologous herpesvirus Infected Cell Polypeptide 0 (ICPO) gene.

2. The recombinant herpesvirus vector genome of claim 1, wherein the recombinant herpesvirus vector genome is a recombinant alphaherpesvirus vector genome.

3. The recombinant herpesvirus vector genome of claim 2, wherein the recombinant alphaherpesvirus vector genome is a recombinant simplexvirus vector genome.

4. The recombinant herpesvirus vector genome of claim 1 , wherein the heterologous herpesvirus ICPO gene is a heterologous alphaherpesvirus ICPO gene.

5. The recombinant herpesvirus vector genome of claim 4, wherein the heterologous alphaherpesvirus ICPO gene is a heterologous varicellovirus ICPO gene.

6. The recombinant herpesvirus vector genome of claim 5, wherein the varicellovirus ICPO is a varicellovirus bovinealphal ICPO gene, a varicellovirus bovinealpha5 ICPO gene, a varicellovirus bubalinealphal ICPO gene, a varicellovirus canidalphal ICPO gene, a varicellovirus caprinealphal ICPO gene, a varicellovirus cercopithecinealpha9 ICPO gene, a varicellovirus cervidalphal ICPO gene, a varicellovirus cervidalpha2 ICPO gene, a varicellovirus cervidalpha3 ICPO gene, a varicellovirus equidalphal ICPO gene, a varicellovirus equidalpha3 ICPO gene, a varicellovirus equidalpha4 ICPO gene, a varicellovirus equidalpha6 ICPO gene, a varicellovirus equidalpha8 ICPO gene, a varicellovirus equidalpha9 ICPO gene, a varicellovirus felidalphal ICPO gene, a varicellovirus humanalpha3 ICPO gene, a varicellovirus monodontidalphal ICPO gene, a varicellovirus phocidalphal ICPO gene, or a varicellovirus suidalphal ICPO gene.

7. The recombinant herpesvirus vector genome of claim 5, wherein the varicellovirus ICPO gene is a varicellovirus bovinealphal ICPO (bICPO) gene.

8. The recombinant herpesvirus vector genome of claim 7, wherein the bICPO gene comprises a polynucleotide sequence according to SEQ ID NO:3 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:3.

9. The recombinant herpesvirus vector genome of claim 7, wherein the bICPO gene encodes a bICPO protein according to SEQ ID NO: 7 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:7.

10. The recombinant herpesvirus vector genome of claim 9, wherein the bICPO gene encodes a bICPO protein comprising a nuclear localization sequence (NLS), preferably KRRR (SEQ ID NO: 10).

11. The recombinant herpesvirus vector genome of claim 5, wherein the varicellovirus ICP0 gene is a varicellovirus equidalphal ICP0 (elCPO) gene.

12. The recombinant herpesvirus vector genome of claim 11, wherein the elCPO gene comprises a polynucleotide sequence according to SEQ ID NO: 5 or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 5.

13. The recombinant herpesvirus vector genome of claim 11, wherein the elCPO gene encodes a elCPO protein at least 80%, at least 90% or at least 95% identical to SEQ ID NO:9.

14. The recombinant herpesvirus vector genome of claim 13, wherein the elCPO protein comprises a nuclear localization sequence (NLS), preferably KRRR (SEQ ID NO: 10).

15. The recombinant herpesvirus vector genome of claim 1, wherein the ICP0 gene is operably linked to a promoter.

16. The recombinant herpesvirus vector genome of claim 15, wherein the promoter is a constitutive promoter, optionally a CbH promoter, a CBA promoter, a SFFV promoter, a MSCV promoter, a SV40 promoter, a hPGK promoter, a RSV promoter, a CMV promoter, an human elongation factor-1 alpha (EF-1 alpha), a mitochondrial heavy-strand promoter, a UBC promoter, a CAG promoter, a GAPDH promoter, an ACTB promoter.

17. The recombinant herpesvirus vector genome of claim 15, wherein the promoter is a synthetic promoter.

18. The recombinant herpesvirus vector genome of claim 15, wherein the promoter is a hybrid promoter.

19. The recombinant herpesvirus vector genome of claim 16, wherein the constitutive promoter is the EF-1 alpha promoter, wherein the EF-1 alpha promoter comprises SEQ ID NO: 14 or is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 14.

20. The recombinant herpesvirus vector genome of claim 15, wherein the promoter is an ICPO promoter, optionally an endogenous ICPO promoter of the recombinant herpesvirus vector genome or a promoter of the heterologous ICPO gene.

21. The recombinant herpesvirus vector genome of claim 15, wherein the promoter is an inducible promoter, optionally a chemically inducible promoter, a temperature inducible promoter, or a light induced promoter22. The recombinant herpesvirus vector genome of claim 1, wherein the recombinant herpesvirus vector genome comprises an inactivating mutation in an endogenous ICPO gene.

23. The recombinant herpesvirus vector genome of claim 22, wherein the inactivating mutation is a partial or complete deletion of the endogenous ICPO gene.

24. The recombinant herpesvirus vector genome of claim 22, wherein the inactivating mutation is an insertion of the varicellovirus ICPO gene into endogenous ICPO gene.

25. The recombinant herpesvirus vector genome of claim 1, wherein the recombinant herpesvirus vector genome comprises inactivating mutations in an ICP4 gene and / or an ICP27 gene.

26. The recombinant herpesvirus vector genome of claim 1, wherein the recombinant herpesvirus vector genome comprises a JOINT region.

27. The recombinant herpesvirus vector genome of claim 1, wherein the vector genome comprises a deletion of the JOINT region.

28. The recombinant herpesvirus vector genome of claim 1, wherein the recombinant herpesvirus vector genome comprises a transgene.

29. The recombinant herpesvirus vector genome of claim 28, wherein the transgene encodes a therapeutic protein.

30. The recombinant herpesvirus vector genome of claim 29, wherein the therapeutic protein is COL7A1 (collagen alpha-1 (VII) chain), SERPINA1 (Alpha-1 Antitrypsin), PKD1 (Polycystin 1), ABCB11 (ATP Binding Cassette Subfamily B Member 11), PKD2 (Polycystin 2), ATP7B (ATPase Copper Transporting Beta), SERPINC1 (Antithrombin III), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), ATP8B1 (ATPase Phospholipid Transporting 8B1), ABCB4 (ATP Binding Cassette Subfamily B Member 4), CFH (Complement Factor H), F8 (Coagulation Factor VIII), APOB (Apolipoprotein B), LDLR (Low Density Lipoprotein Receptor), SMN1 (Survival Motor Neuron 1), RPE65 (Retinal Pigment Epithelium- Specific 65 kDa Protein), F9 (Factor IX), IL-2 (Interleukin-2), a CAR (Chimeric Antigen Receptor), ABCA4 (ATP Binding Cassette Subfamily A Member 4), GAA (Acid Alpha-Glucosidase), GBA (Glucocerebrosidase), SGSH (N-Sulfoglucosamine Sulfohydrolase), IDUA (Alpha-L-Iduronidase), ADA (Adenosine Deaminase), OTC (Ornithine Transcarbamylase), HEXA (Hexosaminidase A), FANCA (Fanconi Anemia Complementation Group A), MECP2 (Methyl CpG Binding Protein 2), ASPA (Aspartoacylase), BTK (Bruton Tyrosine Kinase), HBB (Hemoglobin Subunit Beta), DMD (Dystrophin), CNGB3 (Cyclic Nucleotide Gated Channel Beta 3), PRKAG2 (Protein Kinase AMP- Activated Non-Catalytic Subunit Gamma 2), CLN2 (Ceroid-Lipofuscinosis, Neuronal 2), ARSA (Aryl sulfatase A), GUSB (Beta-Glucuronidase), GALC (Galactosylceramidase), SGCB (Sarcoglycan Beta), MY07A (myosin VIIA), PCHD15 (protocadherin related 15), CDH23 (cadherin related 23), USH2A (usherin), or GPR98 (adhesion G protein-coupled receptor VI).31 . The recombinant herpesvirus vector genome of claim 29, wherein the therapeutic protein is COL7A1 .

32. The recombinant herpesvirus vector genome of claim 1, wherein the recombinant herpesvirus vector genome comprises one or more genes sufficient to package a simplexvirus viral particle.

33. The recombinant herpesvirus vector genome of claim 1, wherein the recombinant herpesvirus vector genome is a Herpes Simplex Virus 1 (HSV-1) vector genome.

34. The recombinant herpesvirus vector genome of claim 1, wherein the recombinant herpesvirus vector genome is a Herpes Simplex Virus 2 (HSV-2) vector genome.

35. A polynucleotide comprising the recombinant herpesvirus vector genome of claim 1, wherein optionally the polynucleotide comprises a bacterial artificial chromosome (BAC).

36. A host cell comprising the polynucleotide of claim 35 and optionally one or more polynucleotides encoding genes sufficient to package a simplexvirus viral particle.

37. The host cell of claim 36, wherein gene sufficient to package a simplexvirus viral particle comprises Human Herpes Simplex Virus 1 ICP4, Human Herpes Simplex Virus 1 ICPO, Human Herpes Simplex Virus 1 ICP27, or a combination thereof.

38. A method of making a viral particle, comprising culturing the host cell of claim 36 under conditions that the host cell to produce the viral particle.

39. A recombinant alphaherpesvirus vector, comprising the recombinant herpesvirus vector genome of claim 1, a viral capsid, a viral envelope, and a viral tegument.

40. The recombinant alphaherpesvirus vector of claim 39, wherein the recombinant alphaherpesvirus vector is a recombinant simplexvirus vector.

41. The recombinant alphaherpesvirus vector of claim 40, wherein the recombinant alphaherpesvirus vector is a recombinant Herpes Simplex Virus 1 (HSV-1) particle.

42. The recombinant alphaherpesvirus vector of claim 40, wherein the recombinant alphaherpesvirus vector is a recombinant Herpes Simplex Virus 2 (HSV-2) particle.

43. The recombinant alphaherpesvirus vector of claim 39, wherein the recombinant alphaherpesvirus vector is capable of transducing a cell.

44. The recombinant alphaherpesvirus vector of claim 39, wherein the recombinant alphaherpesvirus vector is non-toxic to transduced cells.

45. The recombinant alphaherpesvirus vector of claim 39, wherein the recombinant alphaherpesvirus vector is not oncolytic.

46. The recombinant alphaherpesvirus vector of claim 39, wherein the recombinant herpesvirus vector genome comprises a transgene.

47. The recombinant alphaherpesvirus herpesvirus vector of claim 46, wherein the transgene encodes a therapeutic protein.

48. The recombinant herpesvirus vector of claim 47, wherein the therapeutic protein is C0L7A1 (collagen alpha- 1 (VII) chain), SERPINA1 (Alpha- 1 Antitrypsin), PKD1 (Polycystin 1), ABCB11 (ATP Binding Cassette Subfamily B Member 11), PKD2 (Polycystin 2), ATP7B (ATPase Copper Transporting Beta), SERPINC1 (Antithrombin III), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), ATP8B1 (ATPase Phospholipid Transporting 8B1), ABCB4 (ATP Binding Cassette Subfamily B Member 4), CFH (Complement Factor H), F8 (Coagulation Factor VIII), APOB (Apolipoprotein B), LDLR (Low Density Lipoprotein Receptor), SMN1 (Survival Motor Neuron 1), RPE65 (Retinal Pigment Epithelium-Specific 65 kDa Protein), F9 (Factor IX), IL -2 (Interleukin-2), a CAR (Chimeric Antigen Receptor), ABCA4 (ATP Binding Cassette Subfamily A Member 4), GAA (Acid Alpha-Glucosidase), GBA (Glucocerebrosidase), SGSH (N-Sulfoglucosamine Sulfohydrolase), IDUA (Alpha-L-Iduronidase), ADA (Adenosine Deaminase), OTC (Ornithine Transcarbamylase), HEXA (Hexosaminidase A), FANCA (Fanconi Anemia Complementation Group A), MECP2 (Methyl CpG Binding Protein 2), ASPA (Aspartoacylase), BTK (Bruton Tyrosine Kinase), HBB (Hemoglobin Subunit Beta), DMD (Dystrophin), CNGB3 (Cyclic Nucleotide Gated Channel Beta 3), PRKAG2 (Protein Kinase AMP- Activated Non-Catalytic Subunit Gamma 2), CLN2 (Ceroid-Lipofuscinosis, Neuronal 2), ARSA (Aryl sulfatase A), GUSB (Beta-Glucuronidase), GALC (Galactosylceramidase), SGCB(Sarcoglycan Beta), MY07A (myosin VIIA), PCHD15 (protocadherin related 15), CDH23 (cadherin related 23), USH2A (usherin), or GPR98 (adhesion G protein-coupled receptor VI).

49. The recombinant herpesvirus vector genome of claim 48, wherein the therapeutic protein is C0L7A1.

50. The recombinant alphaherpesvirus vector of claim 47, wherein the recombinant alphaherpesvirus vector causes cells transduced with said recombinant alphaherpesvirus vector to express the therapeutic protein at a level and / or for a duration greater than a reference vector having a vector genome lacking the heterologous vari cellovirus ICPO.

51. A method of transducing a population of cells, the method comprising contacting the population of cells with the recombinant alphaherpesvirus vector of claim 39.

52. The method of claim 51, wherein the therapeutic protein is expressed and is detectable after the contacting step.

53. The method of claim 52, wherein the therapeutic protein remains detectable at least 10 days after contacting the population of cells.

54. The method of claim 51, wherein the recombinant alphaherpesvirus vector causes transduced cells to express the therapeutic protein at a level and / or for a duration greater than a reference vector having a vector genome lacking the heterologous vari cellovirus ICPO, optionally at least 10-fold greater level and at least twice the duration.

55. A pharmaceutical composition comprising the recombinant alphaherpesvirus vector of claim 39.

56. A method of treating dystrophic epidermolysis bullosa (DEB) in a subject in need thereof, comprising administering an effective amount of the recombinant alphaherpesvirus vector of claim 39 to the subject.

57. The method of claim 56, wherein the administering is systemic administration, subcutaneous administration, topical administration, or intradermal administration.

58. A method of expressing a therapeutic protein in a subject in need thereof, comprising administering an effective amount of the recombinant alphaherpesvirus vector of claim 39 to the subject by systemic administration, subcutaneous administration, topical administration, or intradermal administration.

59. The method of claim 56, wherein the vector is administered no more than once every two weeks.

60. The method of claim 56, wherein the vector is administered no more than once a month.61 . The method of claim 56, wherein the therapeutic protein is expressed and is detectable after a first administering step.

62. The method of claim 61, wherein the therapeutic protein is detectable for at least 10 days after the administering.

63. The method of claim 58, wherein the vector causes transduced cells to express the therapeutic protein at a level and / or for a duration greater than a reference vector having a vector genome lacking the heterologous vari cellovirus ICPO, optionally at least 10-fold greater level and at least twice the duration.

64. The method of claim 58, wherein the method causes no or substantially no detectable cellular toxicity when administered in a therapeutically effective amount.

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