HSV vector

Abstract

Disclosed herein are highly transduction replication-deficient herpes simplex virus (HSV) vectors and methods of using the same.

Description

[0001] Sequence Listing

[0002] Pursuant to 37 CFR 1.52 (e) (5), this specification refers to a sequence listing (submitted electronically as a txt file named "P113870003WO00-SEQ-ROS"). The .txt file was generated on April 15, 2022 and is 96,682 bytes in size. The sequence listing is incorporated herein by reference in its entirety. Background Art

[0003] Systemic delivery of certain therapeutic agents may be problematic for agents with poor pharmacokinetics and / or risk of off-target adverse reactions. Local injection at a specific target site may require highly invasive techniques or may not be feasible. Delivery of agents by viral vectors allows for specific targeting of cell populations to provide the ability for local production and / or delivery of agents. Summary of the invention

[0004] The present disclosure provides compositions and methods for delivering a viral vector to a target cell. In some embodiments, the present disclosure provides vectors for administering nucleic acids encoding polypeptides for therapeutic or cosmetic indications to specific tissues (eg, skin).

[0005] Aspects of the present disclosure relate to a method of expressing a polypeptide in a tissue of a subject, the method comprising administering to the subject a vector comprising a variant of a herpes simplex virus (HSV) strain, the genome of the variant containing alterations such that the variant is unable to express functional ICP0 and ICP4 proteins (e.g., "ICP0- / ICP4-").

[0006] In some embodiments, the HSV strain is an HSV-1 strain. In some embodiments, the HSV-1 strain is a McKrae strain.

[0007] In some embodiments, the variant is unable to express functional ICP4 and ICPO proteins characterized by the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 17, respectively.

[0008] In some embodiments, the tissue comprises epidermis, dermis or subcutaneous fat or muscle. In some embodiments, the tissue is skin tissue. In some embodiments, the skin tissue comprises fibroblasts, keratinocytes, adipocytes or muscle cells.

[0009] In some embodiments, the vector comprises a tissue-specific promoter. In some embodiments, the tissue is skin. In some embodiments, the vector comprises a collagen 1 promoter. In some embodiments, the vector comprises a human cytomegalovirus (HCMV) enhancer. In some embodiments, the vector comprises a bovine growth hormone (BGH) polyadenylation signal or an HSV viral polyadenylation signal.

[0010] In some embodiments, the vector comprises a nucleic acid encoding a therapeutic polypeptide. In some embodiments, the vector comprises a nucleic acid encoding a therapeutic polypeptide for treating skin.

[0011] In some embodiments, the vector is administered in vivo. In some embodiments, the vector is administered by contact with the skin. In some embodiments, the vector is administered by intradermal injection.

[0012] In some embodiments, the alteration is a disruption or deletion of the ICPO and ICP4 genes. In some embodiments, the ICPO and / or ICP4 genes are disrupted by deleting their respective promoters.

[0013] In some embodiments, the present disclosure provides a variant of the HSV McKrae strain, the genome of the variant containing changes such that the variant cannot express functional ICP0 and ICP4 proteins. In some embodiments, the present disclosure provides an HSV strain comprising a variant HSV strain genome, the variant HSV strain genome containing changes such that the variant cannot express functional ICP4 and ICP0 proteins characterized by the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 17, respectively. In some embodiments, the variant is a replication-deficient variant.

[0014] In some embodiments, vectors comprising a variant HSV strain as described herein are provided.

[0015] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a carrier as described herein and a pharmaceutically acceptable carrier.

[0016] In some embodiments, the present disclosure provides cells transduced with a vector as described herein.

[0017] In some embodiments, provided herein is a method for propagating a vector comprising a variant HSV genome, wherein the variant HSV genome contains changes that render the variant unable to express functional ICP0 and ICP4 proteins, the method comprising the steps of: (i) infecting cultured ICP0 and ICP4 complementing cells containing nucleic acid (e.g., DNA) encoding HSV proteins ICP0 and ICP4 with the vector, and (ii) isolating the supernatant from the culture of step (i).

[0018] In some embodiments, the method comprises the step of purifying the vector in the supernatant by chromatography.In some embodiments, the method comprises the step of concentrating the purified vector by tangential flow filtration.

[0019] In some embodiments, the present invention provides a method for preparing a vector comprising a variant HSV genome, wherein the variant HSV genome contains an alteration such that the variant cannot express functional ICP0 and ICP4 proteins, and wherein the vector expresses a marker element, the method comprising incubating cells transfected with: (a) a first nucleic acid molecule: (i) comprising a portion of the HSV genome, but not encoding functional ICP0 and ICP4 proteins; and (ii) comprising a first homologous region (HR1) and a second homologous region (HR2), and (b) a second nucleic acid molecule, wherein the second nucleic acid molecule comprises a sequence encoding a marker element, wherein the sequence is flanked by a first homologous region (HR1') and a second homologous region (HR2'), wherein HR1 is homologous to HR1' and HR2 is homologous to HR2', such that the sequence encoding the marker element in the second nucleic acid molecule is integrated into the first nucleic acid molecule via homologous recombination.

[0020] In some embodiments, the cell is an ICPO and / or ICP4 complementing cell. In some embodiments, the marker element is a polypeptide. In some embodiments, the polypeptide is a soluble tumor necrosis factor receptor.

[0021] In some embodiments, the polypeptide is quantified by enzyme-linked immunosorbent assay (ELISA).In some embodiments, the polypeptide is detected by fluorescence.

[0022] In some embodiments, the method includes the step of purifying viral plaques expressing the marker element.

[0023] In some embodiments, the present disclosure provides a method for preparing a vector comprising a variant HSV genome, wherein the variant HSV genome contains an alteration such that the variant cannot express functional ICP0 and ICP4 proteins, and wherein the vector expresses a target agent, the method comprising incubating cells transfected with: (a) a first nucleic acid molecule: (i) comprising a portion of the HSV genome, but not encoding functional ICP0 and ICP4 proteins; and (ii) comprising a sequence encoding a marker element, wherein the sequence encoding the marker element is flanked by a first homologous region (HR1) and a second homologous region (HR2); and (b) a second nucleic acid molecule, wherein the second nucleic acid molecule comprises a sequence encoding a target agent, wherein the sequence encoding the target agent is flanked by a first homologous region (HR1') and a second homologous region (HR2'), wherein HR1 is homologous to HR1' and HR2 is homologous to HR2', such that the sequence encoding the target agent is integrated into the first nucleic acid molecule via homologous recombination.

[0024] In some embodiments, the cell is an ICPO and / or ICP4 complementing cell.

[0025] In some embodiments, the method includes the step of purifying viral plaques that do not express the marker element.

[0026] In some embodiments of the methods described herein, the HSV genome is an HSV-1 genome. In some embodiments, the HSV genome is a McKrae strain genome.

[0027] In some embodiments, the present disclosure provides a method for measuring the transduction efficiency of an HSV vector in skin tissue, the method comprising: (a) contacting the skin tissue of an animal with an HSV vector as described herein; (b) removing a portion of the skin tissue from the animal; and (c) determining the number of HSV genomes transduced in the skin tissue.

[0028] In some embodiments, skin tissue includes fibroblasts, keratinocytes, adipocytes, muscle cells, epidermis, dermis, subcutaneous tissue, or underlying subcutaneous fat or muscle.

[0029] In some embodiments, the number of genomes is measured by an amplification technique. In some embodiments, the amplification technique is quantitative polymerase chain reaction (qPCR).

[0030] In some embodiments, the present disclosure provides a method for measuring the transduction efficiency of an HSV vector in skin tissue, wherein the HSV vector contains an expression cassette comprising a polypeptide payload, the method comprising: (a) contacting the skin tissue of an animal with an HSV vector as described herein; (b) removing a portion of the skin tissue from the animal; and (c) determining the amount of the polypeptide encoded by the nucleic acid of the expression cassette.

[0031] In some embodiments, skin tissue includes fibroblasts, keratinocytes, adipocytes, muscle cells, epidermis, dermis, subcutaneous tissue, or underlying subcutaneous fat or muscle.

[0032] In some embodiments, the amount of the polypeptide is measured by immunoassay. In some embodiments, the immunoassay is ELISA or immunohistochemistry (IHC). In some embodiments, ELISA or IHC is performed on tissue of the epidermis, dermis, subcutaneous tissue, subcutaneous fat, underlying muscle, or draining lymph nodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The drawings are for illustrative purposes only and are not intended to be limiting.

[0034] Figure 1 Depicts an exemplary Western blot of ICP0 protein in McKrae HSV-1 mutants (PGN0499i8.1, PGN04 99i8.4, PGN04 99i8.5, PGN04 99i8.6, PGN04 99i8.7, and PGN04 99i8.8) lacking ICP0 and ICP4. The ICP0- / ICP4-mutants contain HCMV-mCherry in the ICP4 locus. Expression of ICP0 was not detected in any of the ICP0- / ICP4-mutants. A control ICP4-mutant is also shown, which does express ICP0. The parent vector of the ICP0- / ICP4-mutant is the ICP4-McKrae strain vector, as previously described in International Publication No. WO2017165813A1, which is incorporated herein by reference in its entirety.

[0035] Figure 2A and 2B Each depicts an exemplary PCR analysis of an ICP4-mutant with sequential deletion of ICP0. Figure 2A Shown are polymerase chain reaction (PCR) experiments for ICP0 (upper part) and UL56 (lower part) of mutants PGN04 99i8.1, PGN04 99i8.2, PGN0499i8.3, PGN04 99i8.4, PGN04 99i8.5, PGN0499i8.6, PGN04 99i8.7, and PGN04 12H2. Figure 2B PCR experiments for ICP0 are shown for mutants PGN04 99i8.1, PGN04 99i8.2, PGN04 99i8.3, PGN0499i8.4, PGN04 99i8.5, PGN04 99i8.6, PGN0499i8.7 and CT1 positive control. Vector PGN04 99.i8 and 12H2 virus were negative for ICP0 DNA.

[0036] Figure 3A and 3B It was shown that green fluorescent protein (GFP) expression was enhanced in the ICP0- / ICP4-mutant (PGN04). Figure 3A Shown are ICP4-mutants (MOI=5, measured 18 hours after infection) in which GFP expression is driven by the human cytomegalovirus (HCMV) promoter. Figure 3B ICP0- / ICP4- mutant (PNG04) (MOI = 5, measured 18 hours after infection), in which GFP expression is driven by the ICP0 promoter. Deletion of multiple immediate early genes, including (1) ICP0- / ICP4- / ICP22- / ICP27-, (2) ICP0- / ICP4- / ICP22-, (3) ICP0- / ICP4- / ICP27-, and (4) ICP0- / ICP22- / ICP27-, results in little gene expression on non-complemented cells. ICP0- / ICP4- mutant (PGN04) ( Figure 3B ) showed good GFP expression similar to that of the virus containing ICP0 ( Figure 3A ). Given that both ICP0- / ICP4- / ICP27- and ICP0- / ICP4- / ICP22- mutants showed poor expression, but ICP0- / ICP4- mutant (PGN04) showed good expression, the data demonstrated that both ICP22 and ICP27 are required for useful gene expression in ICP0- mutants. Therefore, it is not necessary to delete ICP22 and ICP27 to reduce vector toxicity.

[0037] Figures 4A-4D Each depicts an exemplary photograph showing efficient gene expression of GFP driven by the ICP0 promoter even in the absence of ICP0. Figures 4A-4D Shown is the time course of gene expression of the ICP0- / ICP4-mutant (PGN04.6; MOI=10) in Vero cells (dividing) in culture. Figure 4A Gene expression of the ICP0- / ICP4-mutant (PGN04.6; MOI=10) after 2 days is shown. Figure 4BGene expression of the ICP0- / ICP4-mutant (PGN04.6; MOI=10) after 3 days is shown. Figure 4C Gene expression of the ICP0- / ICP4-mutant (PGN04.6; MOI=10) after 4 days is shown. Figure 4D Gene expression of the ICP0- / ICP4-mutant (PGN04.6; MOI=10) after 5 days is shown. ICP0- viruses generally do not show good gene expression; however, the ICP0- / ICP4-mutant (PGN04.6) does show good gene expression (see Figures 4A-4D This retention of gene expression in the ICP0- / ICP4- mutant (PGN04.6) may be due to the retention of ICP22 and ICP27, which play a role in viral evasion of the innate immune response.

[0038] Figure 5 The effect of ICP0 on tissue-specific promoters is demonstrated, and exemplary graphs are depicted showing the relative expression of tumor necrosis factor alpha soluble receptor (sTNFR) in cell lines from epidermal and neuronal lineages, ICP4-mutants with HCMV promoter (ICP0+), compared to ICP4-mutants with large collagen-1 (eCOL1) promoter (ICP0+), compared to ICP0- / ICP4- virus controls (with ICP0-GFP and HCMV-Red) and ICP0- / ICP4-mutants with large collagen-1 promoter (eCOL1). Gene expression results (in picograms / mL sTNFR (HCMV 100x)) for each mutant are shown for three different cell types: HaCaT (keratinocyte cell line), SK-N-MC (neuroblastoma cells), and Rin5F (insulinoma cells). Both SK-N-MC and Rin5F cells express neuronal markers. Specifically, it was shown that in the presence of ICP0, the expression tissue specificity of the promoter sequence was lost. Therefore, ICP0 deletion is necessary to achieve tissue specificity through tissue-specific promoters. In addition, ICP0-mutants containing the eCOL1 promoter showed no activity in keratinocytes (HaCaT) or neuronal cell types (SK-N-MC or Rin5F).

[0039] Fig. 6A and 6BExemplary graphs showing that the PGN04 vector with the eCOL1 promoter is expressed only in fibroblasts and each depicting tissue-specific gene expression of sTNFR from the collagen-1 promoter (eCOL1) in ICP0- / ICP4-mutants in fibroblast tissue cell lines and human fibroblasts and further induction of the promoter by transforming growth factor beta peptide (tgf-b). Fig. 6A Shown is sTNFR expression in BJ fibroblasts (in picograms / mL sTNFR, measured after 3 days). Figure 6B Shown is sTNFR expression in NCTC 2472 cells (mouse fibroblast tumor) (in picograms / mL sTNFR). Fig. 6A and 6B All viruses shown in are ICP0- / ICP4- mutants. Fig. 6A and 6B Expression from the eCOL1 promoter was shown to be restricted to fibroblasts in the ICP0- virus and not expressed in neurons or keratinocytes. This expression pattern is ideal for skin indications and the results show a good safety profile compared to ICP0+ viruses and using promoters such as HCMV. The results also demonstrate that using the eCOL1 promoter will protect neurons from any off-target expression. Finally, the results show that expression from the eCOL1 promoter can be induced by TGF-β in the culture medium and therefore expression is tissue specific and inducible.

[0040] Figure 7 COL7 expression from vectors containing tissue-specific promoters is shown, and an exemplary Western blot showing an ICP0- / ICP4-mutant expressing collagen-7 (COL7) from the big collagen-1 promoter is depicted. Virus isolates were grown on ICP0 / ICP4 complementing cells. Figure 7 The results demonstrated that tissue-specific promoters exhibited some expression concurrently with viral replication.

[0041] Figure 8A-8CM An exemplary HSV McKrae strain nucleotide sequence is depicted (SEQ ID NO: 1), which is identified as Accession No. JQ730035.1.

[0042] Fig. 9 An exemplary HSV McKrae strain ICP4 amino acid sequence is depicted (SEQ ID NO: 2).

[0043] Fig.10 An exemplary HSV McKrae strain ICP22 amino acid sequence is depicted (SEQ ID NO: 3).

[0044] Fig.11 An exemplary HSV McKrae strain ICP47 amino acid sequence is depicted (SEQ ID NO: 4).

[0045] Figures 12A-12C An exemplary HSV McKrae strain ICP4 nucleotide sequence is depicted (SEQ ID NO: 5).

[0046] Fig.13 An exemplary HSV McKrae strain ICP22 nucleotide sequence is depicted (SEQ ID NO: 6).

[0047] Fig.14 An exemplary HSV McKrae strain ICP47 nucleotide sequence is depicted (SEQ ID NO: 7).

[0048] Fig.15 An exemplary human cytomegalovirus enhancer nucleotide sequence is depicted (SEQ ID NO: 8).

[0049] Fig.16 An exemplary calcitonin gene-related peptide promoter nucleotide sequence is depicted (SEQ ID NO: 9).

[0050] Fig.17 An exemplary bovine growth hormone polyadenylation signal nucleotide sequence is depicted (SEQ ID NO: 10).

[0051] Fig.18 An exemplary STPSTTT amino acid sequence is depicted (SEQ ID NO: 11).

[0052] Fig.19 Depicted is an exemplary latency-associated transcript intronic variation 1 (SEQ ID NO: 12).

[0053] Fig. 20 Depicted is an exemplary latency-associated transcript variation 2 (SEQ ID NO: 13).

[0054] Fig.21 Depicted is an exemplary latency-associated transcript variation 3 (SEQ ID NO: 14).

[0055] Fig. 22 An exemplary alanine-rich region of the KOS ICP4 amino acid sequence is depicted (SEQ ID NO: 15).

[0056] Fig.23 An exemplary serine-rich region of the McKrae ICP4 amino acid sequence is depicted (SEQ ID NO: 16).

[0057] Fig.24An exemplary McKrae strain ICPO amino acid sequence is depicted (SEQ ID NO: 17).

[0058] Figures 25A-25C An exemplary McKrae strain ICPO DNA sequence is depicted (SEQ ID NO: 18).

[0059] Figures 26A-26G An exemplary McKrae strain latency-associated transcript nucleotide sequence is depicted (SEQ ID NO: 19).

[0060] Fig.27A and 27B An exemplary human collagen 1 promoter nucleotide sequence (SEQ ID NO: 20) is depicted.

[0061] Fig.28 An exemplary human tumor necrosis factor alpha soluble receptor amino acid sequence is depicted (SEQ ID NO: 21).

[0062] Fig.29 An exemplary McKrae strain ICPO promoter sequence is depicted (SEQ ID NO: 22).

[0063] Fig.30 An exemplary synthetic terminator is depicted (SEQ ID NO: 23).

[0064] Fig.31 An exemplary TAATGARAT sequence (SEQ ID NO: 24) is depicted.

[0065] Figures 32A-32C A schematic diagram depicting an exemplary replication-deficient McKrae strain viral vector. Fig.32A Shown are a complete deletion of both copies of the viral ICP4 gene, and an indel of an expression cassette driven by the human cytomegalovirus (HCMV) immediate early promoter within both copies of the ICP4 locus. Fig.32B Shown is a complete deletion of both copies of the viral ICP4 gene, as well as an extended collagen-1 (COL1) promoter-driven expression cassette within both copies of the indel ICP4 locus. Fig.32C Shown are complete deletions of both copies of the viral ICPO and ICP4 genes, and an extended collagen-1 (COL1) promoter-driven expression cassette within both copies of the indel ICP4 locus. Figures 32A-32C Each expression cassette in contains a payload of interest for expression in target cells.

[0066] definition

[0067] In this application, unless the context clearly indicates otherwise, (i) the term "a" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; and (iii) the terms "comprising" and "including" may be understood to cover the listed components or steps, whether presented by themselves or with one or more other components or steps; and (iv) the terms "about" and "approximately" may be understood to allow for standard variations understood by those of ordinary skill in the art; and (v) where a range is provided, the endpoints are included.

[0068] Administration: As used herein, the term "administering" refers to administering a composition to a subject or system. Administration to an animal subject (e.g., to a human) can be performed by any appropriate route. For example, in some embodiments, administration can be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intra-organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, and vitreous administration. In some embodiments, administration may involve intermittent administration. In some embodiments, administration may involve continuous administration (e.g., perfusion) for at least a selected period of time.

[0069] Agent: As used herein, the term "agent" refers to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, carbohydrates, lipids, small molecules, or combinations thereof. In some embodiments, an agent is or comprises a natural product, because it exists in nature and / or is obtained from nature. In some embodiments, an agent is or comprises one or more artificial entities, because it is designed, engineered, and / or produced by the action of the human hand, and / or does not exist in nature. Some specific embodiments of agents that can be utilized according to the present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNA, shRNA, DNA / RNA hybrids, antisense oligonucleotides, ribozymes), peptides, peptide mimetics, etc.

[0070] Improvement: As used herein, the term "improvement" refers to the prevention, alleviation or alleviation of a subject's condition, or amelioration of said condition. Improvement includes but does not require complete recovery or complete prevention of a disease, disorder or condition.

[0071] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a person of either sex and at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals can be transgenic animals, genetically engineered animals, and / or clones.

[0072] Approximately: As used herein, when applied to one or more target values, the term "approximately" or "about" refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​within a range of (greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction of the stated reference value, unless otherwise stated or otherwise apparent from the context (except where such a numerical value would exceed 100% of the possible value).

[0073] Signature sequence: As used herein, the term "signature sequence" or "conserved sequence" refers to a sequence that is found in all members of a family of polypeptides or nucleic acids, and thus can be used by one of ordinary skill in the art to define members of the family.

[0074] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, two or more agents may be administered simultaneously; in some embodiments, such agents may be administered sequentially; in some embodiments, such agents are administered with overlapping dosing regimens.

[0075] Composition: As used herein, the term "composition" or "pharmaceutical composition" refers to a combination of two or more agents as described herein for co-administration or administration as part of the same regimen. In all embodiments, it is not required that the combination of agents results in a physical mixture, that is, it is possible for each component of the composition to be administered as a separate adjuvant; however, many patients or practitioners in the art may find it advantageous to prepare a composition that is a mixture of two or more ingredients in a pharmaceutically acceptable carrier, diluent or excipient, making it possible to administer the components of the combination simultaneously.

[0076] Engineering: As used herein, the term "engineering" refers to aspects that have been artificially manipulated. For example, when two or more sequences that are not linked together in the order in nature are artificially manipulated to be directly connected to each other in an engineered polynucleotide, the polynucleotide is considered to be "engineered". For example, in some embodiments of the present disclosure, the engineered polynucleotide comprises a regulatory sequence that is found in nature to be operably associated with a first coding sequence, but not operably associated with a second coding sequence, and is artificially connected so that it is operably associated with a second coding sequence. Similarly, if a cell or organism has been manipulated so that its genetic information has changed (for example, new genetic material that did not previously exist has been introduced, for example, by transformation, mating, somatic cell hybridization, transfection, transduction or other mechanisms, or previously existing genetic material, for example, by substitution or deletion mutation or by mating scheme Change or removal), then the cell or organism is considered to be "engineered". By convention and as understood by those skilled in the art, the progeny of engineered polynucleotides or cells are still generally referred to as "engineered", even if the actual manipulation is performed on a previous entity.

[0077] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0078] Functional protein: As used herein, "functional protein", e.g., functional HSV immediate early (IE) protein, e.g., functional ICP0, ICP4, ICP22, ICP27 and / or ICP47 protein, refers to a protein that exhibits at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the activity of the corresponding full-length wild-type protein. For example, a functional ICP4 protein refers to a protein that exhibits at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the activity (e.g., DNA binding activity or transcriptional regulatory activity) of a corresponding full-length wild-type ICP4 protein.

[0079] Homology: As used herein, the term "homology" refers to the overall correlation between polymer molecules, such as nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, if the sequence of a polymer molecule is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical, the polymer molecules are considered to be "homologous" to each other. In some embodiments, if the sequence of a polymer molecule is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% similar, the polymer molecules are considered to be "homologous" to each other.

[0080] Isolated: As used herein, the term "isolated" refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was originally associated when it was produced (whether in nature and / or in an experimental setting), and / or (2) manually designed, produced, prepared, and / or manufactured. An isolated substance and / or entity may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which it was originally associated. In some embodiments, the separated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be appreciated by those skilled in the art, a substance may still be considered "separated" or even "pure" after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the separation percentage or purity of the substance is calculated without including such carriers or excipients. As just one example, in some embodiments, a biopolymer such as a polypeptide or polynucleotide that occurs in nature is considered "isolated" when it a) is not associated with some or all components that accompany it in its native state in nature, depending on its source or source of derivation; b) is substantially free of other polypeptides or nucleic acids of the species in which it is produced in nature; c) is expressed by or otherwise associated with components from a cell or other expression system that is not the species in which it is produced in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system that is different from the cellular system in which it is produced in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered an "isolated" polypeptide to the extent that it has been separated from other components with which it is a) associated in nature; and / or b) associated when originally produced.

[0081] Marker element: As used herein, the term "marker element" refers to a detectable or selectable agent. In some embodiments, a "marker element" is a detectable or selectable nucleic acid sequence. In some embodiments, a "marker element" is an expression product (e.g., RNA or protein) whose presence or absence is detectable and / or selectable in a cell. In some embodiments, the expression product is an enzyme or comprises an enzyme. In some embodiments, the expression product is a fluorophore.

[0082] Nucleic acid: As used herein, the term "nucleic acid" refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, nucleic acids are compounds and / or substances that are or can be incorporated into an oligonucleotide chain via phosphodiester linkages. As will be apparent from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA; in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, nucleic acid is one or more natural nucleic acid residues, comprises one or more natural nucleic acid residues, or consists of one or more natural nucleic acid residues. In some embodiments, nucleic acid is one or more nucleic acid analogs, comprises one or more nucleic acid analogs, or consists of one or more nucleic acid analogs. In some embodiments, nucleic acid analogs differ from nucleic acids in that nucleic acid analogs do not use a phosphodiester backbone. For example, in some embodiments, nucleic acid is one or more "peptide nucleic acids", comprises one or more peptide nucleic acids, or consists of one or more peptide nucleic acids, which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, and are considered to be within the scope of the present disclosure. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages instead of phosphodiester bonds. In some embodiments, the nucleic acid is one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine), comprises one or more natural nucleosides or consists of one or more natural nucleosides. In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those sugars in natural nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product (such as an RNA or a protein). In some embodiments, the nucleic acid includes one or more introns.In some embodiments, the nucleic acid is prepared by one or more of: isolation from a natural source, enzymatic synthesis by complementary template-based polymerization (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues long. In some embodiments, the nucleic acid is single-stranded; in some embodiments, the nucleic acid is double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0083] Operably linked: The term "operably linked" refers to polynucleotide elements being linked in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a gene, the promoter or enhancer is operably linked to a gene. Operably linked nucleotide sequences are generally contiguous. However, because enhancers generally function when separated from the promoter by several thousand bases, and intron sequences can have variable lengths, some polynucleotide elements may be operably linked but not directly flanked, and may even function in trans from different alleles or chromosomes.

[0084] Patient: As used herein, the term "patient" refers to any organism to which a provided composition is or can be applied, e.g., for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. In some embodiments, the patient suffers from or is susceptible to one or more conditions or disorders. In some embodiments, the patient exhibits one or more signs or symptoms of a condition or disorder. In some embodiments, the patient has been diagnosed with one or more conditions or disorders. In some embodiments, the patient is receiving or has received a therapy to diagnose and / or treat a disease, condition, or disorder.

[0085] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that, when administered to a relevant population, shows a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those suitable for: oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue, and the like; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, such as in the form of a sterile solution or suspension or a sustained release formulation, and the like; topical application, such as in the form of a cream, ointment, or controlled release patch or a spray for application to the skin, lungs, or oral cavity, and the like; intravaginal or intrarectal, such as in the form of a pessary, cream, suppository, or foam, and the like; sublingual administration; ocular administration; transdermal administration; or nasal administration, pulmonary administration, and administration to other mucosal surfaces.

[0086] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" when applied to a carrier, diluent or excipient used to formulate a composition as disclosed herein, means that the carrier, diluent or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0087] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle involved in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic, compatible substances used in pharmaceutical formulations.

[0088] Prevent or prevention: As used herein, the terms "Prevent" or "prevention", when used in conjunction with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing a disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of a disease, disorder, or condition. Prevention is considered complete when the onset of a disease, disorder, or condition has been delayed for a predetermined period of time.

[0089] Subject: As used herein, the term "subject" refers to a mammal (e.g., a human, including prenatal human forms in some embodiments). In some embodiments, the subject suffers from a relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person with one or more characteristics that are susceptible to a disease, disorder, or condition or that are at risk of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom diagnosis and / or therapy is being administered and / or has been administered.

[0090] Therapeutic transgene: The term "transgene" refers to an exogenous gene or polynucleotide sequence. The term "therapeutic transgene" refers to a transgene that, when expressed in or by a virus, confers a therapeutic effect in a target cell, body fluid, tissue, organ, physiological system, or subject.

[0091] Treatment: As used herein, the term "treatment" (as well as "treat" or "treating") refers to any administration of a substance that partially or completely alleviates, ameliorates, relieves, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition (e.g., neuropathy). Such treatment may be applied to subjects who do not show signs of the relevant disease, disorder, and / or condition, and / or subjects who only show early signs of the disease, disorder, and / or condition. Alternatively or in addition, such treatment may be applied to subjects who show one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be applied to subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be applied to subjects known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition.

[0092] Vector: As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to the nucleic acid to which it has been connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome or part thereof. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors, additional mammalian vectors, herpes simplex virus (HSV) vectors, etc., having a bacterial origin of replication). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after the vector is introduced into the host cell, and thus replicated together with the host genome. In addition, certain vectors can direct the expression of the gene to which they are operably connected. Such vectors are referred to as "expression vectors" herein.

[0093] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be carried out according to the manufacturer's instructions or as generally implemented in the art or as described herein. The aforementioned techniques and procedures can be carried out according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose. DETAILED DESCRIPTION

[0094] Various aspects of the present invention are described in detail in the following sections. The use of sections is not meant to limit the present invention. Each section may apply to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise stated or it is clear from the context that it is a transition.

[0095] The present disclosure provides, inter alia, compositions comprising HSV vectors and methods of use and production thereof. In some embodiments, HSV vectors are provided for delivering payloads to fibroblasts, keratinocytes, adipocytes, muscle cells, or any other tissue, including the epidermis, dermis, or underlying subcutaneous fat or muscle and accessible by applying the vector into or onto the skin. In some embodiments, McKrae strain HSV vectors are provided. Certain HSV (e.g., McKrae strain) vectors may also be used to express one or more polypeptides in neurons, genetically modify cells using homologous recombination, typically alone or in combination with DNA modifying enzymes, and tumor indications, in which the ability to specifically express several gene products under one or more promoter sequences may be advantageous. Genetically modifying the vector produces a non-toxic, high capacity (>18 kilobase capacity) vector with true tissue specificity and the ability to be inducible.

[0096] Viral vectors and HSV

[0097] Viral vectors can be used to promote nucleic acid transfer into cells. Known viral vectors include those derived from retroviruses, adenoviruses, adeno-associated viruses (AAV), vaccinia viruses, and baculoviruses. Vectors derived from herpes simplex virus (HSV), such as herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2), are particularly suitable for delivering agents to specific targeted tissues. Considerations for selecting specific vectors and delivery systems include, for example, the characteristics of target cells, the required expression lifespan, the toxicity and invasiveness of the vector, and the size of the genetic material to be transferred.

[0098] HSV-1 vectors can usually accommodate up to 25kb of foreign DNA sequences.HSV-1 has a double-stranded linear DNA genome of about 152kb, which can be maintained in the nucleus in the form of episomes.HSV-1 virions have an envelope and are about 110nm in diameter.Viral infection is initiated in epithelial cells of the skin or mucous membranes by the combination of viral envelope glycoproteins with the heparin sulfate moiety on the plasma membrane.HSV is particularly suitable for gene delivery to the nervous system and has a natural tropism to sensory neurons.The virus can establish a latent state, in which the viral genome persists throughout the life of the host in the form of an intranuclear epithelial element.The lifelong persistence of the latent genome in the human trigeminal ganglion without developing sensory loss or histological damage to the ganglion illustrates the effectiveness of the latent mechanism.Wild-type HSV virus can be reactivated from the latent period under the influence of various stresses.However, the recombinant viral vector that causes replication defects retains the ability to establish a persistent quiescent state in neurons, for example, but still cannot be replicated (or reactivated) in the nervous system.

[0099] HSV-1-based vectors may have one or more HSV genes necessary for replication that render them non-functional (e.g., by deletion or destruction). HSV genes necessary for replication may include, for example, immediate early genes, such as ICP0, ICP4, and / or ICP27. In some embodiments, the disclosure provides replication-defective HSV vectors, in which one or more of ICP0, ICP4, ICP22, ICP27, and / or ICP47 are deleted or destroyed. In some embodiments, the disclosure provides HSV vectors (e.g., ICP0- / ICP4-) with non-functional ICP0 and ICP4 genes. In some embodiments, the disclosure provides replication-defective HSV vectors (e.g., ICP0- / ICP4-) in which ICP0 and ICP4 are deleted or destroyed. In some embodiments, ICP0 and / or ICP4 genes are destroyed by deleting their respective promoters.

[0100] In some embodiments, the present disclosure provides HSV vectors with non-functional ICP0, ICP4, ICP22 and ICP47 genes. In some embodiments, the present disclosure provides HSV vectors in which ICP0 and ICP4 are missing and ICP22 and ICP47 are destroyed. In some embodiments, the present disclosure provides HSV vectors in which ICP0 and ICP4 are missing and the expression of ICP22 and ICP47 is destroyed or delayed. In some embodiments, the present disclosure provides HSV vectors in which ICP0 and ICP4 are missing and ICP22, ICP27 and / or ICP47 are not expressed as immediate early proteins. In some embodiments, the present disclosure provides HSV vectors in which ICP0 and ICP4 are missing and ICP22, ICP27 and ICP47 are expressed using undestroyed promoter sequences, thereby providing unique advantages regarding appropriate vector functions based on innate immunity and evasion of cellular and humoral immunity.

[0101] HSV-1 vectors with deleted HSV genes can be produced in cell lines that express defective proteins in trans. In some embodiments, HSV-1 vectors are produced in mammalian cell lines. In some embodiments, the cell line expresses ICP0 and / or ICP4. In some embodiments, the cell line expresses one or more of ICP0, ICP4, ICP22, ICP27, and / or ICP47. In some embodiments, cells supplement ICP0 and / or ICP4 and at least one other viral gene. In some embodiments, the cell line expresses ICP0 and / or ICP4 and at least one other immediate early gene. In some embodiments, the cell line expresses ICP0 and / or ICP4, and also expresses other viral or non-viral genes that modify the innate immune response. In some embodiments, the cell line expresses ICP0 and / or ICP4, and has been modified to remove type I or type III interferon response pathways. In some embodiments, the cell line expresses ICP0 and / or ICP4, and has been modified to remove genes associated with the interferon gene stimulator (STING) pathway.

[0102] In some embodiments, the cell line expresses ICP0, ICP4, ICP22, and ICP47. In some embodiments, the cell line expresses ICP0, ICP4, ICP22, and UL55. In some embodiments, the cell line expresses ICP0, ICP4, ICP27, and UL55. In some embodiments, the cell line expresses ICP0, ICP4, ICP27, and UL55. In some embodiments, the cell line expresses ICP0 and ICP4, and ICP22, ICP27, or ICP27 and ICP27. In some embodiments, the cell line comprises a nucleic acid molecule having a Simian Virus 40 polyadenylation signal (SV40 pA).

[0103] In some embodiments, HSV-1 vector is produced in the mammalian cell line of Vero pedigree. In some embodiments, viral vector is produced in Vero D cell. In some embodiments, viral vector is produced in Vero 6-5C cell. In some embodiments, viral vector is produced in Vero 7240 cell. Cell (for example, the cell of Vero pedigree) can be complementary cell, for example ICP0 and / or ICP4 complementary cell. In some embodiments, cell is ICP0 and / or ICP4 complementary cell. In some embodiments, cell supplements ICP0 and / or ICP4 and at least one other viral gene. In some embodiments, cell supplements ICP0 and / or ICP4 and at least one immediate early gene. In some embodiments, cell is ICP0, ICP4, ICP27 and UL55 complementary cell. In some embodiments, cell is ICP0, ICP4, ICP22 and ICP47 complementary cell.

[0104] In some embodiments, the HSV-1 vector is produced in 1-D4 cells. In some embodiments, the HSV-1 vector is produced in 5B4 cells.

[0105] HSV-1 strain

[0106] At least 17 strains of HSV-1 have been isolated, including but not limited to McKrae, strain 17, strain F, H129, HF10, MacIntyre, strain HF, ATCC 2011, and KOS (for review, see Watson et al., Virology (2012)). In some embodiments, the present disclosure provides HSV viral vectors with gene deletions that render HSV replication defective but do not reduce invasiveness. In some embodiments, the HSV vector is an HSV-1 vector and may comprise any of the following: McKrae, strain 17, strain F, H129, HF10, MacIntyre, strain HF, ATCC 2011, and KOS, or any other HSV-1 strain known in the art.

[0107] Various strains of HSV-1 exhibit certain differences. For example, strain-to-strain differences in HSV-1 peripheral replication and virulence were observed after injection into animals. HSV genes also affect viral characteristics and phenotypes, and each strain may exhibit unique genes and / or non-coding sequences. Therefore, it should be understood that the HSV-1 strain used in accordance with the methods of the present invention may affect the expression characteristics of the mutant vector, for example, by localizing the expression of the target payload to a specific cell or tissue type. The selection of a specific strain may depend on the target payload, the targeted cell or tissue type, the identity of the subject, etc.

[0108] McKrae strain

[0109] In some embodiments, the HSV vector is an HSV-1 vector comprising a variant of the McKrae strain. The McKrae strain was isolated from a patient with herpes simplex keratitis and subsequently passaged in tissue culture. McKrae undergoes spontaneous or induced reactivation at a higher frequency than other known strains and is one of the most virulent HSV-1 strains. McKrae is also more neuroinvasive than other known strains (e.g., strains 17, KOS, F, and H129). The partial genome sequence of McKrae is shown in Fig. 9 (SEQ ID NO: 1) (Accession No. JQ730035).

[0110] There are at least 9 genes and several non-coding sequences that are unique to the McKrae strain. For example, in McKrae, RL1 (ICP34.5) has a PAT repeat extending between residues 159 and 160, which produces 8 iterations, while other strains only contain 3-5 iterations. In addition to those genes related to pathogenesis and latency reactivation (such as RL1, RS1 and RL2), three UL genes (UL36, UL49A, UL56) and three US genes (US7, US10 and US11) are also unique to the McKrae strain. In addition to genetic variations, non-coding sequences (such as LAT, 'a' sequences and miRNAs) also contain variations that are unique to McKrae.

[0111] The McKrae strain also contains an extended repeat element of six iterations of the internal tandem repeat STPSTTT (SEQ ID NO: 11) located within the US07 (gI) coding sequence. In addition, in McKrae, UL36 contains a premature stop codon introduced due to the deletion of a G nucleotide in a single nucleotide string encoding amino acid residue 2453 (nt 72,535), and UL 56 (180aa) contains a single base pair insertion at nucleotide 115,992 (amino acid 97). The McKrae strain also contains an extended ORF in US10, which is derived from a single bp insertion at nucleotide 143,416, and a frameshift causes the loss of the stop codon in McKrae and a unique C-terminal protein sequence. Compared with other strains, McKrae has amino acid differences in UL49A at residues 28 and 51. McKrae has histidine and threonine at residues 28 and 51, respectively, while strain 17 has arginine and threonine, and other strains (e.g., KOS) have histidine and alanine. In addition, the McKrae strain contains a reduced tandem repeat found at the UL-RL junction (49 bp in McKrae compared to 181 bp in strains 17 and KOS), as well as approximately 330 nucleotides deleted immediately following the UL-RL junction repeat. McKrae also contains a unique variation within the 'a' sequence direct repeat 2 (DR2) array. Instead of a complete series of tandem repeats, the McKrae DR2 repeats are interrupted twice by the same guanine-rich sequence.

[0112] The major variation within the LAT intron between strains is due to differences in the repeat element (GCACCCC CACTCCCAC) (SEQ ID NO: 12), which differs in the number of iterations starting at nucleotide 119,482 in the McKrae strain. McKrae contains 13 repeats, while strains F, H129 and 17 contain 9 repeats, and KOS contains 15 repeats. In addition, tandem repeat variation between strains is found in McKrae starting at base 125,520. The McKrae repeat element includes twelve iterations of CCCCAGCCCTCCCCAG (SEQ ID NO: 13) and eight iterations of CCCCTCGCCCCCTCCCG (SEQ ID NO: 14). The first repeat unit differs from the other strains in that it contains a GA transition, and McKrae contains three more iterations than any other strain. The second repeat element of the McKrae strain is collapsed, lacking 188 nucleotides relative to all other strains, and is separated from the upstream repeat by a 105 bp 100% conserved sequence containing miR-H5.

[0113] McKrae further contains a unique coding sequence for ICP4 that is not found in other known strains (see Watson et al., Virology (2012)). ICP4 is an immediate early transcriptional regulator and is associated with reactivation. Although other strains contain an alanine-rich region (AASAPDAADALAAA) (SEQ ID NO: 15) between residues 707 and 720, in McKrae, the alanine-rich region is replaced by a serine-rich sequence (GPRRSSSSSGVA A) (SEQ ID NO: 16). The serine-rich substitution block present in McKrae is adjacent to the nuclear localization signal (NLS) (amino acids 728-734). Conformational changes in this region can change the NLS, and in turn affect not only the localization of ICP4, but also other viral proteins (e.g., ICP0, ICP8) affected by the localization of ICP4 (Knipe and Smith, 1986). Therefore, this region can partially affect the viral phenotype by changing the localization of the protein to the nucleus.

[0114] In some aspects, the HSV vector of the present invention comprises a variant herpes simplex virus (HSV) McKrae strain genome containing an alteration such that the variant is unable to express functional ICPO and ICP4 proteins. In some embodiments, the alteration is a deletion or disruption of the ICPO and ICP4 genes. In some embodiments, the ICPO and / or ICP4 genes are disrupted by deleting their respective promoters.

[0115] In some embodiments, the ICPO protein expressed by the wild-type McKrae strain and not expressed by the variants produced according to the methods described herein is encoded by a cDNA sequence comprising nucleotides 2171-2227, 2977-3642, and 3767-5370 of a polynucleotide sequence comprising:

[0116]

[0117] In some embodiments, the ICPO protein expressed by the wild-type McKrae strain and not expressed by the variants produced according to the methods described herein comprises: (SEQ ID NO: 17).

[0118] In some embodiments, the ICP4 protein expressed by the wild-type McKrae strain and not expressed by the variants produced according to the methods described herein is encoded by a polynucleotide sequence comprising:

[0119]

[0120] In some embodiments, the ICP4 protein expressed by the wild-type McKrae strain and not expressed by the variants produced according to the methods described herein is encoded by a polynucleotide sequence comprising:

[0121]

[0122] In some embodiments, the ICP4 protein expressed by the wild-type McKrae strain and not expressed by the variant produced according to the methods described herein comprises:

[0123]

[0124] Replication-defective McKrae vector

[0125] McKrae Main Chain

[0126] Viral genes are expressed in a strictly regulated ordered cascade, which begins with the production of immediate early (IE) genes. The IE proteins produced, including infected cell proteins ICP0, ICP4, ICP22, ICP27 and ICP47, are responsible for regulating viral gene expression during the subsequent stages of the replication cycle. Replication-deficient variant viruses lack one or more functions necessary for the synthesis and assembly of viral genome replication or viral particles. Such viruses can be propagated in complementary cell lines expressing missing gene products; however, in normal (e.g., non-complementing) cells, viruses express viral gene products but do not replicate to form progeny virions.

[0127] Replication defective virus can be produced by various methods known in the art for modifying genes. In some embodiments, one or more nucleotides are caused to be different from the wild-type sequence. In some embodiments, one or more nucleotides are deleted. In some embodiments, the deletion of one or more nucleotides produces premature termination codons. In some embodiments, the deletion of one or more nucleotides produces genes encoding truncated polypeptides. In some embodiments, the deletion of one or more nucleotides produces genes encoding non-functional polypeptides. In some embodiments, the deletion of one or more nucleotides causes gene non-function by destroying. In some embodiments, gene is destroyed by deleting its promoter.

[0128] In some embodiments, one or more genes are deleted to cause viral replication defects. In some embodiments, the genes encoding ICP0 and ICP4 are each independently completely or partially deleted. In some embodiments, the genes encoding ICP0 and ICP4 are partially deleted. In some embodiments, the genes encoding ICP0 and ICP4 are each completely deleted. In some embodiments, the gene encoding ICP0 is partially deleted, and the gene encoding ICP4 is completely deleted. In some embodiments, the gene encoding ICP0 is completely deleted, and the gene encoding ICP4 is partially deleted. In some embodiments, the gene encoding ICP0 is completely deleted, and the gene encoding ICP4 is partially deleted. In some embodiments, the gene encoding ICP0 and ICP4 is deleted, and the upstream promoter sequence of ICP22 and ICP47 is modified. In some embodiments, the upstream promoter sequence of ICP22 and ICP47 is modified to change the time of gene expression and / or the amount of expressed genes. In some embodiments, the genes encoding ICP0 and ICP4 are deleted, and the gene expression of ICP22, ICP27 and / or ICP47 is modified. In some embodiments, the deletion of genes encoding ICP0 and ICP4 results in modification of ICP22, ICP27 and / or ICP47 expression. In some embodiments, modification of ICP22, ICP27 and / or ICP47 expression improves gene expression, reduces toxicity and / or reduces immune response (e.g., innate immune response, cell-mediated immune response or humoral immune response).

[0129] The HSV-1IE promoter contains one or more copies of the IE-specific regulatory sequence with a consensus of TAATGARAT (SEQ ID NO: 24) (wherein R is a purine). These motifs are usually located within a few hundred base pairs of the proximal IE promoter sequence, but in combination with their flanking sequences, they are discrete functional entities that can confer IE-specific regulation of other proximal promoter elements of different time categories. In some embodiments, replication-deficient viruses are produced by deleting nucleotides in the IE-specific regulatory sequence. In some embodiments, the IE-specific regulatory sequence contains internal deletions. In some embodiments, the IE-specific regulatory sequence contains terminal deletions. In some embodiments, the IE-specific regulatory sequence is completely deleted.

[0130] A schematic diagram of an exemplary replication-deficient McKrae strain viral vector is depicted in Figures 32A-32C middle. Fig.32A Shown are a complete deletion of both copies of the viral ICP4 gene, and an indel of an expression cassette driven by the human cytomegalovirus (HCMV) immediate early promoter within both copies of the ICP4 locus. Fig.32B Shown is a complete deletion of both copies of the viral ICP4 gene, as well as an extended collagen-1 (COL1) promoter-driven expression cassette within both copies of the indel ICP4 locus. Fig.32C Shown are complete deletions of both copies of the viral ICPO and ICP4 genes, and an extended collagen-1 (COL1) promoter-driven expression cassette within both copies of the indel ICP4 locus. Figures 32A-32C Each expression cassette in contains a payload of interest for expression in target cells.

[0131] In some embodiments, the extent of ICP0 and / or ICP4 deletion results in modification (e.g., removal) of upstream promoter sequences of two additional immediate early viral genes: ICP22 and ICP47. In some embodiments, such modifications may alter the timing or level of expression of gene products, but not necessarily alter gene function.

[0132] Payload

[0133] Viral vectors according to the present disclosure may contain nucleic acid molecules that constitute the payload of the vector. In some embodiments, the payload comprises a nucleic acid molecule encoding a protein. In some embodiments, the payload comprises a nucleic acid molecule comprising a sequence complementary to a nucleic acid sequence encoding a protein. In some embodiments, the payload encodes a regulatory nucleic acid molecule. In some embodiments, the payload encodes a small interfering RNA (siRNA) polynucleotide. In some embodiments, the payload encodes a micro RNA (miRNA) polynucleotide.

[0134] In some embodiments, the payload is a nucleic acid molecule encoding a protein that is exogenous to the target tissue or subject to which the vector is administered. In some embodiments, the payload is a nucleic acid molecule encoding a protein that is endogenous to the target tissue or subject to which the vector is administered. In some embodiments, the nucleic acid molecule is codon optimized.

[0135] Regulatory elements

[0136] It is contemplated herein that non-natural regulatory sequences, gene control sequences, promoters, non-coding sequences, introns or coding sequences are included in the nucleic acids disclosed herein. It is further contemplated herein that nucleic acids include nucleic acid tags or signaling sequences, or encoded protein tags or protein signaling sequences. Typically, the coding region is operably linked to one or more regulatory nucleic acid components.

[0137] The promoter included in the nucleic acid of the present disclosure can be a tissue or cell type specific promoter, a promoter specific to a variety of tissues or cell types, an organ specific promoter, a promoter specific to a variety of organs, a systemic or ubiquitous promoter, or an almost systemic or ubiquitous promoter. Promoters with random expression, inducible expression, conditional expression, or otherwise discontinuous, unstable, or unpredictable expression are also included in the scope of the present disclosure. Promoters of the present disclosure may include any of the above-mentioned features or other promoter features known in the art.

[0138] Examples of known promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the CMV / human β3 globin promoter, the glial fibrillary acidic protein (GFAP) promoter, the chicken β-actin (CBA) promoter, the β-glucuronidase (GUSB) promoter, the collagen 1 promoter (eCOL1), and the ubiquitin promoter, such as those isolated from human ubiquitin A, human ubiquitin B, and human ubiquitin C.

[0139] In some embodiments, the promoter is a cell type specific promoter. In some embodiments, the promoter is a neuron specific promoter. In some embodiments, the promoter is a neuron specific promoter, because it has specific expression in neurons, preferential expression in neurons, or usually drives the expression of related coding sequences in neurons or neuron subsets, but does not drive the expression of related coding sequences in one or more other tissues or cell types. Examples of such promoters include calcitonin gene-related peptide (CGRP), synaptic protein I (SYN), calcium / calmodulin-dependent protein kinase II, tubulin αI, neuron-specific enolase, microtubule-associated protein 1B (MAP1B) and platelet-derived growth factor β chain promoter and its derivatives. In some embodiments, the promoter is a calcitonin gene-related peptide (CGRP) promoter or its derivatives.

[0140] In some embodiments, the promoter is a chimera of one or more promoters or regulatory elements found in nature. In some embodiments, the viral vector comprises a payload, the expression of which is driven by a calcitonin gene-related peptide (CGRP) promoter with an HCMV enhancer sequence.

[0141] In some embodiments, the promoter comprises a genomic sequence. In some embodiments, the promoter sequence contains more than two kilobases of genomic sequence. In some embodiments, the promoter contains more than five kilobases of genomic sequence. In some embodiments, the promoter comprises a genomic sequence containing human collagen-1 promoter. In some embodiments, the promoter expresses genes in a manner similar to certain tissues in the body, rather than the uncontrolled gene expression observed in small segments of human genome promoters or viral promoters.

[0142] In some embodiments, the promoter is a tissue-specific promoter (e.g., a promoter that is active only in certain cell types). In some embodiments, the promoter is tissue-specific for fibroblasts. In some embodiments, the tissue-specific promoter for fibroblasts is the collagen-1 (eCOL1) promoter. In some embodiments, the promoter is tissue-specific for fibroblasts and can be induced by small molecules and / or peptides. In some embodiments, the promoter is tissue-specific for cell lines of different lineages found in the skin or underlying subcutaneous tissue (including muscle).

[0143] Other regulatory elements may additionally be operably linked to the payload, such as enhancers and polyadenylation sites. In some embodiments, the enhancer comprises a human cytomegalovirus (HCMV) sequence. In some embodiments, the polyadenylation site comprises a bovine growth hormone (BGH) polyadenylation signal.

[0144] Preparation of vector

[0145] The present disclosure is particularly directed to replication-deficient HSV vectors (e.g., McKrae strain vectors). In some embodiments, the viral vector is produced by the deletion or destruction of one or more immediate early genes. Recombination technology methods known in the art can be used to delete or destroy viral genes. In some embodiments, the viral vectors of the present disclosure may be replication-defective due to homologous recombination events. In some embodiments, the replication-deficient viral vector is produced by the deletion or destruction of the ICP4 gene. In some embodiments, the replication-deficient viral vector is produced by the deletion or destruction of the ICP0 and ICP4 genes. In some embodiments, the ICP0 and / or ICP4 genes are destroyed by deleting their respective promoters. In some embodiments, the replication-deficient viral vector is produced by the deletion of the ICP0 and ICP4 genes and the deletion of the promoter of one or more other immediate early genes (e.g., ICP22 and / or ICP47).

[0146] In some embodiments, the viral vector of the present invention is produced by deleting the locus encoding one or more ICPs (e.g., ICP0 and / or ICP4) by homologous recombination. In some embodiments, the production of the viral vector of the present invention includes the step of homologous recombination. In some embodiments, homologous recombination includes the recombination of a first plasmid and a second plasmid. In some embodiments, the first plasmid contains a nucleic acid sequence homologous to a region of the HSV genome, and the region is adjacent to the nucleic acid region of the HSV genome expected to be replaced. In some embodiments, the first plasmid contains a nucleic acid sequence encoding a target gene between homologous nucleic acid sequences. In some embodiments, the target gene comprises a marker protein that can be detected by fluorescence, chemiluminescence or any other detectable property, and the marker protein can identify the vector produced by successful homologous recombination in some embodiments. In some embodiments, the second plasmid contains the HSV genome or a fragment thereof.

[0147] In some embodiments, homologous recombination includes recombination between a plasmid and a viral genome (e.g., an HSV genome). In some embodiments, the viral vectors of the present disclosure are produced by homologous recombination of a first plasmid with a circular episome or a linear configuration of the HSV McKrae strain genome. In some embodiments, the first plasmid comprises a nucleic acid sequence homologous to a region upstream of the ICP4 promoter, including a viral origin contained within a short inverted repeat region of HSV.

[0148] In some embodiments, the vector is prepared by first replacing two copies of the ICP4 locus by homologous recombination using plasmid SASB3 and screening for plaques expressing green fluorescent protein (GFP). In some embodiments, the SphI to Afl III (Sal I connected) fragment (1928bp) (nucleotides 124485-126413) of the HSV-1KOS strain genome is cloned into pSP72 digested with Sph I / Sal I, followed by inserting a 695bp Bgl II to BamH I fragment (nucleotides 131931 to 132626) (which contains the region upstream of the ICP4 promoter, including the viral start point contained in the short inverted repeat region) into the Bgl II to BamH I site of the vector plasmid to construct a plasmid. In some embodiments, the plasmid is constructed by cloning the HCMV-eGFP fragment in the BamHI site of the plasmid as described above. In some embodiments, the plasmid as described above is then recombined into a specific locus of the wild-type McKrae virus. In some embodiments, the resulting vector is isolated using a stable cell line that expresses one or more genes required for replication that are deleted or disrupted in the HSV genome.

[0149] In some embodiments, the vector is prepared by first replacing two copies of the ICP4 locus by homologous recombination using plasmid SDAXB and screening for plaques expressing green fluorescent protein (GFP). In some embodiments, the Sph I to Afl III fragment (1928bp) of the HSV-1 KOS strain genome (nucleotides 124346 to 126273 of accession number KT899744) is cloned into pSP72 digested with Sph I / Afl III to make SDA, and then the Afl III site is changed to a BamHI site (SDAB) to construct a plasmid. The BamHI to Bgl II DNA PCR fragment (which contains the region upstream of the ICP4 promoter, including the viral start point contained in the short inverted repeat region) (nucleotides 144933 to 145534 of accession number JQ730035) is cloned into the BamHI site of SDAB to make SDAXB. In some embodiments, the plasmid is constructed by cloning the HCMV-eGFP fragment in the BamHI site of the plasmid as described above. In some embodiments, the plasmid as described above is then recombined into a specific locus of the wild-type McKrae virus. In some embodiments, the resulting vector is isolated using a stable cell line that expresses one or more genes that are deleted or disrupted in the HSV genome that are required for replication.

[0150] In some embodiments, the vector is prepared by first replacing two copies of the ICPO locus by homologous recombination using plasmid OE2 and screening for plaques expressing green fluorescent protein (GFP). In some embodiments, the plasmid contains sequences flanking base pairs 1201 to 2165 and 6021 to 6971 of the ICPO gene presented by JQ730035.1 of the McKrae strain genome. In some embodiments, the plasmid as described above is then recombined into a specific locus of the wild-type McKrae virus. In some embodiments, the resulting vector is isolated using a stable cell line that expresses one or more genes that are deleted or disrupted in the HSV genome that are required for replication.

[0151] In some embodiments, the viral vectors of the present disclosure are produced by homologous recombination of a first plasmid (which in some embodiments may contain a promoter element) containing a nucleic acid sequence homologous to a region upstream of the ICP0 gene sequence with a circular episome or linear configuration (e.g., as described above) of the HSV McKrae strain genome lacking ICP4 to produce a viral vector lacking both ICP0 and ICP4. In some embodiments, the viral vectors of the present disclosure are produced by homologous recombination of a first plasmid containing a nucleic acid sequence homologous to a region upstream of the ICP4 promoter (including a viral origin contained within a long inverted repeat region of HSV) with a circular episome or linear configuration (e.g., as described above) of the HSV McKrae strain genome lacking ICP0 to produce a viral vector lacking both ICP0 and ICP4.

[0152] Characterization of vector

[0153] The viral vector according to the present disclosure can be characterized by genome sequencing to determine whether the expected vector has been successfully produced. Any sequencing method known in the art is acceptable for this purpose. Sequencing methods include, for example, nanopore sequencing, single molecule real-time sequencing (SMRT), DNA nanoball (DNB) sequencing, pyrophosphate sequencing, and use DNA arrays.

[0154] The expression of the payload from the viral vector can be detected by any method known in the art for detecting proteins or nucleic acids. The method for detecting protein expression includes immunohistochemistry, flow cytometry, western blot, enzyme-linked immunosorbent assay (ELISA), immunoelectron microscopy, single protein immunoprecipitation (IP), protein complex immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), immunoelectrophoresis, spectrophotometry and bicinchoninic acid assay (BCA). The method for detecting nucleic acid expression includes Southern blotting, Northern blotting, polymerase chain reaction (PCR), quantitative PCR and RT-PCR.

[0155] In some embodiments, the present disclosure provides methods for testing the ability of viral vectors to transduce specific types of cells (e.g., neurons, keratinocytes, muscle cells, etc.). In some embodiments, the present disclosure provides methods for testing the ability of viral vectors to transduce neurons. In some embodiments, neurons are peripheral neurons. In some embodiments, neurons are sensory neurons. In some embodiments, neurons include dorsal root ganglia (DRG). In some embodiments, viral vector preparations can be injected into one or more dermatomes corresponding to segments of DRG (e.g., left and right L4, L5 and L6 DRG). In some embodiments, DRG is removed, and DNA is isolated from DRG, and vector genome copies are analyzed using qPCR assays targeting sequences within HSV-1. In some embodiments, qPCR assays targeting HSV-1 glycoproteins (U L -22) sequences within genes.

[0156] In some embodiments, the present disclosure provides for testing the ability of viral vectors to transduce keratinocytes, fibroblasts, adipocytes, muscle cells, and all other cells that can be transduced via application to the skin (e.g., by topical application or injection). In some embodiments, the vector preparation can be applied to the skin via topical application. In some embodiments, the vector preparation can be applied to the skin via injection. In some embodiments, injection can include intradermal or subcutaneous injection methods. In some embodiments, vector expression in the skin can be measured by ELISA, histology, or analysis of vector genomic DNA or RNA.

[0157] In some embodiments, the present disclosure provides methods for testing the ability of a viral vector to transduce tumor cells.

[0158] In some embodiments, the present disclosure provides for testing the ability of viral vectors to localize to other organs, including the liver, lungs, heart, kidneys, bladder, and prostate.

[0159] Application / Use

[0160] Viral vectors according to the present disclosure can be used for a variety of therapeutic applications. In some embodiments, vectors as described herein can be used to deliver one or more payloads to one or more target cells. In some embodiments, the target cells are present in tissues that are poorly vascularized and difficult to reach through the systemic circulation. In some embodiments, the target cells are cells susceptible to HSV infection. In some embodiments, the target cells are particularly susceptible to infection by the McKrae strain of HSV. In some embodiments, the target cells are neuronal cells. In some embodiments, the target cells are dorsal root ganglion (DRG) cells.

[0161] In some embodiments, the target cells are skin cells. In some embodiments, the target cells are keratinocytes, fibroblasts, adipocytes, and / or muscle cells. In some embodiments, the target cells are all other cells present in the epidermis, dermis, and subcutaneous tissues that can be reached by topical (e.g., applied to the skin) or intradermal administration of the HSV vectors of the present disclosure.

[0162] In some embodiments, the target cell is a tumor cell. In some embodiments, the target cell is present in the liver, lung, heart, kidney, bladder or prostate.

[0163] Gene therapy

[0164] Viral vectors according to the present disclosure can be used in any setting where gene therapy is contemplated. For example, a viral vector comprising a heterologous nucleic acid segment operably linked to a promoter can be used for any disease or clinical condition associated with a reduction or deficiency of a protein encoded by the heterologous nucleic acid segment, or any disease or clinical condition that can be effectively treated by expressing the encoded protein in a subject.

[0165] Viral vectors containing expression cassettes for synthesizing RNAi agents (e.g., one or more siRNAs or shRNAs) can be used to treat any disease or clinical condition associated with overexpression of a transcript or a protein encoded thereby in a subject, or any disease or clinical condition that can be treated by causing a decrease in a transcript or a protein encoded thereby in a subject.

[0166] Viral vectors containing expression cassettes for synthesizing one or more RNAs that self-hybridize or hybridize with each other to form RNAi agents that target transcripts encoding cytokines can be used to modulate immune system responses (e.g., responses responsible for organ transplant rejection, allergies, autoimmune diseases, inflammation, etc.).

[0167] Viral vectors that provide a template for the synthesis of one or more RNAs that self-hybridize or hybridize with each other to form RNAi agents that target transcripts of infectious agents or that target cellular transcripts that encode products that are necessary for or contribute to any aspect of the infectious process can be used to treat infectious diseases.

[0168] Viral vectors containing genomic DNA sequences of DNA modification enzymes can be used to treat genetic diseases. Viral vectors containing genomic DNA sequences can be used to treat genetic diseases. Viral vectors containing genetic material from other viruses can be used as treatments or vaccines.

[0169] Application

[0170] Compositions comprising viral vectors as described herein can be formulated for delivery by any available route, including but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal and vaginal. Preferred delivery routes include intradermal and transdermal. In some embodiments, the pharmaceutical composition comprises a viral vector in combination with a pharmaceutically acceptable carrier. As used herein, the expression "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents compatible with drug administration, etc. Supplementary active compounds may also be incorporated into the composition. In some embodiments, the viral vector is formulated in glycerol. In some embodiments, the viral vector is formulated in approximately 10% glycerol in phosphate buffered saline (PBS).

[0171] It is advantageous to formulate the composition in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of viral vector calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.

[0172] The pharmaceutical composition may be administered at different intervals and over different time periods as desired, e.g., once a week for between about 1 and 10 weeks, between about 2 and 8 weeks, between about 3 and 7 weeks, between about 4, 5 or 6 weeks, etc. Those skilled in the art will appreciate that certain factors may affect the dosage and time course required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatment, the general health and / or age of the subject, and / or other diseases present in the subject. Treatment of a subject with a viral vector may include a single treatment, or in many cases may include a series of treatments.

[0173] Composition

[0174] In some embodiments, the active agent (e.g., a viral vector of the present disclosure and / or other agents to be administered with a viral vector of the present disclosure) is prepared with a carrier that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such compositions will be clear to those skilled in the art. In some embodiments, the compositions target specific cell types or cells infected by the virus.

[0175] Combination therapy

[0176] According to the present disclosure, the compositions provided can be administered in combination with one or more other active agents and / or treatments, such as known therapeutic agents and / or independently active biologically active agents. In certain embodiments, the compositions provided include one or more such other active agents; in some embodiments, such other active agents are provided as part of different compositions. In some embodiments, combination therapy involves simultaneous administration of one or more doses or units of two or more different active agents and / or treatments; in some embodiments, combination therapy involves simultaneous exposure to two or more different active agents and / or treatments, for example, by overlapping dosing regimens.

[0177] In some embodiments, provided compositions comprise or are administered in combination with one or more additional active agents useful for treating related diseases, disorders, and / or conditions.

[0178] List of implementation plans

[0179] Certain implementations are set forth in the clauses listed below.

[0180] Item 1. A variant of a herpes simplex virus (HSV) strain, the genome of which contains an alteration such that the variant is unable to express one or more functional ICPO and ICP4 proteins.

[0181] Clause 2. The variant HSV strain as described in Clause 1, wherein the HSV strain is an HSV-1 strain.

[0182] Clause 3. The variant HSV strain as described in Clause 1, wherein the HSV strain is a McKrae strain.

[0183] Item 4. A variant herpes simplex virus (HSV) strain comprising a variant HSV strain genome, wherein the variant HSV strain genome contains changes such that the variant is unable to express functional ICP4 and ICP0 proteins characterized by the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 17, respectively.

[0184] Clause 5. A variant HSV strain as described in any of Clauses 1-4, wherein the HSV strain comprises a deletion of substantially all or all nucleic acids encoding the ICP4 and / or ICPO proteins.

[0185] Clause 6. A variant HSV strain as described in any of Clauses 1-4, wherein the HSV strain contains an alteration such that the variant is unable to express a functional ICP47 protein, for example, wherein the HSV strain comprises a deletion of substantially all or all of the nucleic acid encoding the ICP47 protein.

[0186] Clause 7. A variant HSV strain as described in any of Clauses 1-5, wherein the HSV strain expresses one or more functional ICP22, ICP27 and / or ICP47 proteins, for example, wherein the ICP22, ICP27 and / or ICP47 proteins are substantially wild-type proteins or are wild-type proteins.

[0187] Clause 8. A vector comprising the variant HSV strain of any one of clauses 1-7.

[0188] Item 9. The vector of Item 8, wherein the vector comprises a cell- or tissue-specific promoter.

[0189] Clause 10. The carrier of Clause 9, wherein the tissue is skin.

[0190] Item 11. The vector of Item 9, wherein the tissue-specific promoter is a collagen 1 promoter.

[0191] Clause 12. The vector of any one of Clauses 8-11, wherein the vector comprises a human cytomegalovirus (HCMV) enhancer.

[0192] Clause 13. The vector of any one of Clauses 8-12, wherein the vector comprises a bovine growth hormone (BGH) polyadenylation signal or an HSV viral polyadenylation signal.

[0193] Clause 14. The vector of any one of Clauses 8 to 13, further comprising a nucleic acid encoding one or more therapeutic transgenes.

[0194] Clause 15. The vector of any of clauses 8 to 14, wherein the nucleic acid encoding one or more therapeutic transgenes is at least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, about 1 kb to about 35 kb, about 1 kb to about 35 kb, about 1 kb to about 30 kb, about 1 kb to about 25 kb, about 1 kb to about 20 kb, about 1 kb to about 15 kb, about 1 kb to about 10 kb, about 1 kb to about 35 kb, about 1 kb to about 35 kb, about 1 kb to about 30 kb, about 1 kb to about 25 kb, about 1 kb to about 20 kb, about 1 kb to about 15 kb, about 1 kb to about 10 kb, about 1 kb to about 35 kb, about 1 kb to about 35 kb, about 1 kb to about 35 kb, about 1 kb to about 30 kb. kb to about 25kb, about 5kb to about 20kb, about 5kb to about 15kb, about 5kb to about 10kb, about 10kb to about 35kb, about 10kb to about 30kb, about 10kb to about 25kb, about 10kb to about 20kb, about 10kb to about 15kb, about 15kb to about 35kb, about 15kb to about 30kb, about 15kb to about 25kb, about 15kb to about 20kb, about 20kb to about 35kb, about 20kb to about 30kb, about 20kb to about 25kb, about 25kb to about 35kb, about 25kb to about 30kb, or about 30kb to about 25kb.

[0195] Clause 16. The vector of any one of Clauses 8 to 15, wherein the one or more therapeutic transgenes encode one or more therapeutic polypeptides.

[0196] Clause 17. The vector of any one of Clauses 8-16, wherein the tissue-specific promoter is operably linked to the one or more therapeutic transgenes.

[0197] Clause 18. The vector of any one of Clauses 8-17, wherein the vector is capable of expressing the one or more therapeutic transgenes in a target cell, body fluid, tissue, organ or physiological system of a subject.

[0198] Clause 19. The vector of any one of clauses 8 to 18, wherein the vector is capable of expressing the one or more therapeutic transgenes in the target cell, body fluid, tissue, organ or physiological system of the subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 28 or 30 days.

[0199] Clause 20. The vector of any one of Clauses 8-19, wherein the vector is non-toxic to cells, body fluids, tissues, organs or physiological systems of a subject.

[0200] Clause 21. The vector of any one of clauses 8-20, wherein the vector is less toxic to the cells, body fluids, tissues, organs or physiological systems of the subject relative to an HSV vector that does express one or more functional ICPO and ICP4 proteins.

[0201] Item 22. A cell transduced with a vector according to any one of items 8-21.

[0202] Clause 23. A pharmaceutical composition comprising a variant of a herpes simplex virus (HSV) strain according to any one of clauses 1 to 7 or a vector according to any one of clauses 8 to 22 and a pharmaceutically acceptable carrier.

[0203] Clause 24. A method for propagating a vector comprising a variant herpes simplex virus (HSV) genome, wherein the variant HSV genome contains an alteration such that the variant is unable to express functional ICP0 and ICP4 proteins, the method comprising the steps of:

[0204] (a) infecting cultured ICP0 and ICP4 complementing cells containing nucleic acid (e.g., DNA) encoding HSV proteins ICP0 and ICP4 with the vector, and

[0205] (b) isolating the supernatant from the culture of step (a).

[0206] Clause 25. The method according to Clause 24, further comprising the step of purifying the vector in the supernatant by chromatography.

[0207] Clause 26. The method of Clause 24 or Clause 25, further comprising the step of concentrating the purified vector by tangential flow filtration.

[0208] Clause 27. A method of making a vector comprising a variant herpes simplex virus (HSV) genome, the variant HSV genome containing an alteration such that the variant is unable to express functional ICPO and ICP4 proteins, and wherein the vector expresses a marker element, the method comprising incubating cells transfected with:

[0209] (a) First Nucleic Acid Molecule:

[0210] (i) contains a portion of the HSV genome but does not encode functional ICP0 and ICP4 proteins; and

[0211] (ii) comprises a first homology region (HR1) and a second homology region (HR2), and

[0212] (b) a second nucleic acid molecule comprising a sequence encoding a marker element, wherein the sequence is flanked by a first homology region (HR1') and a second homology region (HR2'),

[0213] Wherein HR1 is homologous to HR1' and HR2 is homologous to HR2', such that the sequence encoding the marker element in the second nucleic acid molecule is integrated into the first nucleic acid molecule via homologous recombination.

[0214] Clause 28. The method of Clause 27, wherein the cell is an ICPO and / or ICP4 complementing cell.

[0215] Item 29. The method of Item 27 or 28, wherein the marker element is a polypeptide.

[0216] Clause 30. The method of Clause 29, wherein the polypeptide is a soluble tumor necrosis factor receptor.

[0217] Clause 31. The method of Clause 29, wherein the polypeptide is quantified by enzyme-linked immunosorbent assay (ELISA).

[0218] Clause 32. The method of any one of Clauses 29-31, wherein the polypeptide is detected by fluorescence.

[0219] Clause 33. The method of any one of Clauses 27-32, further comprising the step of purifying viral plaques expressing the marker element.

[0220] Clause 34. A method of preparing a vector comprising a variant herpes simplex virus (HSV) genome, the variant HSV genome containing an alteration such that the variant is unable to express functional ICPO and ICP4 proteins, and wherein the vector expresses an agent of interest, the method comprising incubating cells transfected with:

[0221] (a) First Nucleic Acid Molecule:

[0222] (i) contains a portion of the HSV genome but does not encode functional ICP0 and ICP4 proteins; and

[0223] (ii) comprising a sequence encoding a marker element, wherein said sequence encoding said marker element is flanked by a first homology region (HR1) and a second homology region (HR2); and

[0224] (b) a second nucleic acid molecule comprising a sequence encoding an agent of interest, wherein the sequence encoding the agent of interest is flanked by a first homology region (HR1′) and a second homology region (HR2′),

[0225] wherein HR1 is homologous to HR1' and HR2 is homologous to HR2', such that the sequence encoding the target agent is integrated into the first nucleic acid molecule via homologous recombination.

[0226] Clause 35. The method of Clause 34, wherein the cell is an ICPO and / or ICP4 complementing cell.

[0227] Clause 36. The method of Clause 34 or Clause 35, further comprising the step of purifying viral plaques that do not express the marker element.

[0228] Clause 37. The method of any one of Clauses 24-36, wherein the HSV genome is an HSV-1 genome.

[0229] Clause 38. The method of any one of Clauses 24-36, wherein the HSV genome is a McKrae strain genome.

[0230] Clause 39. A method of expressing a transgene (e.g., a polypeptide) in a target cell, body fluid, tissue, organ or physiological system of a subject, the method comprising administering to the subject a variant of a herpes simplex virus (HSV) strain according to any one of clauses 1-7 or a vector according to any one of clauses 8-21.

[0231] Clause 40. The method of Clause 39, wherein the tissue is selected from skin tissue, eye tissue, or tumor tissue.

[0232] Clause 41. The method of Clause 39, wherein the tissue is skin tissue.

[0233] Clause 42. The method of Clause 39, wherein the tissue comprises epidermis, dermis, or subcutaneous fat or muscle.

[0234] Clause 43. The method of Clause 41, wherein the skin tissue comprises fibroblasts, keratinocytes, adipocytes, or muscle cells.

[0235] Clause 44. The method of any one of Clauses 39-43, wherein the vector is administered in vivo.

[0236] Clause 45. The method of any one of Clauses 39-43, wherein the carrier is applied by contact with the skin.

[0237] Clause 46. The method of any one of Clauses 39-43, wherein the vector is administered by intradermal injection.

[0238] Clause 47. A method for measuring the transduction efficiency of an HSV vector in skin tissue, the method comprising:

[0239] (a) contacting skin tissue of an animal with a carrier according to any one of clauses 8 to 21;

[0240] (b) removing a portion of the skin tissue from the animal; and

[0241] (c) determining the amount of transduced HSV genomes in the skin tissue.

[0242] Clause 48. The method of Clause 47, wherein the skin tissue comprises fibroblasts, keratinocytes, adipocytes, muscle cells, epidermis, dermis, subcutaneous tissue, or underlying subcutaneous fat or muscle.

[0243] Clause 49. The method of Clause 47 or Clause 48, wherein the number of genomes is measured by an amplification technique.

[0244] Clause 50. The method of Clause 49, wherein the amplification technique is quantitative polymerase chain reaction (qPCR).

[0245] Clause 51. A method of measuring the transduction efficiency of an HSV vector in skin tissue, the HSV vector comprising an expression cassette comprising a polypeptide payload, the method comprising:

[0246] (a) contacting skin tissue of an animal with a carrier according to any one of clauses 8 to 21;

[0247] (b) removing a portion of the skin tissue from the animal; and

[0248] (c) determining the amount of the polypeptide encoded by the nucleic acid of the expression cassette.

[0249] Clause 52. The method of Clause 51, wherein the skin tissue comprises fibroblasts, keratinocytes, adipocytes, muscle cells, epidermis, dermis, subcutaneous tissue, or underlying subcutaneous fat or muscle.

[0250] Clause 53. The method of Clause 51 or Clause 52, wherein the amount of the polypeptide is measured by immunoassay.

[0251] Clause 54. The method of Clause 53, wherein the immunoassay is enzyme-linked immunosorbent assay (ELISA) or immunohistochemistry (IHC).

[0252] Clause 55. The method of Clause 53, wherein the ELISA or IHC is performed on tissue of the epidermis, dermis, subcutaneous tissue, subcutaneous fat, underlying muscle, or draining lymph nodes.

[0253] Example

[0254] Example 1: Western blot demonstrates that ICPO is missing from HSV ICP4-mutants.

[0255] This example shows an exemplary method for testing the expression of ICPO protein from infected cell cultures using Western blot.

[0256] The viral vector preparation was added to non-complementing Vero cells for one hour, and then full culture medium was added to the cells. After 24 hours, the cells were washed with phosphate buffered saline (PBS), then lysed and collected in RIPA buffer. Thirty (30) μL of RIPA buffer lysate was added to 10 μL of 4×LDS sample buffer (Invitrogen) and heated at 95°C for 5 minutes, and then PAGE electrophoresis was performed on a 7% Tris acetate PAGE gel. The PAGE gel was run at 140V for 90 minutes.

[0257] Next, the protein is transferred to the transfer membrane (immobilon membrane), and then incubated in 2% skim milk powder in Tris buffered saline (TBS). At room temperature, add the first antibody (mouse anti-ICP0) overnight with a dilution of 1:3000. On the second day, the membrane was washed three times in TBS, and then incubated in 2% skim milk powder in TBS for 15 minutes. The second anti-mouse antibody conjugated with alkaline phosphatase is added to the incubation with a dilution of 1:10000, and then incubated at room temperature for 2 hours. Then the membrane is washed 3 times in 20mL TBS in 30 minutes.

[0258] The membrane was developed by adding NBT / BCIP solution in 0.5 M Tris buffer (pH 9.5) for 30 minutes to visualize the bands. After visualization, none of the McKrae HSV-1 mutants lacking ICP0 and ICP4 (PGN04 99i8.1, PGN0499i8.4, PGN04 99i8.5, PGN04 99i8.6, PGN04 99i8.7, and PGN04 99i8.8) expressed ICP0 ( Figure 1 ). A control ICP4-variant is also shown as a control, which does express ICPO. These results demonstrate that the ICP0- / ICP4-mutants developed according to the present disclosure do not express ICPO.

[0259] Example 2: PCR confirmation of deletion of ICPO DNA sequence.

[0260] This example demonstrates PCR analysis of viral vector DNA preparations confirming the deletion of the ICPO sequence in the ICP0- / ICP4- mutants developed according to the present disclosure.

[0261] The viral vector preparation was added to non-complementing Vero cells for one hour, and then the full culture medium was added to the cells. After 24 hours, the vector and genomic DNA were collected using the New England Biolabs Monarch genomic DNA kit as described in the user manual. PCR analysis was performed on each DNA sample using PCR primers specific to the ICP0 sequence (as a test sequence) and UL56 primers (to confirm the presence of viral DNA). The ICP4 mutant vector CT1 was included as a positive control for the virus containing ICP0.

[0262] PCR results demonstrated that the ICP0- / ICP4- mutant developed according to the present disclosure did not contain the ICP0 DNA sequence ( FIG. 2 ).

[0263] Example 3: Preparation of carrier

[0264] This example describes methods for making and formulating exemplary vectors for use in gene therapy.

[0265] Genetic structure of the vector

[0266] The vector is prepared by first using a plasmid to replace two copies of the ICP0 locus in the McKrae strain ICP4-mutant by homologous recombination and screening for plaques expressing marker elements. The plasmid is constructed by cloning a fragment of the HSV-1 genome, which contains the ICP0 promoter region and the sequence downstream of the 3' end of the ICP0 gene. The plasmid is further modified by cloning a marker element (e.g., eGFP) fragment into the plasmid. The plasmid is then recombined into the ICP0 locus of the McKrae strain ICP4 mutant HSV virus. The resulting vector is isolated using a stable Vero cell line (such as 7240) expressing ICP0 and ICP4. Vero 7240 cells are complementary cells that express ICP0, ICP4, ICP27, and UL55.

[0267] To prepare a vector capable of expressing the gene of interest, a plasmid was constructed by cloning the promoter and gene sequence into a plasmid containing ICP0 or ICP4 flanking sequences. Plaques that did not express the marker element were isolated and tested for GOI expression by ELISA. The same method was used to recombinant human tissue-specific promoters in the ICP4 locus of ICP0- / ICP4- mutant viruses.

[0268] The plasmid used to generate the human genomic promoter sequence was generated by PCR of human genomic DNA cloned into a plasmid containing the ICP4 flanking sequences.

[0269] Production of crude vector

[0270] ICP0 / ICP4 or ICP0 / ICP4 / ICP27 / UL55 complemented Vero cells were cultured in tissue culture flasks using complete medium (DMEM supplemented with FBS, HEPES, and Pen Strep) and plated at 3-4 × 10 4 cells / cm 2 The inoculation density was expanded into 6-12×T175 flasks. The culture flasks were incubated at 37°C / 7.5% CO2 for 3-4 days.

[0271] When the cells are confluent, the cells are infected with a known concentration of virus stock at a multiplicity of infection (MOI) of about 0.1. Infection is initiated by removing the culture supernatant from each flask and infecting with a total of 2.5 mL of complete medium containing the appropriate amount of virus stock. The virus is adsorbed on the cell monolayer by incubating the culture for 1.5-2 hours and shaking and rotating the flask every 15-20 minutes. After the adsorption step, an additional 10 mL of complete medium is added to each flask and the culture is incubated again at 37°C / 7.5% CO2.

[0272] Approximately 72 hours after the start of infection, the flasks were observed microscopically to confirm that the cells showed signs of cytopathic effect and detached from the flask surface. At this time, the cells and supernatant were harvested, pooled together, and centrifuged at about 1500×g for about 10 minutes. The supernatant was removed from the cell pellet and saved separately for subsequent processing.

[0273] The cell pellet was suspended in 4-5 mL of complete medium, homogenized, and then frozen at -80°C. After the cell suspension was frozen for >20 minutes, it was thawed and centrifuged at about 1500×g for about 10 minutes. The supernatant of this second cell pellet was removed and combined with the supernatant of the first collection.

[0274] The pooled supernatant is aliquoted into centrifuge tubes. The virus is then centrifuged at about 40,000 × g for about 30 minutes at 2°C-8°C to allow the virus to form a mass. After the centrifugation step is complete, the supernatant in the tube is removed and discarded. The next day, the virus mass is homogenized by pipetting and pooled together. The resuspended virus stock is then aliquoted into frozen vials, usually in a volume of about 120 μL per vial. Complete medium (200-300 μL) is added to the virus mass to cover them with liquid, and the cells are stored overnight at 2°C-8°C to loosen the virus particles. The vials are then labeled and frozen at -80°C. Subsequently, the frozen vials are thawed for viral plaque titration assays to determine the concentration of the prepared virus stock before being used in any in vivo or in vitro studies.

[0275] Preparation of clarified vector

[0276] Cell thawing and expansion

[0277] Vero cells (e.g., ICP0 / ICP4 or ICP0 / ICP4 / ICP27 / UL55 complementation) from a working cell bank were thawed at 37°C, transferred to a conical tube, and pooled. Cells were plated at approximately 1.0 × 10 7 viable cells / mL / tube into a vial. Dilute the cells gradually with complete medium and remove samples to obtain viable cell counts. 4 cells / cm 2 The density was plated in tissue culture flasks.

[0278] Cells were incubated at 37°C, 7.5% CO2 and examined periodically by phase contrast microscopy. Cells were passaged when subconfluent. Complete medium was removed, rinsed with PBS, and cells were dissociated. Flasks were incubated until cells detached and then resuspended in complete medium, pooled, counted, and counted at 1.0-4.0×10 4 cells / cm 2 The cells were expanded and allowed to grow to 1-2 days post-confluence before infection.

[0279] Infection with vector

[0280] When the cells reach the required confluence, the model flask is subcultured and the cells are counted to estimate the number of cells in each cell factory. The virus library vector inoculum is prepared by thawing to obtain the appropriate volume required for the multiplicity of infection (MOI) of 0.1 and diluting the stock solution with complete medium until the target volume required for infection. The cell factory is infected by the initial adsorption phase, and then the incubation in complete medium is used for the first day of infection. After about 24 hours, the culture medium is removed and replaced with an equal volume of serum-free medium. The culture is monitored daily and the cytopathic effect percentage is estimated by visual inspection.

[0281] Crude virus harvest and clarification

[0282] The infection was stopped by placing the cell factory in a biosafety cabinet and pooling the supernatant and cell debris into sterile bags. The batch of unclarified harvest was sampled for adventitious factors. After sampling, the sodium chloride level of the harvest was increased and then mixed. The harvest was then aliquoted into centrifuge tubes and cell debris was removed by centrifugation. The supernatant was pooled into sterile bags. After pre-treating the filter capsule with sterile water to clarify, the supernatant containing the virus was pumped through the filter capsule into another sterile bag, followed by sterile water, to recover the remaining virus in the capsule. The bags were mixed and the filtrate was stored at 4°C overnight.

[0283] The filtrate was then heated and adjusted to approximately 2 mM MgCl2 by adding 2 volumes of 3 mM MgCl2 in sterile water.The diluted filtrates were mixed and treated with the endonuclease.

[0284] Cation exchange column chromatography

[0285] The BPG 400 column was filled with SP high performance resin, sterilized with 0.5 N NaOH, and equilibrated with wash buffer (PBS, pH 7.0) and stripping buffer (1 M NaCl-PBS, pH 7.0), and then loaded with the endonuclease-treated virus.

[0286] Use a pipe welding machine to connect a processing bag containing the filtrate treated with endonucleases to the inlet, and load the virus onto the column. The effluent is collected in a sterile bag. Wash with PBS after the virus capture step until the UV light returns to the baseline. Stop the pump and connect a processing bag containing 0.45M NaCl-PBS (pH 7.0) to the inlet. Transfer the outlet pipe to a sterile container in a biosafety cabinet. Pump the buffer into the column, and when the UV absorbance begins to increase sharply, transfer the column outlet to a new sterile container to collect the eluted virus. Stop collecting after the UV absorbance returns to near the baseline. This is the purified virus eluate fraction. Connect a processing bag containing stripping buffer to the inlet, and transfer the end of the outlet pipe to a sterile bottle to collect the stripping fraction. Pump the buffer through the column until the UV absorbance reaches a peak and returns to near the baseline. The collected eluate is stored overnight at 4°C.

[0287] Tangential Flow Filtration

[0288] The tangential flow filtration system using a 0.1 micron pore size hollow fiber filter cartridge is prepared by assembling tubing and filter cartridge and sterilizing the system by autoclaving. Rinse the system with sterile PBS (pH 7.0), and add the virus eluate fraction to the system reservoir, and balance by recirculation. After balance, open the permeate collection pump and collect the filtrate. The system is run until the load volume is reduced to about 500mL. Use continuous constant volume diafiltration, dilute the retentate in the reservoir with DPBS (pH 7.0), and when the permeate conductivity is within 10% of the diafiltration buffer (DPBS pH 7.0), reclaim the product in the retentate.

[0289] Formulation, final filtration and packaging

[0290] The recovered retentate was adjusted to 10% final volume with sterile glycerol and mixed thoroughly before filtering through a 0.45 μm disk filter unit. The product was dispensed into labeled cryovials for storage at ≤ 65 °C.

[0291] Example 4: Analysis of ICP0- / ICP4-replication-deficient HSV viral vector.

[0292] This example demonstrates that HSV strains with ICP0 and ICP4 deletions (ICP0- / ICP4-) are more suitable for skin application. It was found that the ICP0- / ICP4- mutant was less toxic to skin cells and more suitable for delivery to the skin than other mutants (e.g., ICP4-(ICP0+)). In addition, the ICP0- / ICP4- mutant (but not the ICP4-(ICP0+)) mutant maintained the tissue specificity of a tissue-specific promoter (e.g., collagen 1 promoter).

[0293] ICP0- / ICP4- mutants provide enhanced payload expression

[0294] To evaluate the role of ICP0 in payload expression, GFP expression was measured in ICP4-(ICP0+) and ICP0- / ICP4- mutants (MOI=5). GFP expression was quantified 18 hours after infection. In ICP4-(ICP0+) mutants, GFP expression was driven by the HCMV promoter. In ICP0- / ICP4- mutants, GFP expression was driven by the ICP0 promoter. Relative to the GFP expression observed in ICP4-(ICP0+) mutants ( Figure 3A ), loss of ICP0 did not reduce GFP expression ( Figure 3B ). These results demonstrate that despite the loss of ICP0, strong gene expression is retained in the ICP0- / ICP4- mutant.

[0295] In addition, the role of ICP22 and ICP27 in gene expression was evaluated. Deletion of multiple immediate early genes, including (1) ICP0- / ICP4- / ICP22- / ICP27-, (2) ICP0- / ICP4- / ICP22-, (3) ICP0- / ICP4- / ICP27-, and (4) ICP0- / ICP22- / ICP27-, resulted in little gene expression in non-complemented cells. Whereas both ICP0- / ICP4- / ICP27- and ICP0- / ICP4- / ICP22- mutants showed poor expression, the ICP0- / ICP4- mutant (PGN 04) showed good expression ( Figure 3B ), the data suggest that both ICP22 and ICP27 can be used for strong gene expression in the ICP0- mutant and do not need to be deleted to reduce vector toxicity.

[0296] ICP0- / ICP4- mutants provide sustained expression

[0297] The persistence of gene expression was assessed by measuring GFP expression in ICP0- / ICP4-mutants (PGN04.6; MOI = 10) in five-day-old Vero cells (split) ( Figures 4A-4D ). GFP expression is driven by the ICP0 promoter. ICP0- viruses generally do not show good gene expression; however, the ICP0- / ICP4- mutant (PGN04.6) does show good gene expression (see Figures 4A-4D This retention of gene expression in the ICP0- / ICP4- mutant (PGN04.6) may be due to the retention of ICP22 and ICP27, which play a role in viral evasion of the innate immune response.

[0298] ICP0- / ICP4- mutants promote tissue-specific delivery

[0299] The ICP0- / ICP4- mutant, designed to express a payload driven by the collagen-1 promoter, was found to express the payload only in skin cells (fibroblasts), whereas no expression was observed in neurons or keratinocytes ( Fig. 6A and 6B ). This selective expression pattern makes ICP0- / ICP4- mutants very suitable for skin indications.

[0300] Data for ICP4-(ICP0+) mutants with HCMV promoter, ICP4-(ICP0+) mutants with large collagen-1 (eCOL1) promoter, ICP0- / ICP4 virus controls (with ICP0-GFP and HCMV-Red) and ICP0- / ICP4- mutants with large collagen-1 promoter (eCOL1) in cell lines of epidermal and neuronal lineages were collected. Gene expression results (in picograms / mL sTNFR (HCMV 100x)) for each mutant were measured in three different cell types: HaCaT (keratinocyte cell line), SK-N-MC (neuroblastoma cells) and Rin5F (insulinoma cells). Both SK-N-MC and Rin5F cells express neuronal markers. ICP0- mutants containing eCOL1 promoters showed no activity in keratinocytes (HaCaT) or neuronal cell types (SK-N-MC or Rin5F). However, in the presence of ICP0, the tissue specificity of the promoter was lost ( Figure 5 ). Therefore, ICP0 deficiency promotes tissue-specific expression through tissue-specific promoters.

[0301] ICP0- / ICP4- provide inducible and tissue-specific expression

[0302] Tissue-specific expression and inducibility of ICP0- / ICP4- mutants were evaluated by expressing sTNFR using a fibroblast-specific promoter (collagen 1 promoter). Expression was measured in BJ fibroblasts and NCTC 2472 cells (mouse fibroblast tumor). sTNFR expression was measured as picograms / mL sTNFR. Expression from the collagen 1 promoter (eCOL1) promoter was restricted to fibroblasts in ICP0- / ICP4- mutants and was not expressed in neurons or keratinocytes ( Fig. 6A and 6B ). Thus, the use of ICP0- / ICP4- mutants with the eCOL1 promoter protects neurons from any off-target expression. These data also show that expression from the eCOL1 promoter can be induced by TGF-β in the culture medium, providing tissue-specific and inducible expression.

[0303] Equivalent solutions

[0304] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein.The scope of the present invention is not intended to be limited to the above description, but rather is set forth in the following claims.

Claims

1. A method for expressing a polypeptide in a tissue of a subject, the method comprising administering to the subject a vector comprising a variant of a herpes simplex virus (HSV) strain, the genome of the variant containing an alteration such that the variant is unable to express functional ICP0 and ICP4 proteins.

2. The method of claim 1, wherein the HSV strain is an HSV-1 strain.

3. The method of claim 1, wherein the HSV strain is the McKrae strain.

4. The method of claim 1, wherein the variant is unable to express functional ICP4 and ICPO proteins characterized by the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 17, respectively.

5. The method of claim 1, wherein the tissue comprises epidermis, dermis or subcutaneous fat or muscle. The method of claim 1 , wherein the vector comprises a tissue-specific promoter.

7. The method of claim 6, wherein the tissue is skin. The method of claim 6 , wherein the tissue-specific promoter is a collagen 1 promoter.

9. The method of claim 1, wherein the vector comprises a human cytomegalovirus (HCMV) enhancer.

10. The method of claim 1, wherein the vector comprises a bovine growth hormone (BGH) polyadenylation signal or an HSV viral polyadenylation signal.

11. The method of claim 1, further comprising a nucleic acid encoding a therapeutic polypeptide.

12. The method of claim 1, wherein the tissue is skin tissue.

13. The method of claim 12, wherein the skin tissue comprises fibroblasts, keratinocytes, adipocytes, or muscle cells.

14. The method of claim 1, wherein the vector is administered in vivo.

15. The method of claim 1, wherein the carrier is administered by contact with the skin.

16. The method of claim 1, wherein the vector is administered by intradermal injection.

17. A method for propagating a vector comprising a variant herpes simplex virus (HSV) genome, wherein the variant HSV genome contains an alteration such that the variant is unable to express functional ICPO and ICP4 proteins, the method comprising the steps of: (a) infecting cultured ICP0 and ICP4 complementing cells containing DNA encoding HSV proteins ICP0 and ICP4 with the vector, and (b) isolating the supernatant from the culture of step (a).

18. The method of claim 17, further comprising the step of purifying the vector in the supernatant by chromatography.

19. The method of claim 18, further comprising the step of concentrating the purified vector by tangential flow filtration.

20. A method for preparing a vector comprising a variant herpes simplex virus (HSV) genome, the variant HSV genome containing an alteration such that the variant is unable to express functional ICPO and ICP4 proteins, and wherein the vector expresses a marker element, the method comprising incubating cells transfected with: (a) First Nucleic Acid Molecule: (i) contains a portion of the HSV genome but does not encode functional ICP0 and ICP4 proteins; and (ii) comprises a first homology region (HR1) and a second homology region (HR2), and (b) a second nucleic acid molecule comprising a sequence encoding a marker element, wherein the sequence is flanked by a first homology region (HR1') and a second homology region (HR2'), Wherein HR1 is homologous to HR1' and HR2 is homologous to HR2', such that the sequence encoding the marker element in the second nucleic acid molecule is integrated into the first nucleic acid molecule via homologous recombination.

21. The method of claim 20, wherein the cells are ICPO and / or ICP4 complementing cells.

22. The method of claim 20, wherein the marker element is a polypeptide.

23. The method of claim 22, wherein the polypeptide is a soluble tumor necrosis factor receptor.

24. The method of claim 22, wherein the polypeptide is quantified by enzyme-linked immunosorbent assay (ELISA).

25. The method of claim 22, wherein the polypeptide is detected by fluorescence.

26. The method of claim 20, further comprising the step of purifying viral plaques expressing the marker element.

27. A method for preparing a vector comprising a variant herpes simplex virus (HSV) genome, the variant HSV genome containing changes such that the variant is unable to express functional ICPO and ICP4 proteins, and wherein the vector expresses an agent of interest, the method comprising incubating cells transfected with: (a) First Nucleic Acid Molecule: (i) contains a portion of the HSV genome but does not encode functional ICP0 and ICP4 proteins; and (ii) comprising a sequence encoding a marker element, wherein said sequence encoding said marker element is flanked by a first homology region (HR1) and a second homology region (HR2); and (b) a second nucleic acid molecule comprising a sequence encoding an agent of interest, wherein the sequence encoding the agent of interest is flanked by a first homology region (HR1′) and a second homology region (HR2′), wherein HR1 is homologous to HR1' and HR2 is homologous to HR2', such that the sequence encoding the target agent is integrated into the first nucleic acid molecule via homologous recombination.

28. The method of claim 27, wherein the cells are ICPO and / or ICP4 complementing cells.

29. The method of claim 27, further comprising the step of purifying viral plaques that do not express the marker element.

30. The method of any one of claims 17-29, wherein the HSV genome is an HSV-1 genome.

31. The method of any one of claims 17-29, wherein the HSV genome is a McKrae strain genome.

32. A variant of a herpes simplex virus (HSV) strain, the genome of which contains an alteration such that the variant is unable to express one or more functional ICPO and ICP4 proteins.

33. A variant HSV strain as described in claim 32, wherein the HSV strain is an HSV-1 strain.

34. The variant HSV strain of claim 32, wherein the HSV strain is a McKrae strain.

35. A variant herpes simplex virus (HSV) strain comprising a variant HSV strain genome, wherein the variant HSV strain genome contains changes such that the variant is unable to express functional ICP4 and ICP0 proteins characterized by the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 17, respectively.

36. A vector comprising the variant HSV strain described in any one of claims 32-35.

37. The vector of claim 36, wherein the vector comprises a tissue-specific promoter.

38. The vector of claim 37, wherein the tissue is skin.

39. The vector of claim 37, wherein the tissue-specific promoter is a collagen 1 promoter.

40. The vector of any one of claims 36-39, wherein the vector comprises a human cytomegalovirus (HCMV) enhancer.

41. The vector of any one of claims 36-40, wherein the vector comprises a bovine growth hormone (BGH) polyadenylation signal or an HSV viral polyadenylation signal.

42. The vector of any one of claims 36-41, further comprising a nucleic acid encoding a therapeutic polypeptide.

43. A method for measuring the transduction efficiency of an HSV vector in skin tissue, the method comprising: (a) contacting the skin tissue of an animal with a carrier according to any one of claims 36 to 42; (b) removing a portion of the skin tissue from the animal; and (c) determining the amount of transduced HSV genomes in the skin tissue.

44. The method of claim 43, wherein the skin tissue comprises fibroblasts, keratinocytes, adipocytes, muscle cells, epidermis, dermis, subcutaneous tissue, or underlying subcutaneous fat or muscle.

45. The method of claim 43 or claim 44, wherein the number of genomes is measured by an amplification technique.

46. ​​The method of claim 45, wherein the amplification technique is quantitative polymerase chain reaction (qPCR).

47. A method for measuring the transduction efficiency of an HSV vector in skin tissue, the HSV vector comprising an expression cassette comprising a polypeptide payload, the method comprising: (a) contacting the skin tissue of an animal with a carrier according to any one of claims 36 to 42; (b) removing a portion of the skin tissue from the animal; and (c) determining the amount of the polypeptide encoded by the nucleic acid of the expression cassette.

48. The method of claim 47, wherein the skin tissue comprises fibroblasts, keratinocytes, adipocytes, muscle cells, epidermis, dermis, subcutaneous tissue, or underlying subcutaneous fat or muscle.

49. The method of claim 47 or claim 48, wherein the amount of the polypeptide is measured by immunoassay.

50. The method of claim 49, wherein the immunoassay is enzyme-linked immunosorbent assay (ELISA) or immunohistochemistry (IHC).

51. The method of claim 50, wherein the ELISA or IHC is performed on tissue of the epidermis, dermis, subcutaneous tissue, subcutaneous fat, underlying muscle, or draining lymph nodes.

52. A cell transduced with the vector of any one of claims 36-42.

53. A pharmaceutical composition comprising the carrier according to any one of claims 36-41 and a pharmaceutically acceptable carrier.

54. The method of any one of claims 1-31 or 43-51, the variant of any one of claims 32-34, the HSV strain of claim 35, the vector of any one of claims 36-42, the cell of claim 52, or the pharmaceutical composition of claim 53, wherein the alteration is a disruption or deletion of the ICP0 and ICP4 genes.

55. The method, variant, HSV strain, vector, cell or pharmaceutical composition of claim 54, wherein the ICPO and / or ICP4 gene is disrupted by deleting its respective promoter.

56. The method of any one of claims 1-31, 43-51, 54 or 55, the variant of any one of claims 32-34, 54 or 55, the HSV strain of any one of claims 35, 54 or 55, the vector of any one of claims 36-42, 54 or 55, the cell of any one of claims 52, 54 or 55, or the pharmaceutical composition of any one of claims 53, 54 or 55, wherein the variant is a replication-deficient variant.

Images