Viral Vectors for Cancer Therapy

Abstract

The disclosure provides recombinant nucleic acids comprising one or more polynucleotides encoding immune-modulating polypeptides (e.g., proinflammatory cytokines such as human IL-2 or IL-12 polypeptides); viruses comprising the recombinant nucleic acids; compositions and formulations comprising the recombinant nucleic acids and / or viruses; methods of their use (e.g., for the treatment of cancer, such as lung cancer); and articles of manufacture or kits thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 170,103, filed April 2, 2021, which is incorporated by reference in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CFR) (Filename 761342001540SeqList.txt, Recorded: April 1, 2022, Size: 86,233 bytes).

[0003] FIELD OF THEINVENTION The present disclosure relates, in part, to recombinant nucleic acids comprising one or more polynucleotides encoding immune modulating polypeptides, viruses comprising them, pharmaceutical compositions and formulations thereof, and methods of their use (e.g., to treat cancer, such as lung cancer). [Background technology]

[0004] background Cancer is among the leading causes of death worldwide. Despite significant advances in clinical practice and treatment methods, more effective cancer treatment options remain needed to extend survival and reduce cancer mortality.

[0005] All references cited herein, including patent applications, patent publications, non-patent literature, and NCBI / UniProtKB / Swiss-Prot accession numbers, are incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0006] overview To address these and other needs, provided herein are recombinant nucleic acids (e.g., recombinant herpesvirus genomes) encoding one or more polypeptides (e.g., one or more immune modulating polypeptides such as cytokines and chemokines) for use in viruses (e.g., herpes viruses), pharmaceutical compositions and formulations, medicaments, and / or methods useful for treating cancer in a subject in need thereof.

[0007] Thus, certain aspects of the present disclosure relate to a recombinant herpesvirus genome comprising one or more polynucleotides encoding immunomodulatory polypeptides. In some embodiments, the recombinant herpesvirus genome comprises two or more polynucleotides encoding immunomodulatory polypeptides. In some embodiments, the recombinant herpesvirus genome is replication-competent. In some embodiments, the recombinant herpesvirus genome is replication-deficient. In some embodiments that may be combined with any of the above embodiments, the recombinant herpesvirus genome comprises one or more polynucleotides encoding immunomodulatory polypeptides in one or more viral loci. In some embodiments that may be combined with any of the above embodiments, the recombinant herpesvirus genome is selected from a recombinant herpes simplex virus genome, a recombinant varicella zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, an Epstein-Barr virus genome, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any combination or derivative thereof.

[0008] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes virus genome is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any combination or derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome.

[0009] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome is engineered to reduce or eliminate expression of one or more herpes simplex virus genes (e.g., one or more toxic herpes simplex virus genes). In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation. In some embodiments, the inactivating mutation is present in a herpes simplex virus gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of a herpes simplex virus gene. In some embodiments, the herpes simplex virus gene is selected from infected cell protein (ICP)0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL)41, and UL55. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP4 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP0 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP47 gene. In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in one or both copies of the ICP34.5 gene.In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome does not contain an inactivating mutation in the UL36 gene.

[0010] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide in one or more viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide in one or both ICP4 viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide in the ICP22 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide in the UL41 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide in one or both ICP0 viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide in the ICP27 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome includes one or more polynucleotides encoding an immunomodulatory polypeptide within the UL55 viral locus.

[0011] In some aspects that may be combined with any of the preceding aspects, the immunomodulatory polypeptide is a human immunomodulatory polypeptide. In some aspects that may be combined with any of the preceding aspects, the immunomodulatory polypeptide is a secreted immunomodulatory polypeptide. In some aspects that may be combined with any of the preceding aspects, the immunomodulatory polypeptide is a cytokine or a chemokine. In some aspects that may be combined with any of the preceding aspects, the cytokine is a proinflammatory cytokine. In some embodiments that may be combined with any of the preceding embodiments, the cytokine is selected from interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-28 (IL-28), interleukin-32 (IL-32), interleukin-33 (IL-33), interleukin-34 (IL-34), tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), granulocyte colony stimulating factor (G-CSF), and granulocyte macrophage colony stimulating factor (GM-CSF). In some embodiments that may be combined with any of the preceding embodiments, the cytokine is IL-2. In some embodiments that may be combined with any of the preceding embodiments, the cytokine is IL-12. In some embodiments that may be combined with any of the preceding embodiments, the cytokine is not GM-CSF. In some embodiments that may be combined with any of the preceding embodiments, the chemokine is a proinflammatory chemokine.In some embodiments that may be combined with any of the preceding embodiments, the chemokine is selected from chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 2 (CSCL2), chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-X-C motif) ligand 9 (CXCL9), chemokine (C-X-C motif) ligand 11 (CXCL11), chemokine (C-X-C motif) ligand 16 (CXCL16), CC-motif chemokine ligand 2 (CCL2), CC-motif chemokine ligand 3 (CCL3), CC-motif chemokine ligand 4 (CCL4), CC-motif chemokine ligand 5 (CCL5), and CC-motif chemokine ligand 11 (CCL11). In some embodiments that may be combined with any of the preceding embodiments, the immunomodulatory polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-30. In some embodiments, the immunomodulatory polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-19. In some embodiments, the immune modulatory polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs:20-30.In some embodiments, the immunomodulatory polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the immunomodulatory polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 5-6.

[0012] In some aspects that may be combined with any of the preceding aspects, the recombinant herpesvirus genome has reduced cytotoxicity when introduced into a target cell compared to a corresponding wild-type herpesvirus genome. In some aspects, the target cell is a human cell. In some aspects, the target cell is a respiratory cell. In some aspects, the target cell is an airway epithelial cell.

[0013] Other aspects of the present disclosure relate to a herpesvirus comprising any of the recombinant herpesvirus genomes described herein. In some aspects, the herpesvirus is replication-competent. In some aspects, the herpesvirus is replication-deficient. In some aspects that may be combined with any of the above aspects, the herpesvirus is not oncolytic. In some aspects that may be combined with any of the above aspects, the herpesvirus is a pseudotyped virus. In some aspects that may be combined with any of the above aspects, the herpesvirus is not a pseudotyped virus. In some aspects that may be combined with any of the above aspects, the herpesvirus is not a pseudotyped oncolytic virus. In some aspects that may be combined with any of the above aspects, the herpesvirus has reduced cytotoxicity compared to the corresponding wild-type herpesvirus. In some embodiments that may be combined with any of the above embodiments, the herpes virus is selected from herpes simplex virus, varicella zoster virus, human cytomegalovirus, herpes virus 6A, herpes virus 6B, herpes virus 7, Epstein-Barr virus, Kaposi's sarcoma-associated herpes virus, and any combination or derivative thereof. In some embodiments that may be combined with any of the above embodiments, the herpes virus is a herpes simplex virus. In some embodiments, the herpes simplex virus is not oncolytic. In some embodiments, the herpes simplex virus is HSV-1, HSV-2, or any combination or derivative thereof. In some embodiments, the herpes simplex virus is HSV-1. In some embodiments, the HSV-1 is not oncolytic.

[0014] Another aspect of the present disclosure relates to a pharmaceutical composition comprising any of the recombinant herpesvirus genomes and / or any of the recombinant herpesviruses described herein and a pharma- ceutically acceptable carrier or excipient. In some embodiments that may be combined with any of the above-mentioned embodiments, the pharmaceutical composition is suitable for topical, transdermal, subcutaneous, intradermal, oral, intranasal, intratracheal, sublingual, oral, rectal, intravaginal, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, topical, or epicutaneous administration. In some embodiments that may be combined with any of the above-mentioned embodiments, the pharmaceutical composition is suitable for oral, intranasal, intratracheal, or inhalation administration. In some embodiments, the pharmaceutical composition is suitable for intranasal or inhalation administration. In some embodiments, the pharmaceutical composition is suitable for inhalation administration. In some embodiments that may be combined with any of the preceding aspects, the pharmaceutical composition is suitable for use in a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, an electrohydrodynamic aerosol device, or any combination thereof. In some embodiments that may be combined with any of the preceding aspects, the pharmaceutical composition is suitable for use in a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer. In some embodiments that may be combined with any of the preceding aspects, the pharmaceutical composition comprises a phosphate buffer. In some embodiments that may be combined with any of the preceding aspects, the pharmaceutical composition comprises glycerol. In some embodiments that may be combined with any of the preceding aspects, the pharmaceutical composition comprises a lipid carrier. In some embodiments that may be combined with any of the preceding aspects, the pharmaceutical composition comprises a nanoparticle carrier.

[0015] Other aspects of the present disclosure relate to the use of any of the recombinant nucleic acids (e.g., recombinant herpes virus genomes), recombinant viruses (e.g., recombinant herpes viruses), and / or pharmaceutical compositions described herein as pharmaceuticals.

[0016] Other aspects of the present disclosure relate to the use of any of the recombinant nucleic acids (e.g., recombinant herpes virus genomes), recombinant viruses (e.g., recombinant herpes viruses), and / or pharmaceutical compositions described herein in therapeutic methods.

[0017] Other aspects of the present disclosure relate to the use of any of the recombinant nucleic acids (e.g., recombinant herpes virus genomes), recombinant viruses (e.g., recombinant herpes viruses), and / or pharmaceutical compositions described herein in the preparation of a medicament for treating cancer (e.g., lung cancer).

[0018] Another aspect of the present disclosure relates to a method of expressing, enhancing, increasing, enhancing, and / or replenishing the level of an immunomodulatory polypeptide in one or more cells of a subject, comprising administering to the subject an effective amount of any of the recombinant herpesviruses and / or pharmaceutical compositions described herein. In some embodiments, the one or more cells are one or more cells of the respiratory tract, airway epithelium, and / or lung. In some embodiments that may be combined with any of the above embodiments, the subject is a human. In some embodiments that may be combined with any of the above embodiments, the herpesvirus or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, bucally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, intratumorally, topically, or via inhalation. In some embodiments that may be combined with any of the above embodiments, the herpesvirus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the herpes virus or pharmaceutical composition is administered to the subject intranasally or via inhalation. In some embodiments, the herpes virus or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the herpes virus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpes virus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0019] Another aspect of the present disclosure relates to a method of providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of cancer in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant herpesviruses and / or pharmaceutical compositions described herein. In some embodiments, the cancer is selected from solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, renal cancer, salivary gland cancer, skin cancer, stomach cancer, thymic epithelial cancer, and thyroid cancer. In some embodiments, the cancer is small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is osteosarcoma. In some embodiments that may be combined with any of the above embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, bucally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, intratumorally, topically, or via inhalation. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the herpes virus or pharmaceutical composition is administered to the subject intranasally, or via inhalation. In some embodiments, the herpes virus or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the herpes virus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpes virus or pharmaceutical composition is administered using a nebulizer.In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0020] Another aspect of the present disclosure relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant herpesviruses and / or pharmaceutical compositions described herein. In some embodiments, the cancer is selected from carcinoma, lymphoma, blastoma, sarcoma, neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, leukemia, and lymphoid malignancies. In some embodiments, the cancer is selected from solid tumors, hematological cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, renal cancer, salivary gland cancer, skin cancer, stomach cancer, thymic epithelial cancer, and thyroid cancer. In some embodiments, the cancer is small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is osteosarcoma. In some embodiments that may be combined with any of the above embodiments, the subject is a human. In some embodiments that may be combined with any of the above embodiments, the herpes virus or pharmaceutical composition is administered to the subject topically, percutaneously, subcutaneously, epicutaneously, intradermally, orally, sublingually, bucally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, intratumorally, topically, or via inhalation. In some embodiments that may be combined with any of the above embodiments, the herpes virus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the herpes virus or pharmaceutical composition is administered to the subject intranasally or via inhalation. In some embodiments, the herpes virus or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the herpes virus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpes virus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0021] Another aspect of the present disclosure relates to a method of delivering a polypeptide (e.g., an immunomodulatory polypeptide) to one or more cells of the respiratory tract of a subject, comprising administering to the subject a pharmaceutical composition comprising (a) a herpesvirus comprising a recombinant herpesvirus genome, the recombinant herpesvirus genome comprising one or more polynucleotides encoding a polypeptide, and (b) a pharmaceutically acceptable carrier. In some embodiments, the subject suffers from a disease or condition affecting one or more cells of the respiratory tract, such as a cancer or neoplasm affecting the airways and / or lungs, originating from the respiratory tract or metastasizing from other tissues or organs. In some embodiments, the subject does not suffer from a genetic lung disease. In some embodiments, the subject does not suffer from a disease selected from alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and pulmonary fibrosis. In some embodiments, the lack or reduced level of expression and / or activity of the polypeptide in the subject is not associated with a genetic lung disease. In some embodiments, the lack or reduced level of expression and / or activity of the polypeptide in the subject is not associated with a disease selected from alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and pulmonary fibrosis.

[0022] In some embodiments that may be combined with any of the preceding embodiments, the herpes virus is a pseudotyped virus. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus is not a pseudotyped virus. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus is not a pseudotyped oncolytic virus.

[0023] Other aspects of the disclosure relate to articles of manufacture or kits comprising any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein and instructions for their administration. [Brief description of the drawings]

[0024] [Figure 1-1] Schematic diagrams of wild-type and modified herpes simplex virus genomes are shown in Figure 1A. Figure 1B shows a modified herpes simplex virus genome that includes a deletion of the coding sequence of ICP4 (both copies), where an expression cassette contains a nucleic acid encoding an immunomodulatory polypeptide integrated into each of the ICP4 loci. [Figure 1-2] Schematic diagrams of wild-type and modified herpes simplex virus genomes are shown. Figure 1C shows a modified herpes simplex virus genome comprising a deletion of the coding sequence of ICP4 (both copies) and UL41, wherein the expression cassette contains a nucleic acid encoding an immunomodulatory polypeptide integrated into each of the ICP4 loci. Figure 1D shows a modified herpes simplex virus genome comprising a deletion of the coding sequence of ICP4 (both copies) and UL41, wherein the expression cassette contains a nucleic acid encoding an immunomodulatory polypeptide integrated into the UL41 locus. [Figure 1-3] Schematic diagrams of wild-type and modified herpes simplex virus genomes are shown. Figure 1E shows a modified herpes simplex virus genome comprising a deletion of the coding sequence of ICP4 (both copies) and ICP22, wherein the expression cassette contains a nucleic acid encoding an immunomodulatory polypeptide integrated into each of the ICP4 loci. Figure 1F shows a modified herpes simplex virus genome comprising a deletion of the coding sequence of ICP4 (both copies) and ICP22, wherein the expression cassette contains a nucleic acid encoding an immunomodulatory polypeptide integrated into the ICP22 locus. [Figure 1-4]Schematic diagrams of wild-type and modified herpes simplex virus genomes are shown. Figure 1G shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies), UL41 and ICP22, wherein the expression cassettes contain nucleic acids encoding immunomodulatory polypeptides integrated into the ICP4 locus, respectively. Figure 1H shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies), UL41 and ICP22, wherein the expression cassettes contain nucleic acids encoding immunomodulatory polypeptides integrated into the UL41 locus, respectively. [Figure 1-5] Schematic diagrams of wild-type and modified herpes simplex virus genomes are shown. Figure 1I shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP4 (both copies), UL41 and ICP22, where the expression cassette contains a nucleic acid encoding an immunomodulatory polypeptide integrated into the ICP22 locus. [Diagram 2] Western blot detection of human IL-12 and IL-2 in uninfected control cells (mock) or cells infected with modified herpes simplex viruses encoding human IL-12 (Figure 2A) or IL-2 (Figure 2B) transgenes at a multiplicity of infection (MOI) of 1. Recombinant human IL-12 (Figure 2A) or IL-2 (Figure 2B) were used as positive controls. Figure 2A: Lane 1 ladder; lane 2 recombinant IL-12; lanes 3-6 HSV-IL12; lane 7 mock. Figure 2B: Lane 1 ladder; lane 2 recombinant IL-2; lanes 3-5 HSV-IL2; lane 6 mock. [Diagram 3] In vitro bioactivity assays of human (Figure 3A) and mouse (Figure 3B) HSV-IL12 are shown as a function of IFNγ release from human PBMCs (Figure 3A) or mouse splenocytes (Figure 3B). Data represent cells assayed in triplicate and are presented as mean ± standard deviation (SD), n=1. [Figure 4-1]In vivo evaluation of HSV-IL12 in healthy mice. Figure 4A shows animal weights after intratracheal inoculation of HSV-IL12. Data are presented as mean ± standard error of the mean (SEM). n = 2-3 animals per group for each time point. Figures 4B-4C show genome (Figure 4B) and transcript (Figure 4C) levels in the lungs of BALB / c mice after intratracheal inoculation of HSV-IL12. qPCR (Figure 4B) and qRT-PCR (Figure 4C) were performed to measure il12 genome and transcript, respectively. Data show samples performed in duplicate and are displayed as mean ± SEM of n = 2-3 animals per group. Figures 4D-4E show IL-12 protein concentrations in serum (Figure 4D) and bronchoalveolar lavage fluid (BALF; Figure 4E) analyzed by ELISA. Serum samples are shown as mean ± SD of one animal sample assayed in duplicate. BALF data are presented as mean ± SEM for n = 2-3 animals per group. Figure 4F shows IL-12 concentrations in lung homogenates after intratracheal challenge with HSV-IL12. Biopsies were cut from frozen lung tissue, homogenized, and assayed in duplicate by ELISA. Protein concentrations of homogenates were measured by BCA assay, and IL-12 concentrations were normalized to total protein. Data are presented as mean ± SEM for n = 2-3 animals per group. Figure 4G shows animal weights after weekly intratracheal challenge with HSV-IL12. Animals were weighed at the indicated time points before HSV-IL12 challenge. Data are presented as mean ± SEM for n = 4-6 animals per group. Figures 4H-4I show il12 genome (Figure 4H) and transcript (Figure 4I) levels in lungs of BALB / c mice after three consecutive weeks of weekly treatment. Data represent samples performed in duplicate. Values ​​from individual animals are shown with the mean ± SEM of n = 2-3 animals per group. Figures 4J-4K show IL-12 protein concentrations in BALF and lung homogenates. BALF (Figure 4J) and lung homogenates (Figure 4K) were analyzed by ELISA for mIL-12 protein concentration. All samples were assayed in duplicate.Values ​​from individual animals are shown along with the mean ± SEM of n = 2-3 animals per group. Figures 4L-4M show analysis of BALF cells as a measure of inflammation. Biopsies were dissected from frozen lung tissue, homogenized, and assayed in duplicate by ELISA. Data are presented as the mean ± SEM of n = 2-3 animals per group. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 4-4] See description of Figure 4-1. [Figure 4-5] See description of Figure 4-1. [Figure 5-1]In vivo evaluation of HSV-IL2 in healthy mice. Figure 5A shows animal weights after intratracheal challenge with HSV-IL2. Data are presented as mean ± SEM. n = 2-3 animals per group for each time point. Figures 5B-5C show il2 genomic (Figure 5B) and transcript (Figure 5C) levels in the lungs of BALB / c animals after intratracheal challenge with HSV-IL2. Data are presented as mean ± SEM for n = 2-3 animals per group, showing samples performed in duplicate. Figures 5D-5E show IL-2 protein concentrations in serum (Figure 5D) and bronchoalveolar lavage fluid (BALF; Figure 5E) analyzed by ELISA. Serum samples are shown as mean ± SEM for 2-3 animals per group when samples were assayed in duplicate. BALF data are shown as mean ± SEM for n = 2-3 animals per group. Figure 5F shows IL-2 concentrations in lung homogenates following intratracheal challenge with HSV-IL2. Biopsies were cut from frozen lung tissue, homogenized, and assayed in duplicate by ELISA. Protein concentrations of homogenates were measured by BCA assay, and IL-2 concentrations were normalized to total protein. Data are presented as mean ± SEM of n = 2–3 animals per group. Figures 5G–5I show IL-2 pharmacokinetics in serum (Figure 5G), bronchoalveolar lavage fluid (BALF; Figure 5H), and lysates (Figure 5I). Data are presented as mean ± SEM of n = 2–3 animals per group. [Figure 5-2] See description of Figure 5-1. [Figure 5-3] See description of Figure 5-1. [Figure 6-1]In vivo evaluation of HSV-GMCSF in healthy mice. Figure 6A shows animal weights after intratracheal administration of HSV-GMCSF. Data are presented as mean ± SEM. n = 2-3 animals per group for each time point. Figures 6B-6C show gmcsf genome (Figure 6B) and transcript (Figure 6C) levels in the lungs of BALB / c animals after intratracheal administration of HSV-GMCSF. qPCR (Figure 6B) and qRT-PCR (Figure 6C) were performed to measure gmcsf genome and transcript, respectively. Data represent samples performed in duplicate and are presented as mean ± SEM of n = 2-3 animals per group. Figures 6D-6E show GM-CSF protein concentrations in serum (Figure 6D) and bronchoalveolar lavage fluid (BALF; Figure 6E) analyzed by ELISA. Serum and BALF data are presented as mean ± SEM of n = 2-3 animals per group. Figure 6F shows GM-CSF concentrations in lung homogenates after intratracheal administration of HSV-GMCSF. Figure 6G shows animal weights after weekly intratracheal administration of HSV-GMCSF. Animals were weighed at the indicated time points before HSV-GMCSF administration. Data are presented as mean ± SEM. n = 2-3 animals per group. Figures 6H-6I show gmcsf genomic (Figure 6H) and transcript (Figure 6I) levels in lungs of BALB / c animals after three consecutive weeks of weekly treatment. Data represent samples performed in duplicate. Values ​​from individual animals are displayed with mean ± SEM for n = 2-3 animals per group. Figures 6J-6L show GMCSF protein concentrations in serum (Figure 6J), BALF (Figure 6K) and lung homogenates (Figure 6L). All samples were assayed in duplicate. Values ​​from individual animals are displayed with mean ± SEM for n = 2-3 animals per group. Figures 6M-6N show the analysis of BALF cells as a measure of inflammation. Cells isolated from BALF were counted by use of a hemocytometer and viability was determined based on the trypan blue dye exclusion method. Data are presented as the mean ± SEM of n = 2-3 animals per group. [Figure 6-2] See description of Figure 6-1. [Figure 6-3] See description of Figure 6-1. [Figure 6-4] See description of Figure 6-1. [Figure 6-5] See description of Figure 6-1. [Figure 7-1] Establishment of an in vivo osteosarcoma lung metastasis model in immunocompetent mice. Figure 7A shows the body weight of control (vehicle) vs. K7M2 (mouse tumor cell line) injected mice. Data are presented as mean ± SEM. n = 2-5 animals per group for each time point. Figures 7B-7C show lung weight of control vs. tumor cell line exposed mice at 3 weeks (Figure 7B) or 6 weeks (Figure 7C) after inoculation of K7M2 cells. Values ​​from individual animals are shown along with the mean ± SEM of n = 2-5 animals per group. Figures 7D-7F show hematoxylin and eosin (H&E) histological staining of lung sections at 6 weeks after inoculation of K7M2 cells. Magnifications are 2.5x (Figure 7D), 20x (Figure 7E), 10x (Figure 7F) and 20x (Figure 7G). [Figure 7-2] See description of Figure 7-1. [Figure 8] Figure 8 shows HSV-IL12 efficacy in an in vivo mouse model of osteosarcoma. Figure 8A shows body weights of control vs. HSV-IL12 treated mice. Data are presented as mean ± SEM. n = 5 animals per group for each time point. Figure 8B shows lung weights of control vs. HSV-IL12 treated mice. Values ​​from individual animals are shown with mean ± SEM. n = 3-5 animals per group. [Figure 9-1]Figure 9 shows in vivo evaluation of weekly combinatorial HSV-IL12 / GMCSF intratracheal administration in healthy mice. Figure 9A shows animal weights after weekly combinatorial HSV-IL12 / GMCSF intratracheal administration. Animals were weighed before HSV-IL12 / GMCSF administration, on days 0 and 7, and at sacrifice on day 8. Data are presented as mean ± SEM. n = 3-4 animals per group. Figures 9B-9E show IL-12 and GMCSF protein concentrations in BALF and lung homogenates. BALF (Figures 9B-9C) and lung homogenates (Figures 9D-9E) were analyzed by ELISA for mIL-12 and mGM-CSF protein concentrations. All samples were assayed in duplicate. Values ​​from individual animals are shown along with the mean ± SEM for n = 3-4 animals per group. Figure 9F shows analysis of BALF cells as a measure of inflammation. Cells isolated from BALF were counted by use of a hemocytometer and viability was determined based on the trypan blue dye exclusion method. Data are presented as mean ± SEM for n = 3–4 animals per group. [Figure 9-2] See description of Figure 9-1. [Figure 10] Figure 10 shows combinatorial HSV-IL12 and HSV-GMCSF efficacy in an in vivo mouse model of osteosarcoma. Figure 10A shows the body weights of control vs. HSV-IL12 only, HSV-GMCSF only, and combinatorial HSV-IL12 and HSV-GMCSF administered mice. Data are presented as mean ± SEM. n = 2-5 animals per group for each time point. Figure 10B shows survival curves of control vs. HSV-IL12 only, HSV-GMCSF only, and combinatorial HSV-IL12 and HSV-GMCSF administered mice. Data are presented as mean ± SEM. n = 5 animals per group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description The following description sets forth example methods, parameters, etc. However, it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of example embodiments.

[0026] I. General techniques The techniques and procedures described or referenced herein are generally well understood and may be readily adapted to practice conventional methodology by those of skill in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); and Short Protocols in Molecular Biology (Wiley and Sons, 1999) are commonly employed.

[0027] II. Definition Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0028] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "molecule" optionally includes combinations of two or more such molecules, and so forth.

[0029] As used herein, the term "and / or" may include any and all combinations of one or more of the associated listed items. For example, the term "a and / or b" may refer to "a only," "b only," "a or b," or "a and b;" the term "a, b, and / or c" may refer to "a only," "b only," "c only," "a or b," "a or c," "b or c," "a, b, or c," "a and b," "a and c," "b and c," or "a, b, and c," etc.

[0030] As used herein, the term "about" refers to the normal error range for the respective value, which is readily known to one of ordinary skill in the art. Reference to a value or parameter herein includes (and describes) the embodiment directed to that value or parameter itself.

[0031] It is understood that aspects and embodiments of the present disclosure include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0032] As used herein, the terms "polynucleotide", "nucleic acid sequence", "nucleic acid" and variations thereof are intended to refer collectively to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and other polymers containing a non-nucleotide backbone, provided that the polymer contains nucleobases in a configuration that allows for base pairing and base stacking as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, such as the replacement of one or more naturally occurring nucleotides with analogs, and internucleotide modifications.

[0033] As used herein, a nucleic acid is "operatively linked" or "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" or "operably linked" means that the DNA or RNA sequences being linked are contiguous.

[0034] As used herein, the term "vector" refers to a separate element used to introduce heterologous nucleic acid into cells for either its expression or replication. Expression vectors include vectors capable of expressing such nucleic acid that are operably linked with a regulatory sequence, such as a promoter region, capable of causing the expression of the nucleic acid. Thus, an expression vector can refer to a DNA or RNA construct, such as a plasmid, a phage, a recombinant virus, or other vector, that results in the expression of a nucleic acid when introduced into a suitable host cell. Suitable expression vectors are well known to those skilled in the art and include those that can replicate in eukaryotic cells and those that remain episomal or are integrated into the host cell genome.

[0035] As used herein, "open reading frame" or "ORF" refers to a continuous stretch of nucleic acid, either DNA or RNA, that encodes a protein or polypeptide. Typically, the nucleic acid includes a translation initiation signal or start codon, such as ATG or AUG, and a stop codon.

[0036] As used herein, "untranslated region" or "UTR" refers to the untranslated nucleic acid at the 5' and / or 3' end of an open reading frame. Inclusion of one or more UTRs in a polynucleotide can affect post-transcriptional regulation, mRNA stability, and / or translation of the polynucleotide.

[0037] As used herein, the term "transgene" refers to a polynucleotide that, once introduced into a cell, can be transcribed into RNA, translated and / or expressed under appropriate conditions, which in some aspects confers a desired property to the cell into which it is introduced, or otherwise leads to a desired therapeutic or diagnostic outcome.

[0038] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably and may refer to a polymer of two or more amino acids.

[0039] As used herein, "subject," "host," or "individual" refers to any animal classified as a mammal, including humans, domestic and farm animals, and exhibition, sport, or pet animals, such as dogs, horses, cats, cows, as well as animals used in research, such as mice, rats, hamsters, rabbits, and non-human primates. In some embodiments, the mammal is a human.

[0040] As used herein, the term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptable and toxic to the subject to which the composition or formulation is administered. A "pharmaceutical acceptable" excipient (e.g., vehicle, additive) is one that can reasonably be administered to a mammalian subject to provide an effective dosage of the active ingredient employed.

[0041] As used herein, an "effective amount" is at least the minimum amount necessary to affect a measurable improvement or prevention of one or more symptoms of a particular disorder. An "effective amount" may vary depending on factors such as the patient's condition, age, sex, and weight. An effective amount is also an amount in which any toxic or adverse effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of, reducing the severity of, or delaying the onset of, the disease, its complications, and intermediate pathological phenotypes manifested during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms resulting from the disease, enhancing the quality of life of people suffering from the disease, reducing the dose of other medications used to treat the symptoms of the disease, slowing the progression of the disease, and / or extending survival. An effective amount can be administered in one or more administrations. For purposes of this disclosure, an effective amount of a recombinant nucleic acid, virus, and / or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As will be understood in a clinical context, an effective amount of a recombinant nucleic acid, virus, and / or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if a desired result can or is achieved in conjunction with one or more other agents.

[0042] As used herein, "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. The desired effects of treatment include reducing the rate of progression of the disease / disorder / defect, reversing or alleviating the condition of the disease / disorder / defect, and remission or amelioration of progression.

[0043] As used herein, the term "delaying the progression" of a disease / disorder / defect refers to postponing, preventing, slowing, retarding, stabilizing, and / or postponing the onset of the disease / disorder / defect. This delay can be of varying length or time depending on the history of the disease / disorder / defect and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.

[0044] Throughout this disclosure, various aspects are presented in the form of a range. It should be understood that the description in the form of a range is merely for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered as specifically disclosing all possible subranges and individual numerical values ​​within that range. For example, when a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value in the stated range, is included within the disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also included within the disclosure, subject to any specifically excluded limit value in the stated range. When a stated range includes one or both limit values, ranges excluding one or both of those included limits are also included in the disclosure. This is true regardless of the breadth of the range.

[0045] III. Recombinant Nucleic Acids Certain aspects of the present disclosure relate to recombinant nucleic acids (e.g., isolated recombinant nucleic acids) that include one or more (e.g., one or more, two or more, three or more, four or more, five or more, ten or more, etc.) polynucleotides encoding immune regulating polypeptides. In some embodiments, the recombinant nucleic acid includes one polynucleotide encoding an immune regulating polypeptide. In some embodiments, the recombinant nucleic acid includes two polynucleotides encoding immune regulating polypeptides. In some embodiments, the recombinant nucleic acid includes three or more polynucleotides encoding immune regulating polypeptides. In some embodiments, the recombinant nucleic acid includes one or more polynucleotides encoding two or more immune regulating polypeptides. In some embodiments, the recombinant nucleic acid includes two or more polynucleotides encoding two or more immune regulating polypeptides. In some embodiments, the two or more immune regulating polypeptides are identical. In some embodiments, the two or more immune regulating polypeptides are different.

[0046] In some embodiments, the present disclosure relates to a recombinant nucleic acid comprising a polynucleotide encoding a chimeric polypeptide comprising a first immunomodulatory polypeptide, a linker polypeptide, and a second immunomodulatory polypeptide. In some embodiments, the first and second immunomodulatory polypeptides are the same. In some embodiments, the first and second immunomodulatory polypeptides are different. In some embodiments, the linker polypeptide is a cleavable linker polypeptide. In some embodiments, the linker polypeptide is a non-cleavable linker polypeptide.

[0047] In some embodiments, the recombinant nucleic acid is a vector. In some embodiments, the recombinant nucleic acid is a viral vector. In some embodiments, the recombinant nucleic acid is a herpes virus vector. In some embodiments, the recombinant nucleic acid is a herpes simplex virus amplicon. In some embodiments, the recombinant nucleic acid is a recombinant herpes virus genome. In some embodiments, the recombinant herpes virus genome is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome.

[0048] Polynucleotides encoding immunomodulatory polypeptides In some aspects, the present disclosure relates to recombinant nucleic acids comprising one or more polynucleotides encoding one or more immune modulating polypeptides (e.g., one or more human immune modulating polypeptides). Any suitable immune modulating polypeptide described herein or known in the art, including, for example, human cytokines and chemokines, e.g., human proinflammatory cytokines and chemokines, can be encoded by a polynucleotide of the present disclosure.

[0049] In some aspects, the polynucleotides of the present disclosure comprise the wild-type coding sequence of any immune-modulating gene (including any isoforms thereof) described herein or known in the art. In some aspects, the polynucleotides of the present disclosure comprise the codon-optimized variant of the wild-type coding sequence of any immune-modulating gene described herein or known in the art. In some aspects, the use of the codon-optimized variant of the coding sequence of a gene increases the stability and / or yield of the heterologous expression (RNA and / or protein) of the encoded polypeptide in a target cell compared to the stability and / or yield of the heterologous expression of the corresponding non-codon-optimized wild-type sequence. Any suitable method known in the art can be used to codon-optimize a sequence for expression in one or more target cells (e.g., one or more human cells), for example, by the method described by Fath et al. (PLoS One. 2011 Mar 3;6(3): e17596).

[0050] In some aspects, the present disclosure relates to recombinant nucleic acids comprising one or more polynucleotides comprising a coding sequence for a human cytokine. For example, the IL1A gene (see, e.g., NCBI Gene ID:3552; SEQ ID NO:31), the IL1B gene (see, e.g., NCBI Gene ID:3553; SEQ ID NO:32), the IL2 gene (see, e.g., NCBI Gene ID:3558; SEQ ID NO:33), the IL3 gene (see, e.g., NCBI Gene ID:3562), the IL4 gene (see, e.g., NCBI Gene ID:3565), the IL5 gene (see, e.g., NCBI Gene ID:3567), the IL6 gene (see, e.g., NCBI Gene ID:3569), the IL7 gene (see, e.g., NCBI Gene ID:3574; SEQ ID NO:34), the IL9 gene (see, e.g., NCBI Gene ID:3578), and the IL10 gene (see, e.g., NCBI Gene ID:3579). ID:3586), IL11 gene (see, e.g., NCBI Gene ID:3589), IL12A gene (see, e.g., NCBI Gene ID:3592; SEQ ID NO:35), IL12B gene (see, e.g., NCBI Gene ID:3593; SEQ ID NO:36), IL13 gene (see, e.g., NCBI Gene ID:3596; SEQ ID NO:317), IL15 gene (see, e.g., NCBI Gene ID:3600; SEQ ID NO:38), IL17A gene (see, e.g., NCBI Gene ID:3605;SEQ ID NO:39), IL17B gene (see, e.g., NCBI Gene ID:27190), IL17C gene (see, e.g., NCBI Gene ID:27189), IL17D gene (see, e.g., NCBI Gene ID:53342), IL25 gene (see, e.g., NCBI Gene ID:64806), IL17F gene (see, e.g., NCBI Gene ID:112744), IL18 gene (see, e.g., NCBI Gene ID:3606; SEQ ID NO:40), IFNL2 gene (see, e.g., NCBI Gene ID:282616; SEQ ID NO:41), IFNL3 gene (see, e.g., NCBI Gene ID:282617; SEQ ID NO:42), IFNL1 gene (see, e.g., NCBI Gene ID:282618; SEQ ID NO:43), IFNL2 gene (see, e.g., NCBI Gene ID:282619; SEQ ID NO:44), IFNL3 gene (see, e.g., NCBI Gene ID:282618; SEQ ID NO:45), IFNL4 gene (see, e.g., NCBI Gene ID:282619; SEQ ID NO:46), IFNL5 gene (see, e.g., NCBI Gene ID:282620; SEQ ID NO:47), IFNL6 gene (see, e.g., NCBI Gene ID:282621; SEQ ID NO:48), IFNL7 gene (see, e.g., NCBI Gene ID:282622; SEQ ID NO:49), IFNL8 gene (see, e.g., NCBI Gene ID:282623; SEQ ID NO:49), ID:282618), IL32 gene (see, e.g., NCBI Gene ID:9235; SEQ ID NO:43), IL33 gene (see, e.g., NCBI Gene ID:90865; SEQ ID NO:44), IL34 gene (see, e.g., NCBI Gene ID:146433;SEQ ID NO:45), IL36A gene (see, e.g., NCBI Gene ID:27179), IL36B gene (see, e.g., NCBI Gene ID:27177), IL36G gene (see, e.g., NCBI Gene ID:56300), IFNA1 gene (see, e.g., NCBI Gene ID:3439), IFNA13 gene (see, e.g., NCBI Gene ID:3447), IFNA2 gene (see, e.g., NCBI Gene ID:3440), IFNA4 gene (see, e.g., NCBI Gene ID:3441), IFNA5 gene (see, e.g., NCBI Gene ID:3442), IFNA6 gene (see, e.g., NCBI Gene ID:3443), IFNA7 gene (see, e.g., NCBI Gene ID:3444), IFNA8 gene (see, e.g., NCBI Gene ID:3445), IFNA9 gene (see, e.g., NCBI Gene ID:3446), IFNA10 gene (see, e.g., NCBI Gene ID:3447), IFNA11 gene (see, e.g., NCBI Gene ID:3448), IFNA12 gene (see, e.g., NCBI Gene ID:3449), IFNA13 gene (see, e.g., NCBI Gene ID:3450), IFNA14 gene (see, e.g., NCBI Gene ID:3451), IFNA15 gene (see, e.g., NCBI Gene ID:3452), IFNA16 gene (see, e.g., NCBI Gene ID:3453), IFNA17 gene (see, e.g., NCBI Gene ID:3454), IFNA18 gene (see ID:3445), IFNA10 gene (see, e.g., NCBI Gene ID:3446), IFNA14 gene (see, e.g., NCBI Gene ID:3448), IFNA16 gene (see, e.g., NCBI Gene ID:3449), IFNA17 gene (see, e.g., NCBI Gene ID:3451), IFNA21 gene (see, e.g., NCBI Gene ID:3452), IFNB1 gene (see, e.g., NCBI Gene ID:3456), IFNB3 gene (see, e.g., NCBI Gene ID:618946), IFNG gene (see, e.g., NCBI Gene ID:3458; SEQ ID NO:46), TNF gene (see, e.g., NCBI Gene ID:7124; SEQ ID NO:47), LTA gene (see, e.g., NCBI Gene ID:3459; SEQ ID NO:48), TNF gene (see, e.g., NCBI Gene ID:3456; SEQ ID NO:48), TNF gene (see, e.g., NCBI Gene ID:3459; SEQ ID NO:49 ... ID:4049), CSF3 gene (e.g., NCBI Gene ID:1440; SEQ ID NO:48), CSF2 gene (e.g., NCBI Gene ID:1437;Any suitable human cytokine gene known in the art (including any isoforms thereof) may be encoded by the polynucleotides of the present disclosure, including the CSF1 gene (see, e.g., SEQ ID NO:49), the CSF1 gene (see, e.g., NCBI Gene ID:1435), and the like. In some embodiments, the polynucleotides of the present disclosure do not include sequences encoding the IL4 gene. In some embodiments, the polynucleotides of the present disclosure do not include sequences encoding the IL10 gene. In some embodiments, the polynucleotides of the present disclosure do not include sequences encoding the GM-CSF gene. In some embodiments, the polynucleotides of the present disclosure do not include sequences encoding the IL4, IL10, and / or GM-CSF genes. In some embodiments, the polynucleotides of the present disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any of the human cytokine genes (and / or their coding sequences) described herein or known in the art. In some embodiments, the cytokine is a proinflammatory cytokine. In some embodiments, the cytokine is selected from human IL-1, IL-2, IL-7, IL-12, IL-13, IL-15, IL-17, IL-18, IL-28 (e.g., IL-28α and / or IL-28β), IL-32, IL-33, IL-34, TNFα, IFNγ, G-CSF, and / or GM-CSF;

[0051] In some embodiments, the polynucleotide of the present disclosure encodes an IL-1α polypeptide. In some embodiments, the IL-1α polypeptide is a human IL-1α polypeptide (e.g., UniProt Accession No.: P01583). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type IL1A gene (e.g., see NCBI Gene ID: 3552, SEQ ID NO: 31), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the IL-1α polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide encoding an IL-1α polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1.

[0052] In some embodiments, the polynucleotide encoding an IL-1α polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 1. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, but fewer than 271 consecutive amino acids of SEQ ID NO: 1.

[0053] In some embodiments, the polynucleotide of the present disclosure encodes an IL-1β polypeptide. In some embodiments, the IL-1β polypeptide is a human IL-1β polypeptide (see, e.g., UniProt Accession No.: P01584). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type IL1B gene (see, e.g., NCBI Gene ID: 3553, SEQ ID NO: 32), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding an IL-1β polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2. In some embodiments, the polynucleotide encoding an IL-1β polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

[0054] In some embodiments, the polynucleotide encoding an IL-1β polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO:2. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, but fewer than 269 consecutive amino acids of SEQ ID NO:2.

[0055] In some embodiments, the polynucleotides of the disclosure encode an IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is a human IL-2 polypeptide (see, e.g., UniProt Accession No.: P60568). In some embodiments, the polynucleotide coding sequence comprises a wild-type IL2 gene (see, e.g., NCBI Gene ID: 3558, SEQ ID NO: 33), or a codon-optimized variant thereof (see, e.g., SEQ ID NO: 61). In some embodiments, the polynucleotide encoding the IL-2 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide encoding an IL-2 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:3.

[0056] In some embodiments, the polynucleotide encoding an IL-2 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO:3. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 153 consecutive amino acids of SEQ ID NO:3.

[0057] In some embodiments, the polynucleotides of the present disclosure encode an IL-7 polypeptide. In some embodiments, the IL-7 polypeptide is a human IL-7 polypeptide (see, e.g., UniProt Accession No.: P13232). In some embodiments, the polynucleotide coding sequence comprises a wild-type IL7 gene (see, e.g., NCBI Gene ID: 3574, SEQ ID NO: 34), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding an IL-7 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide encoding an IL-7 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:4.

[0058] In some embodiments, the polynucleotide encoding an IL-7 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO:4. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 177 consecutive amino acids of SEQ ID NO:4.

[0059] In some embodiments, the polynucleotides of the present disclosure encode an IL-12 subunit alpha polypeptide. In some embodiments, the IL-12 subunit alpha polypeptide is a human IL-12 subunit alpha polypeptide (see, e.g., UniProt Accession No.: P29459). In some embodiments, the polynucleotides comprise the coding sequence of a wild-type IL12A gene (see, e.g., NCBI Gene ID: 3592, SEQ ID NO: 35), or a codon-optimized variant thereof (see, e.g., SEQ ID NO: 62). In some embodiments, a polynucleotide encoding an IL-12 subunit alpha polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:5. In some embodiments, a polynucleotide encoding an IL-12 subunit alpha polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:5.

[0060] In some embodiments, the polynucleotide encoding the IL-12 subunit alpha polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO:5. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, but fewer than 219 consecutive amino acids of SEQ ID NO:5.

[0061] In some embodiments, the polynucleotides of the present disclosure encode an IL-12 subunit beta polypeptide. In some embodiments, the IL-12 subunit beta polypeptide is a human IL-12 subunit beta polypeptide (see, e.g., UniProt Accession No.: P29460). In some embodiments, the polynucleotides comprise a coding sequence for a wild-type IL12B gene (see, e.g., NCBI Gene ID: 3593, SEQ ID NO: 36) or a codon-optimized variant thereof (see, e.g., SEQ ID NO: 63). In some embodiments, a polynucleotide encoding an IL-12 subunit beta polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:6. In some embodiments, a polynucleotide encoding an IL-12 subunit beta polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6.

[0062] In some embodiments, the polynucleotide encoding an IL-12 subunit β polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO:6. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, but fewer than 328 consecutive amino acids of SEQ ID NO:6.

[0063] In some embodiments, the polynucleotides of the present disclosure encode an IL-13 polypeptide. In some embodiments, the IL-13 polypeptide is a human IL-13 polypeptide (see, e.g., UniProt Accession No.: P35225). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type IL13 gene (see, e.g., NCBI Gene ID: 3596, SEQ ID NO: 37), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding an IL-13 polypeptide encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 7. In some embodiments, the polynucleotide encoding an IL-13 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

[0064] In some embodiments, the polynucleotide encoding an IL-13 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO:7. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 146 consecutive amino acids of SEQ ID NO:7.

[0065] In some embodiments, the polynucleotides of the present disclosure encode IL-15 polypeptides. In some embodiments, the IL-15 polypeptides are human IL-15 polypeptides (see, e.g., UniProt Accession No.: P40933). In some embodiments, the polynucleotides comprise the coding sequence of a wild-type IL15 gene (see, e.g., NCBI Gene ID: 3600, SEQ ID NO: 38), or a codon-optimized variant thereof. In some embodiments, the polynucleotides encoding IL-15 polypeptides are polynucleotides encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide encoding an IL-15 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:8.

[0066] In some embodiments, the polynucleotide encoding an IL-15 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO:8. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 162 consecutive amino acids of SEQ ID NO:8.

[0067] In some embodiments, the polynucleotide of the present disclosure encodes an IL-17A polypeptide. In some embodiments, the IL-17A polypeptide is a human IL-17A polypeptide (see, e.g., UniProt Accession No.: Q16552). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type IL17A gene (see, e.g., NCBI Gene ID: 3605, SEQ ID NO: 39), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding an IL-17A polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 9. In some embodiments, the polynucleotide encoding an IL-17A polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:9.

[0068] In some embodiments, the polynucleotide encoding an IL-17A polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO:9. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 155 consecutive amino acids of SEQ ID NO:9.

[0069] In some embodiments, the polynucleotide of the present disclosure encodes an IL-18 polypeptide. In some embodiments, the IL-18 polypeptide is a human IL-18 polypeptide (see, e.g., UniProt Accession No.: Q14116). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type IL18 gene (see, e.g., NCBI Gene ID: 3606, SEQ ID NO: 40), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding an IL-18 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 10. In some embodiments, the polynucleotide encoding an IL-18 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:10.

[0070] In some embodiments, the polynucleotide encoding an IL-18 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 10. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, but fewer than 193 consecutive amino acids of SEQ ID NO:10.

[0071] In some embodiments, the polynucleotides of the present disclosure encode IL-28A polypeptides. In some embodiments, the IL-28A polypeptide is a human IL-28A polypeptide (see, e.g., UniProt Accession No.: Q8IZJ0). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type IFNL2 gene (see, e.g., NCBI Gene ID: 282616, SEQ ID NO: 41), or a codon-optimized variant thereof. In some embodiments, the polynucleotides encoding IL-28A polypeptides are polynucleotides encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 11. In some embodiments, the polynucleotide encoding an IL-28A polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:11.

[0072] In some embodiments, a polynucleotide encoding an IL-28A polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO:11. The N-terminal truncation, C-terminal truncation, or fragment can comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, but fewer than 200 consecutive amino acids of SEQ ID NO:11.

[0073] In some embodiments, the polynucleotide of the present disclosure encodes an IL-28B polypeptide. In some embodiments, the IL-28B polypeptide is a human IL-28B polypeptide (see, e.g., UniProt Accession No.: Q8IZI9). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type IFNL3 gene (see, e.g., NCBI Gene ID: 282617, SEQ ID NO: 42), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding an IL-28B polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 12. In some embodiments, the polynucleotide encoding an IL-28B polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:12.

[0074] In some embodiments, a polynucleotide encoding an IL-28B polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 12. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 196 consecutive amino acids of SEQ ID NO:12.

[0075] In some embodiments, the polynucleotides of the present disclosure encode an IL-32 polypeptide. In some embodiments, the IL-32 polypeptide is a human IL-32 polypeptide (see, e.g., UniProt Accession No.: P24001). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type IL32 gene (see, e.g., NCBI Gene ID: 9235, SEQ ID NO: 43), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding IL-32 is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. In some embodiments, the polynucleotide encoding an IL-32 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:13.

[0076] In some embodiments, a polynucleotide encoding an IL-32 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 13. The N-terminal truncation, C-terminal truncation, or fragment can include at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, but fewer than 234 consecutive amino acids of SEQ ID NO:13.

[0077] In some embodiments, the polynucleotides of the present disclosure encode an IL-33 polypeptide. In some embodiments, the IL-33 polypeptide is a human IL-33 polypeptide (see, e.g., UniProt Accession No.: O95760). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type IL33 gene (see, e.g., NCBI Gene ID: 90865, SEQ ID NO: 44), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the IL-33 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 14. In some embodiments, the polynucleotide encoding an IL-33 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:14.

[0078] In some embodiments, a polynucleotide encoding an IL-33 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 14. The N-terminal truncation, C-terminal truncation, or fragment can include at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, but fewer than 270 consecutive amino acids of SEQ ID NO:14.

[0079] In some embodiments, the polynucleotides of the present disclosure encode IL-34 polypeptides. In some embodiments, the IL-34 polypeptides are human IL-34 polypeptides (see, e.g., UniProt Accession No.: Q6ZMJ4). In some embodiments, the polynucleotides comprise the coding sequence of a wild-type IL34 gene (see, e.g., NCBI Gene ID: 146433, SEQ ID NO: 45), or a codon-optimized variant thereof. In some embodiments, the polynucleotides encoding IL-34 polypeptides are polynucleotides encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 15. In some embodiments, the polynucleotide encoding an IL-34 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:15.

[0080] In some embodiments, a polynucleotide encoding an IL-34 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 15. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, but fewer than 242 consecutive amino acids of SEQ ID NO:15.

[0081] In some embodiments, the polynucleotide of the present disclosure encodes a TNFα polypeptide. In some embodiments, the TNFα polypeptide is a human TNFα polypeptide (see, e.g., UniProt Accession No.: P01375). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type TNF gene (see, e.g., NCBI Gene ID: 7124, SEQ ID NO: 46), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the TNFα polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 16. In some embodiments, the polynucleotide encoding a TNFα polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:16.

[0082] In some embodiments, the polynucleotide encoding the TNFα polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 16. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, but fewer than 233 consecutive amino acids of SEQ ID NO:16.

[0083] In some embodiments, the polynucleotide of the present disclosure encodes an IFNγ polypeptide. In some embodiments, the IFNγ polypeptide is a human IFNγ polypeptide (see, e.g., UniProt Accession No.: P01579). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type IFNG gene (see, e.g., NCBI Gene ID: 3458, SEQ ID NO: 47), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the IFNγ polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 17. In some embodiments, the polynucleotide encoding an IFNγ polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:17.

[0084] In some embodiments, the polynucleotide encoding an IFNγ polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 17. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 166 consecutive amino acids of SEQ ID NO:17.

[0085] In some embodiments, the polynucleotide of the present disclosure encodes a G-CSF polypeptide. In some embodiments, the G-CSF polypeptide is a human G-CSF polypeptide (see, e.g., UniProt Accession No.: P09919). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type CSF3 gene (see, e.g., NCBI Gene ID: 1440, SEQ ID NO: 48), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the G-CSF polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide encoding a G-CSF polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:18.

[0086] In some embodiments, the polynucleotide encoding the G-CSF polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 18. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, but fewer than 207 consecutive amino acids of SEQ ID NO: 18.

[0087] In some embodiments, the polynucleotide of the present disclosure encodes a GM-CSF polypeptide. In some embodiments, the GM-CSF polypeptide is a human GM-CSF polypeptide (see, e.g., UniProt Accession No.: P04141). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CSF2 gene (see, e.g., NCBI Gene ID: 1437, SEQ ID NO: 49), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the GM-CSF polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 19. In some embodiments, the polynucleotide encoding a GM-CSF polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:19.

[0088] In some embodiments, the polynucleotide encoding a GM-CSF polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 19. The N-terminal truncation, C-terminal truncation, or fragment can include at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 144 consecutive amino acids of SEQ ID NO:19.

[0089] In some embodiments, the present disclosure relates to recombinant nucleic acids comprising one or more polynucleotides comprising a coding sequence for a human chemokine, such as the CCL1 gene (see, e.g., NCBI Gene ID:6346), the CCL2 gene (see, e.g., NCBI Gene ID:6347; SEQ ID NO:56), the CCL3 gene (see, e.g., NCBI Gene ID:6348; SEQ ID NO:57), the CCL4 gene (see, e.g., NCBI Gene ID:6351; SEQ ID NO:58), the CCL5 gene (see, e.g., NCBI Gene ID:6352; SEQ ID NO:59, the CCL7 gene (see, e.g., NCBI Gene ID:6354), the CCL8 gene (see, e.g., NCBI Gene ID:6355), the CCL11 gene (see, e.g., NCBI Gene ID:6356;SEQ ID NO:60), CCL13 gene (see, e.g., NCBI Gene ID:6357), CCL14 gene (see, e.g., NCBI Gene ID:6358), CCL15 gene (see, e.g., NCBI Gene ID:6359), CCL16 gene (see, e.g., NCBI Gene ID:6360), CCL17 gene (see, e.g., NCBI Gene ID:6361), CCL18 gene (see, e.g., NCBI Gene ID:6362), CCL19 gene (see, e.g., NCBI Gene ID:6363), CCL20 gene (see, e.g., NCBI Gene ID:6364), CCL21 gene (see, e.g., NCBI Gene ID:6366), CCL22 gene (see, e.g., NCBI Gene ID:6367), CCL23 gene (see, e.g., NCBI Gene ID:6369), CCL31 gene (see, e.g., NCBI Gene ID:6370), CCL32 gene (see, e.g., NCBI Gene ID:6371), CCL33 gene (see, e.g., NCBI Gene ID:6372), CCL34 gene (see, e.g., NCBI Gene ID:6373), CCL35 gene (see, e.g., NCBI Gene ID:6374), CCL36 gene (see, e.g., NCBI Gene ID:6375), CCL37 gene (see, e.g., NCBI Gene ID:6376), CCL38 gene (see, e.g., NCBI Gene ID:6377), CCL39 gene (see, e.g., NCBI Gene ID:6388), CCL40 gene (see, e.g., NCBI Gene ID:4078 ID:6368), CCL24 gene (see, e.g., NCBI Gene ID:6369), CCL25 gene (see, e.g., NCBI Gene ID:6370), CCL26 gene (see, e.g., NCBI Gene ID:10344), CCL27 gene (see, e.g., NCBI Gene ID:10850), CCL28 gene (see, e.g., NCBI Gene ID:56477), CXCL1 gene (see, e.g., NCBI Gene ID:2919; SEQ ID NO:50), CXCL2 gene (see, e.g., NCBI Gene ID:2920; SEQ ID NO:51), CXCL3 gene (see, e.g., NCBI Gene ID:2921), CXCL4 gene (see, e.g., NCBI Gene ID:5196), CXCL5 gene (see, e.g., NCBI Gene ID:5201), CXCL6 gene (see, e.g., NCBI Gene ID:5202), CXCL7 gene (see, e.g., NCBI Gene ID:5203), CXCL8 gene (see, e.g., NCBI Gene ID:5204), CXCL9 gene (see, e.g., NCBI Gene ID:5205), CXCL10 gene (see, e.g., NCBI Gene ID:5206), CXCL11 gene (see, e.g., NCBI Gene ID:5207), CXCL12 gene (see, e.g., NCBI Gene ID:5208), CXCL13 gene (see, e.g., NCBI Gene ID:5209), CXCL14 gene (see, e.g., NCBI Gene ID:5210), CXCL15 gene (see, e.g., NCBI Gene ID:5211 ID:6374), the CXCL6 gene (see, e.g., NCBI Gene ID:6372), the PPBP gene (also known as the CXCL7 gene, see, e.g., NCBI Gene ID:5473), the CXCL8 gene (see, e.g., NCBI Gene ID:3576;See, e.g., NCBI Gene ID: 4283; SEQ ID NO: 53), CXCL10 gene (see, e.g., NCBI Gene ID: 3627), CXCL11 gene (see, e.g., NCBI Gene ID: 6373; SEQ ID NO: 54), CXCL12 gene (see, e.g., NCBI Gene ID: 6387), CXCL13 gene (see, e.g., NCBI Gene ID: 10563), CXCL14 gene (see, e.g., NCBI Gene ID: 9547), CXCL16 gene (see, e.g., NCBI Gene ID: 58191;Any suitable human chemokine gene known in the art (including any isoforms thereof) may be encoded by the polynucleotides of the present disclosure, including the CXCL1 gene (see, e.g., SEQ ID NO:55), the CXCL17 gene (see, e.g., NCBI Gene ID:284340), the XCL1 gene (see, e.g., NCBI Gene ID:6375), the XCL2 gene (see, e.g., NCBI Gene ID:6846), the CX3CL1 gene (see, e.g., NCBI Gene ID:6376), and the like. In some embodiments, the polynucleotides of the disclosure (e.g., one or more first polynucleotides and / or one or more second polynucleotides) comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any of the human chemokine genes (and / or their coding sequences) described herein or known in the art. In some embodiments, the chemokine is a proinflammatory chemokine. In some embodiments, the chemokine is selected from human CXCL1, CXCL2, CXCL8, CXCL9, CXCL11, CXCL16, CCL2, CCL3, CCL4, CCL5, and / or CCL11;

[0090] In some embodiments, the polynucleotide of the present disclosure encodes a CXCL1 polypeptide. In some embodiments, the CXCL1 polypeptide is a human CXCL1 polypeptide (see, e.g., UniProt Accession No.: P09341). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CXCL1 gene (see, e.g., NCBI Gene ID: 2919, SEQ ID NO: 50), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CXCL1 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide encoding a CXCL1 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:20.

[0091] In some embodiments, the polynucleotide encoding a CXCL1 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO:20. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 107 consecutive amino acids of SEQ ID NO:20.

[0092] In some embodiments, the polynucleotide of the present disclosure encodes a CXCL2 polypeptide. In some embodiments, the CXCL2 polypeptide is a human CXCL2 polypeptide (see, e.g., UniProt Accession No.: P19875). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CXCL2 gene (see, e.g., NCBI Gene ID: 2920, SEQ ID NO: 51), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CXCL2 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 21. In some embodiments, the polynucleotide encoding a CXCL2 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:21.

[0093] In some embodiments, the polynucleotide encoding a CXCL2 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO:21. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 107 consecutive amino acids of SEQ ID NO:21.

[0094] In some embodiments, the polynucleotide of the present disclosure encodes a CXCL8 polypeptide. In some embodiments, the CXCL8 polypeptide is a human CXCL8 polypeptide (see, e.g., UniProt Accession No.: P10145). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CXCL8 gene (see, e.g., NCBI Gene ID: 3576, SEQ ID NO: 52), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CXCL8 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 22. In some embodiments, the polynucleotide encoding a CXCL8 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:22.

[0095] In some embodiments, the polynucleotide encoding a CXCL8 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 22. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, but fewer than 99 consecutive amino acids of SEQ ID NO:22.

[0096] In some embodiments, the polynucleotide of the present disclosure encodes a CXCL9 polypeptide. In some embodiments, the CXCL9 polypeptide is a human CXCL9 polypeptide (see, e.g., UniProt Accession No.: Q07325). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CXCL9 gene (see, e.g., NCBI Gene ID: 4283, SEQ ID NO: 53), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CXCL9 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 23. In some embodiments, the polynucleotide encoding a CXCL9 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:23.

[0097] In some embodiments, the polynucleotide encoding a CXCL9 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 23. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 125 consecutive amino acids of SEQ ID NO:23.

[0098] In some embodiments, the polynucleotide of the present disclosure encodes a CXCL11 polypeptide. In some embodiments, the CXCL11 polypeptide is a human CXCL11 polypeptide (see, e.g., UniProt Accession No.: O14625). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CXCL11 gene (see, e.g., NCBI Gene ID: 6373, SEQ ID NO: 54), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CXCL11 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 24. In some embodiments, the polynucleotide encoding a CXCL11 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:24.

[0099] In some embodiments, the polynucleotide encoding a CXCL11 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of SEQ ID NO: 24. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, but fewer than 94 consecutive amino acids of SEQ ID NO:24.

[0100] In some embodiments, the polynucleotide of the present disclosure encodes a CXCL16 polypeptide. In some embodiments, the CXCL16 polypeptide is a human CXCL16 polypeptide (see, e.g., UniProt Accession No.: Q9H2A7). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CXCL16 gene (see, e.g., NCBI Gene ID: 58191, SEQ ID NO: 55), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CXCL16 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 25. In some embodiments, the polynucleotide encoding a CXCL16 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:25.

[0101] In some embodiments, the polynucleotide encoding a CXCL16 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 25. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, but fewer than 254 consecutive amino acids of SEQ ID NO:25.

[0102] In some embodiments, the polynucleotide of the present disclosure encodes a CCL2 polypeptide. In some embodiments, the CCL2 polypeptide is a human CCL2 polypeptide (see, e.g., UniProt Accession No.: P13500). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CCL2 gene (see, e.g., NCBI Gene ID: 6347, SEQ ID NO: 56), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CCL2 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 26. In some embodiments, the polynucleotide encoding a CCL2 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:26.

[0103] In some embodiments, the polynucleotide encoding a CCL2 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 26. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, but fewer than 99 consecutive amino acids of SEQ ID NO:26.

[0104] In some embodiments, the polynucleotide of the present disclosure encodes a CCL3 polypeptide. In some embodiments, the CCL3 polypeptide is a human CCL3 polypeptide (see, e.g., UniProt Accession No.: P10147). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CCL3 gene (see, e.g., NCBI Gene ID: 6348, SEQ ID NO: 57), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CCL3 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 27. In some embodiments, the polynucleotide encoding a CCL3 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:27.

[0105] In some embodiments, the polynucleotide encoding a CCL3 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 27. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, but fewer than 92 consecutive amino acids of SEQ ID NO:27.

[0106] In some embodiments, the polynucleotide of the present disclosure encodes a CCL4 polypeptide. In some embodiments, the CCL4 polypeptide is a human CCL4 polypeptide (see, e.g., UniProt Accession No.: P13236). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CCL4 gene (see, e.g., NCBI Gene ID: 6351, SEQ ID NO: 58), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CCL4 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 28. In some embodiments, the polynucleotide encoding a CCL4 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:28.

[0107] In some embodiments, the polynucleotide encoding a CCL4 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 28. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, but fewer than 92 consecutive amino acids of SEQ ID NO:28.

[0108] In some embodiments, the polynucleotide of the present disclosure encodes a CCL5 polypeptide. In some embodiments, the CCL5 polypeptide is a human CCL5 polypeptide (see, e.g., UniProt Accession No.: P13501). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CCL5 gene (see, e.g., NCBI Gene ID: 6352, SEQ ID NO: 59), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CCL5 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 29. In some embodiments, the polynucleotide encoding a CCL5 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:29.

[0109] In some embodiments, the polynucleotide encoding a CCL5 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 29. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, but fewer than 91 consecutive amino acids of SEQ ID NO:29.

[0110] In some embodiments, the polynucleotide of the present disclosure encodes a CCL11 polypeptide. In some embodiments, the CCL11 polypeptide is a human CCL11 polypeptide (see, e.g., UniProt Accession No.: P51671). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type CCL11 gene (see, e.g., NCBI Gene ID: 6356, SEQ ID NO: 60), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CCL11 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide encoding a CCL11 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:30.

[0111] In some embodiments, the polynucleotide encoding a CCL11 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 30. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, but fewer than 97 consecutive amino acids of SEQ ID NO:30.

[0112] In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 1-30. In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-30.

[0113] In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 1-19. In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-19.

[0114] In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs:20-30. In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs:20-30.

[0115] In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs:3, 5, and 6. In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs:3, 5, and 6.

[0116] In some aspects, the polynucleotides of the disclosure are selected from the group consisting of interleukin-1 alpha (IL-1α) peptides, interleukin-1 beta (IL-1β) peptides, interleukin-2 (IL-2) peptides, interleukin-3 (IL-3) peptides, interleukin-4 (IL-4) peptides, interleukin-5 (IL-5) peptides, interleukin-6 (IL-6) peptides, interleukin-7 (IL-7) peptides, interleukin-8 (IL-8) peptides, interleukin-9 (IL-9) peptides, interleukin-10 (IL-11) peptides, interleukin-11 peptides, interleukin-12 peptides, interleukin-13 peptides, interleukin-14 peptides, interleukin-15 peptides, interleukin-16 peptides, interleukin-17 peptides, interleukin-18 peptides, interleukin-19 peptides, interleukin-19 peptides, interleukin-19 peptides, interleukin-10 peptides, interleukin-10 peptides, interleukin-11 peptides, interleukin-12 peptides, interleukin-13 peptides, interleukin-14 peptides, interleukin-15 peptides, interleukin-16 peptides, interleukin-17 peptides, interleukin-18 peptides, interleukin-19 ... -leukin-10 (IL-10) peptides, interleukin-11 (IL-11) peptides, interleukin-12 subunit alpha (IL-12α) peptides, interleukin-12 subunit beta (IL-12β) peptides, interleukin-13 (IL-13) peptides, interleukin-15 (IL-15) peptides, interleukin-17 (IL-17) peptides, interleukin-17B (IL-17B) peptides, interleukin-17C (IL-17C) peptides, interleukin-17D (IL-1 7D) peptides, interleukin-25 (IL-25) peptides, interleukin-17F (IL-17F) peptides, interleukin-18 (IL-18) peptides, interleukin-28A (IL-28A) peptides, interleukin-28B (IL-28B) peptides, interleukin-29 (IL-29) peptides, interleukin-32 (IL-32) peptides, interleukin-33 (IL-33) peptides, interleukin-34 (IL-34) peptides, interleukin-36 alpha (IL-36α) peptides tide, interleukin-36 (IL-36β) beta peptide, interleukin-36 gamma (IL-36γ) peptide, interferon alpha-1 (IFNα-1) peptide, interferon alpha-2 (IFNα-2) peptide, interferon alpha-4 (IFNα-4) peptide, interferon alpha-5 (IFNα-5) peptide, interferon alpha-6 (IFNα-6) peptide, interferon alpha-7 (IFNα-7) peptide, interferon alpha-8 (IFNα-8) peptide,Interferon alpha-10 (IFNα-10) peptides, interferon alpha-14 (IFNα-14) peptides, interferon alpha-16 (IFNα-16) peptides, interferon alpha-17 (IFNα-17) peptides, interferon alpha-21 (IFNα-21) peptides, interferon beta-1 (IFNβ-1) peptides, interferon beta-3 (IFNβ-3) peptides, interferon gamma (IFNγ) peptides, tumor necrosis factor alpha (TNFα) peptides , tumor necrosis factor beta (TNFβ) peptide, granulocyte colony-stimulating factor (G-CSF) peptide, granulocyte-macrophage colony-stimulating factor (GM-CSF) peptide, macrophage colony-stimulating factor (M-CSF) peptide, CC motif chemokine 1 (CCL1) peptide, CC motif chemokine 2 (CCL2) peptide, CC motif chemokine 3 (CCL3) peptide, CC motif chemokine 4 (CCL4) peptide, CC motif chemokine 5 (CCL5) peptide, CC motif chemokine 7 (CCL7) peptide tide, CC motif chemokine 8 (CCL8) peptide, CC motif chemokine 11 (CCL11) peptide, CC motif chemokine 13 (CCL13) peptide, CC motif chemokine 14 (CCL14) peptide, CC motif chemokine 15 (CCL15) peptide, CC motif chemokine 16 (CCL16) peptide, CC motif chemokine 17 (CCL17) peptide, CC motif chemokine 18 (CCL18) peptide, CC motif chemokine 19 (CCL19) peptide, CC motif chemokine 2 0 (CCL20) peptide, CC motif chemokine 21 (CCL21) peptide, CC motif chemokine 22 (CCL22) peptide, CC motif chemokine 23 (CCL23) peptide, CC motif chemokine 24 (CCL24) peptide, CC motif chemokine 25 (CCL25) peptide, CC motif chemokine 26 (CCL26) peptide, CC motif chemokine 27 (CCL27) peptide, CC motif chemokine 28 (CCL28) peptide, CXC motif chemokine 1 (CXCL1) peptide,CXC motif chemokine 2 (CXCL2) peptide, CXC motif chemokine 3 (CXCL3) peptide, CXC motif chemokine 4 (CXCL4) peptide, CXC motif chemokine 5 (CXCL5) peptide, CXC motif chemokine 6 (CXCL6) peptide, CXC motif chemokine 7 (CXCL7) peptide, CXC motif chemokine 9 (CXCL9) peptide, CXC motif chemokine 10 (CXCL10) peptide, CXC motif chemokine 11 (CXCL11) peptide, CXC motif chemokine 12 (CXCL12) peptide, CXC motif chemokine 13 (CXCL14) peptide, CXC motif chemokine 14 (CXCL15) peptide, CXC motif chemokine 15 (CXCL16) peptide, CXC motif chemokine 16 (CXCL17) peptide, CXC motif chemokine 17 (CXCL18) peptide, CXC motif chemokine 18 (CXCL19) peptide, CXC motif chemokine 19 (CXCL20) peptide, CXC motif chemokine 20 (CXCL21) peptide, CXC motif chemokine 21 (CXCL22) peptide, CXC motif chemokine 22 (CXCL23) peptide, CXC motif chemokine 23 (CXCL24) peptide, CXC motif chemokine 24 (CXCL25) peptide, CXC motif chemokine 25 (CXCL26) peptide, CXC motif chemokine 26 (CXCL27) peptide, CXC motif chemokine 27 (CXCL28) peptide, CXC motif chemokine 29 (CXCL29) peptide, CXC motif chemokine 30 (CXCL30) peptide, CXC motif chemokine 31 ( In some embodiments, the polynucleotides of the present disclosure encode any one or more of a CXC motif chemokine 13 (CXCL13) peptide, a CXC motif chemokine 14 (CXCL14) peptide, a CXC motif chemokine 16 (CXCL16) peptide, a CXC motif chemokine 17 (CXCL17) peptide, a C motif chemokine 1 (XCL1) peptide, a C motif chemokine 2 (XCL2) peptide, a C-X3-C motif chemokine 1 (CX3CL1) peptide, and / or any chimeric polypeptides thereof in any suitable combination. In some embodiments, the polynucleotides of the present disclosure do not encode an interleukin-4 (IL-4) peptide. In some embodiments, the polynucleotides of the present disclosure do not encode an interleukin-10 (IL-10) peptide. In some embodiments, the polynucleotides of the present disclosure do not encode a granulocyte macrophage colony stimulating factor (GM-CSF) peptide. In some embodiments, the polynucleotides of the present disclosure do not encode interleukin-4 (IL-4) peptides, interleukin-10 (IL-10) peptides, and / or granulocyte-macrophage colony-stimulating factor (GM-CSF) peptides.

[0117] A polynucleotide of the present disclosure that encodes a polypeptide (e.g., an immunomodulatory polypeptide) may further encode additional coding and non-coding sequences. Examples of additional coding and non-coding sequences may include, but are not limited to, sequences encoding additional polypeptide tags (e.g., encoded in frame with the polypeptide to produce a fusion protein), introns (e.g., native, modified, or heterologous introns), 5' and / or 3' UTRs (e.g., native, modified, or heterologous 5' and / or 3' UTRs), etc. Examples of suitable polypeptide tags may include, but are not limited to, purification tags, such as his-tags, flag-tags, maltose binding protein and glutathione-S-transferase tags, detection tags, such as tags that can be photometrically detected (e.g., green fluorescent protein, red fluorescent protein, etc.) and tags with detectable enzymatic activity (e.g., alkaline phosphatase, etc.), any combination of tags containing secretion sequences, signal sequences, leader sequences, and / or stabilizing sequences, protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites, etc.), etc. In some embodiments, the 5' and / or 3' UTRs increase the stability, localization, and / or translation efficiency of the polynucleotide. In some embodiments, the 5' and / or 3' UTRs improve the level and / or duration of protein expression. In some embodiments, the 5' and / or 3' UTRs contain elements (e.g., one or more miRNA binding sites, etc.) that can block or reduce off-target expression (e.g., inhibit expression in a particular cell type (e.g., neuronal cell), at a particular time during the cell cycle, at a particular developmental stage, etc.). In some embodiments, the 5' and / or 3' UTRs contain elements (e.g., one or more miRNA binding sites, etc.) that can enhance expression of the encoded polypeptide in a particular cell type.

[0118] In some aspects, a polynucleotide of the present disclosure encoding a polypeptide (e.g., an immunomodulatory polypeptide) is operably linked to one or more (e.g., one or more, two or more, three or more, four or more, five or more, ten or more, etc.) regulatory sequences. The term "regulatory sequence" can include enhancers, insulators, promoters, and other expression control elements (e.g., polyadenylation signals). Any suitable enhancer known in the art can be used, including, for example, enhancer sequences from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin, etc.), enhancer sequences from eukaryotic cell viruses (e.g., SV40 enhancer on the late side of the replication origin (bp 100-270), cytomegalovirus early promoter enhancer, polyoma enhancer on the late side of the replication origin, adenovirus enhancers, etc.), and any combination thereof. Any suitable insulator known in the art may be used, including, for example, the HSV chromatin boundary (CTRL / CTCF binding / insulator) elements CTRL1 and / or CTRL2, the chicken hypersensitive region 4 insulator (cHS4), the human HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE), the scaffold / matrix attachment region (S / MAR) from the human interferon beta gene (IFNB1), and any combination thereof.Any suitable promoter known in the art (e.g., suitable for transcription in a mammalian host cell) may be used, including, for example, promoters obtained from the genomes of viruses (e.g., polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, simian virus 40 (SV40), etc.), promoters from heterologous mammalian genes (e.g., from actin promoters (e.g., β-actin promoter), ubiquitin promoters (e.g., ubiquitin C (UbC) promoter), phosphoglycerate kinase (PGK) promoter, immunoglobulin promoters, heat shock promoters, etc.), promoters from homologous mammalian genes, synthetic promoters (such as the CAG promoter), and any combination thereof, provided that such promoters are compatible with the host cell. Regulatory sequences may include sequences that direct constitutive expression of the nucleic acid as well as tissue-specific regulatory sequences and / or inducible or repressible sequences.

[0119] In some embodiments, the polynucleotide of the present disclosure is functionally linked to one or more heterologous promoters. In some embodiments, the one or more heterologous promoters are one or more of a constitutive promoter, a tissue-specific promoter, a temporal promoter, a spatial promoter, an inducible promoter, and a repressible promoter. In some embodiments, the one or more heterologous promoters are one or more of a human cytomegalovirus (HCMV) immediate early promoter, a human elongation factor-1 (EF1) promoter, a human β-actin promoter, a human UbC promoter, a human PGK promoter, a synthetic CAGG promoter, and any combination thereof. In some embodiments, the polynucleotide of the present disclosure that encodes a polypeptide (e.g., an immunomodulatory polypeptide) is functionally linked to a HCMV promoter.

[0120] In some embodiments, when a polynucleotide of the disclosure encoding a polypeptide (e.g., an immune modulating polypeptide such as IL-2 and / or IL-12) is delivered to one or more target cells of a subject (e.g., one or more cells of the respiratory tract, airway, lungs, etc. of a subject), the polynucleotide expresses the polypeptide. In some embodiments, expression of the polypeptide (e.g., an immune modulating polypeptide such as IL-2 and / or IL-12) enhances, increases, enhances, and / or replenishes the level, function, and / or activity of the polypeptide in one or more target cells of the subject (e.g., compared to before expression of the polypeptide, compared to the level of endogenous polypeptide expressed in the cell, etc.). In some embodiments, expression of a polypeptide (e.g., an immune modulating polypeptide such as IL-2 and / or IL-12) provides preventative, palliative, or therapeutic alleviation (e.g., compared to before expression of the polypeptide) of one or more signs or symptoms of cancer (e.g., solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, renal cancer, salivary gland cancer, skin cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, etc.) in a subject.

[0121] Chimeric Polypeptides In some embodiments, the polynucleotide of the present disclosure encodes a chimeric polypeptide comprising a first immunomodulatory polypeptide and a second immunomodulatory polypeptide. In some embodiments, the first and second immunomodulatory polypeptides are the same. In some embodiments, the first and second immunomodulatory polypeptides are different. In some embodiments, the chimeric polypeptide further comprises a linker polypeptide linking the first and second immunomodulatory polypeptides. In some embodiments, the chimeric polypeptide comprises, from N-terminus to C-terminus, first immunomodulatory polypeptide-linker polypeptide-second immunomodulatory polypeptide. The first and / or second immunomodulatory polypeptide can be any of the immunomodulatory polypeptides described herein or known in the art.

[0122] In some embodiments, the linker polypeptide is a cleavable linker polypeptide. Any cleavable linker polypeptide known in the art may be used in the chimeric polypeptides of the present disclosure, including, for example, T2A linker, P2A linker, E2A linker, and F2A linker. In some embodiments, the linker polypeptide is a T2A linker polypeptide. An exemplary nucleic acid sequence encoding a T2A linker polypeptide is provided as SEQ ID NO:64. An exemplary amino acid sequence of a T2A linker polypeptide is provided as SEQ ID NO:68. In some embodiments, the linker polypeptide is a P2A linker polypeptide. An exemplary nucleic acid sequence encoding a P2A linker polypeptide is provided as SEQ ID NO:65. An exemplary amino acid sequence of a P2A linker polypeptide is provided as SEQ ID NO:69. In some embodiments, the linker polypeptide is an E2A linker polypeptide. An exemplary nucleic acid sequence encoding an E2A linker polypeptide is provided as SEQ ID NO:66. An exemplary amino acid sequence of an E2A linker polypeptide is provided as SEQ ID NO:70. In some embodiments, the linker polypeptide is an F2A linker polypeptide. An exemplary nucleic acid sequence encoding an F2A linker polypeptide is provided as SEQ ID NO:67. An exemplary amino acid sequence of an F2A linker polypeptide is provided as SEQ ID NO:71. In some embodiments, the linker polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs:68-71. In some embodiments, the linker polypeptide comprises a sequence selected from SEQ ID NOs:68-71.

[0123] In some embodiments, the linker polypeptide is a non-cleavable linker polypeptide. For example, Any non-cleavable linker polypeptide known in the art may be used in the chimeric polypeptides of the present disclosure, including, for example, TIFF2024513826000001.tif48165. In some embodiments, the linker polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 72-83. In some embodiments, the linker polypeptide comprises a sequence selected from SEQ ID NOs: 72-83.

[0124] An exemplary nucleic acid sequence encoding a chimeric polypeptide comprising a first human immunomodulatory polypeptide, a linker polypeptide, and a second human immunomodulatory polypeptide is provided as SEQ ID NO:84. An exemplary amino acid sequence of a chimeric polypeptide comprising a first human immunomodulatory polypeptide, a linker polypeptide, and a second human immunomodulatory polypeptide is provided as SEQ ID NO:85. In some aspects, a polynucleotide of the present disclosure encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:85. In some aspects, a polynucleotide of the present disclosure encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:85.

[0125] In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a collagen alpha-1 (VII) chain polypeptide (COL7) (e.g., a transgene encoding the same). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a lysyl hydroxylase 3 polypeptide (LH3) (e.g., a transgene encoding the same). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a keratin type I cytoskeleton 17 polypeptide (KRT17) (e.g., a transgene encoding the same). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a transglutaminase (TGM) polypeptide (e.g., a human transglutaminase polypeptide such as a human TGM1 polypeptide and / or a human TGM5 polypeptide) (e.g., a transgene encoding the same). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a cosmetic protein (e.g., collagen protein, fibronectin, elastin, lumican, vitronectin / vitronectin receptor, laminin, neuromodulators, fibrillin, additional skin extracellular matrix proteins, etc.) (e.g., a transgene encoding the same). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for an antibody (e.g., a full-length antibody, an antibody fragment, etc.) (e.g., a transgene encoding the same). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a Kazal-type serine protease inhibitor (SPINK) polypeptide (e.g., a human SPINK polypeptide, such as a SPINK5 polypeptide) (e.g., a transgene encoding the same). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a filaggrin or filaggrin 2 polypeptide (e.g., a human filaggrin or filaggrin 2 polypeptide) (e.g., a transgene encoding the same). In some aspects, the polynucleotides of the disclosure do not include a coding sequence for (e.g., a transgene encoding) a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide (e.g., a human CFTR polypeptide). In some aspects, the polynucleotides of the disclosure do not include a coding sequence for (e.g., a transgene encoding) an ichthyosis-associated polypeptide (e.g.,ATP-binding cassette subfamily A member 12 polypeptide, 1-acylglycerol-3-phosphate O-acyltransferase ABHD5 polypeptide, aldehyde dehydrogenase family 3 member A2 polypeptide, arachidonate 12-lipoxygenase type 12R polypeptide, hydroperoxide isomerase ALOXE3 polypeptide, AP-1 complex subunit sigma-1A polypeptide, arylsulfatase E polypeptide, caspase-14 polypeptide, corneodesmosin polypeptide, ceramide synthase 3 polypeptide, carbohydrate sulfotransferase 8 polypeptide, claudin-1 polypeptide, cystatin-A polypeptide, cytochrome P450 4F22 polypeptide, 3-beta-hydroxysteroid-delta(8),delta(7)-isomerase polypeptide, very long chain fatty acid elongation protein 4 polypeptide, filaggrin polypeptide, filaggrin 2 polypeptide, gap junction beta-2 polypeptide, gap junction beta-3 polypeptide, gap junction beta-4 polypeptide, gap junction beta-6 polypeptide, 3-ketodihydrosphingosine reductase polypeptide, keratin, type II cytoskeleton 1 polypeptide, keratin, type II cytoskeleton 2 epithelial polypeptide, keratin, type I cytoskeleton 9 polypeptide, keratin, type I cytoskeleton 10 polypeptide, lipase member N polypeptide, loricrin polypeptide, membrane-bound transcription factor site-2 protease polypeptide, magnetite calcium transporter NIPA4 polypeptide, sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylation polypeptide, peroxisomal targeting signal 2 receptor polypeptide, D-3-phosphoglycerate dehydrogenase polypeptide, phytanoyl-CoA dioxygenase, peroxisomal polypeptide, patatin-like phospholipase domain-containing protein 1 polypeptide, proteasome maturation protein polypeptide, phosphoserine aminotransferase polypeptide, short-chain dehydrogenase / reductase family 9C member 7 polypeptide, serpin B8 polypeptide, long-chain fatty acid transport protein 4 polypeptide, synaptosomal associated protein 29 polypeptide, tumorigenicity suppressor 14 protein polypeptide,In some embodiments, the polynucleotides of the present disclosure do not include coding sequences for (e.g., transgenes encoding) collagen alpha-1 (VII) chain polypeptides, lysyl hydroxylase 3 polypeptides, keratin type I cytoskeleton 17 polypeptides, and / or any chimeric polypeptides thereof. In some embodiments, the polynucleotides of the disclosure are selected from the group consisting of collagen alpha-1 (VII) chain polypeptides, lysyl hydroxylase 3 polypeptides, keratin type I cytoskeleton 17 polypeptides, transglutaminase (TGM) polypeptides, filaggrin polypeptides, cosmetic proteins, antibodies, SPINK polypeptides, CFTR polypeptides, ichthyosis-associated polypeptides, alpha-1-antitrypsin polypeptides, sodium-dependent phosphate transport protein 2B polypeptides, dynein heavy chain 5 axonemal polypeptides, dynein heavy chain 11 axonemal polypeptides, coiled-coil domain-containing protein 39 polypeptides, dynein intermediate chain 1 axonemal polypeptides, coiled-coil domain-containing protein 40 polypeptides, coiled-coil domain-containing protein 103 polypeptides, sperm associated antigen 1 polypeptides, zinc finger MYND domain-containing protein 10 polypeptide, armadillo repeat-containing protein 4 polypeptide, coiled-coil domain-containing protein 151 polypeptide, dynein intermediate chain 2 axonemal polypeptide, radial spokehead 1 homolog polypeptide, coiled-coil domain-containing protein 114 polypeptide, radial spokehead protein 4 homolog A polypeptide, dynein assembly factor 1 axonemal polypeptide, dynein assembly factor 2 axonemal polypeptide, leucine-rich repeat-containing protein 6 polypeptide, pulmonary surfactant-associated protein B polypeptide, pulmonary surfactant-associated protein C polypeptide, homeobox protein Nkx-2.1 polypeptide, ATP-binding cassette subfamily A member 3 polypeptide, cytokine receptor common subunit beta polypeptide,Granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide, Bone morphogenetic protein receptor type 2 polypeptide, Sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide, Serine / threonine-protein kinase receptor R3 polypeptide, Endoglin polypeptide, Mothers against decapentaplegic decapentaplegic homolog 9 polypeptide, caveolin-1 polypeptide, potassium channel subfamily K member 3 polypeptide, eIF-2-alpha kinase GCN2 polypeptide, pulmonary surfactant associated protein A2 polypeptide, telomerase reverse transcriptase polypeptide, dyskerin polypeptide, telomere elongation regulator helicase 1 polypeptide, poly(A)-specific ribonuclease PARN polypeptide, TERF1-interacting nuclear factor 2 polypeptide, H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide, mucin-5B polypeptide, desmoplakin polypeptide, CST complex subunit STN1 polypeptide, dipeptidyl peptidase 9 polypeptide, and / or any chimeric polypeptide thereof (e.g., a transgene encoding the same).

[0126] Recombinant Nucleic Acids In some aspects, the disclosure relates to a recombinant nucleic acid comprising any one or more of the polynucleotides described herein. In some aspects, the recombinant nucleic acid is a vector (e.g., an expression vector, a display vector, etc.). In some aspects, the vector is a DNA vector or an RNA vector. Generally, any suitable vector can be used to maintain, propagate, and / or express the polynucleotide to produce one or more polypeptides in a subject. Examples of suitable vectors can include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, Sindbis virus vectors, measles vectors, herpes virus vectors, lentivirus vectors, retrovirus vectors, etc.). In some aspects, the vector is a herpes virus vector. In some aspects, the vector is capable of autonomous replication in a host cell. In some aspects, the vector is incapable of autonomous replication in a host cell. In some aspects, the vector can be integrated into host DNA. In some embodiments, the vector is not capable of integrating into the host DNA (e.g., is episomal). Methods for producing vectors containing one or more polynucleotides of interest, including, for example, by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid (e.g., by genetic engineering techniques), are well known to those of skill in the art.

[0127] In some embodiments, the recombinant nucleic acid of the present disclosure is a herpes simplex virus (HSV) amplicon. Herpes virus amplicons, including structural features, and methods for producing them are generally known to those skilled in the art (see, for example, de Silva S. and Bowers W. "Herpes Virus Amplicon Vectors". Viruses 2009, 1, 594-629). In some embodiments, the herpes simplex virus amplicon is an HSV-1 amplicon. In some embodiments, the herpes simplex virus amplicon is an HSV-1 hybrid amplicon. Examples of HSV-1 hybrid amplicons can include, but are not limited to, HSV / AAV hybrid amplicons, HSV / EBV hybrid amplicons, HSV / EBV / RV hybrid amplicons, and / or HSV / Sleeping Beauty hybrid amplicons. In some embodiments, the amplicon is an HSV / AAV hybrid amplicon. In some embodiments, the amplicon is an HSV / Sleeping Beauty hybrid amplicon.

[0128] In some embodiments, the recombinant nucleic acid of the present disclosure is a recombinant herpesvirus genome.The recombinant herpesvirus genome can be a recombinant genome from any member of the DNA virus known in the art of the Herpesviridae family, including, for example, recombinant herpes simplex virus genome, recombinant varicella zoster virus genome, recombinant human cytomegalovirus genome, recombinant herpesvirus 6A genome, recombinant herpesvirus 6B genome, recombinant herpesvirus 7 genome, recombinant Epstein-Barr virus genome, recombinant Kaposi's sarcoma-associated herpesvirus genome, and any combination or derivative thereof.In some embodiments, the recombinant herpesvirus genome comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) inactivating mutations. As used herein, "inactivating mutation" may refer to any mutation that results in a gene or regulon product (RNA or protein) with reduced, undetectable, or eliminated amount and / or function (e.g., compared to a corresponding sequence lacking the inactivating mutation). Examples of inactivating mutations may include, but are not limited to, deletions, insertions, point mutations, and rearrangements in the transcriptional control sequences (promoters, enhancers, insulators, etc.) and / or coding sequences of a given gene or regulon. Any suitable method of measuring the amount of a gene or regulon product known in the art may be used, including, for example, qPCR, Northern blot, RNAseq, Western blot, ELISA, etc. In some embodiments, the one or more inactivating mutations are in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) herpesvirus genes. In some embodiments, the recombinant herpesvirus genome is attenuated (e.g., compared to a corresponding wild-type herpesvirus genome). In some embodiments, the recombinant herpesvirus genome is replication-competent. In some embodiments, the recombinant herpesvirus genome is replication-deficient.In some embodiments, the recombinant herpesvirus genome is not oncolytic.

[0129] In some embodiments, the recombinant nucleic acid is a recombinant herpes simplex virus (HSV) genome. In some embodiments, the recombinant herpes simplex virus genome comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome is attenuated (e.g., compared to the corresponding wild-type herpes simplex virus genome). In some embodiments, the recombinant herpes simplex virus genome is replication-competent. In some embodiments, the recombinant herpes simplex virus genome is replication-deficient. In some embodiments, the recombinant herpes simplex virus genome is not oncolytic.

[0130] In some embodiments, the recombinant herpesvirus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. In some embodiments, the recombinant HSV-1 genome is, for example, 17, Ty25, R62, S25, Ku86, S23, R11, Ty148, Ku47, H166 syn, 1319-2005, F-13, M-12, 90237, F-17, KOS, 3083-2008, F12g, L2, CD38, H193, M-15, India 2011, 0116209, F-11I, 66-207, 2762, 369-2007, 3355, MacIntyre, McKrae, 7862, 7-h se, HF10, 1394,2005, 270-2007, OD4, SC16, M-19, 4J1037, 5J1060, J1060, KOS79, 132- The recombinant HSV-1 genome may be derived from any HSV-1 strain known in the art, including 1988, 160-1982, H166, 2158-2007, RE, 78326, F18g, F11, 172-2010, H129, F, E4, CJ994, F14g, E03, E22, E10, E06, E11, E25, E23, E35, E15, E07, E12, E14, E08, E19, E13, ATCC 2011 strains, etc. (see, e.g., Bowen et al. J Virol. 2019 Apr 3;93(8)). In some embodiments, the recombinant HSV-1 genome is derived from a KOS strain. In some embodiments, the recombinant HSV-1 genome is not derived from a McKrae strain. In some embodiments, the recombinant HSV-1 genome is attenuated (e.g., compared to a corresponding wild-type HSV-1 genome). In some embodiments, the recombinant HSV-1 genome is replication-competent. In some embodiments, the recombinant HSV-1 genome is replication-deficient. In some embodiments, the recombinant HSV-1 genome is not oncolytic.

[0131] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the infected cell protein (or infected cell polypeptide) (ICP)0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL)41, and / or UL55 herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP34.5 (one or both copies) and / or ICP47 herpes simplex virus genes (e.g., to avoid production of immunostimulatory virus). In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP34.5 (one or both copies) herpes simplex virus gene. In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP47 herpes simplex virus gene. In some embodiments, the recombinant herpes simplex virus genome does not contain inactivating mutations in ICP34.5 (one or both copies) and ICP47 herpes simplex virus genes.In some embodiments, the recombinant herpes simplex virus genome is not oncolytic.In some embodiments, the recombinant herpes simplex virus genome does not have conditional replication capability.In some embodiments, the recombinant herpes simplex virus genome does not have conditional replication capability in cancerous cells.

[0132] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and further comprises an inactivating mutation in the ICP4 (one or both copies), ICP22, ICP27, ICP47, UL41, and / or UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and an inactivating mutation in the ICP4 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), and an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, and / or UL41 genes.In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP27, ICP47, and / or UL55 gene.

[0133] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies) and further comprises an inactivating mutation in the ICP0 (one or both copies), ICP22, ICP27, ICP47, UL41, and / or UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies) and an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies) and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies), an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP4 (one or both copies), ICP22, and / or UL41 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 (one or both copies), ICP27, ICP47, and / or UL55 genes.

[0134] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene and further comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP27, ICP47, UL41, and / or UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene and an inactivating mutation in the UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP22 and / or UL41 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP27, ICP47, and / or UL55 genes.

[0135] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene and further comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP47, UL41, and / or UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP27 gene.

[0136] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene and further comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, UL41, and / or UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP47 gene.

[0137] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene and further comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, and / or UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the UL41 gene.

[0138] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene and further comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, and / or UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the UL55 gene.

[0139] In some embodiments, the recombinant herpes simplex virus genome comprises a long internal repeat (IR L ) regions and short internal repeats (IR SIn some embodiments, the inactivation (e.g., deletion) of the joint region eliminates one copy of each of the ICP4 and ICP0 genes. In some embodiments, the inactivation (e.g., deletion) of the joint region further inactivates (e.g., deletes) the promoters for the ICP22 and ICP47 genes. If desired, expression of one or both of these genes can be restored by insertion of an immediate early promoter into the recombinant herpes simplex virus genome (see, e.g., Hill et al. (1995). Nature 375(6530): 411-415; Goldsmith et al. (1998). J Exp Med 187(3): 341-348). Without wishing to be bound by theory, inactivation (e.g., deletion) of the joint region may contribute to the stability of the recombinant herpes simplex virus genome and / or enable the recombinant herpes simplex virus genome to accommodate more and / or larger transgenes.

[0140] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutation in the ICP4 (one or both copies), ICP27, and / or UL55 genes is a deletion of the coding sequence of the ICP4 (one or both copies), ICP27, and / or UL55 genes. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions in the promoter regions of the ICP22 and ICP47 genes (e.g., the ICP22 and ICP47 coding sequences are intact but not transcriptionally active). In some embodiments, the recombinant herpes simplex virus genome comprises deletions in the coding sequences of the ICP4 (one or both copies), ICP27, and UL55 genes, and deletions in the promoter regions of the ICP22 and ICP47 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 (one or both copies) and / or UL41 genes.

[0141] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 (one or both copies) gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 (one or both copies) and ICP4 (one or both copies) genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27 and / or UL55 gene comprises a deletion of the coding sequence of the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27 and / or UL55 gene. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP47 and / or UL41 gene.

[0142] In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure in one, two, three, four, five, six, seven or more viral loci. Examples of suitable viral loci may include, but are not limited to, ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, tk, UL41 and UL55 herpes simplex virus loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure in one or both viral ICP4 loci (e.g., recombinant viruses comprising a polynucleotide encoding a polypeptide (such as an immunomodulatory polypeptide) in one or both ICP4 loci). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure in the viral ICP22 locus (e.g., recombinant viruses comprising a polynucleotide encoding a polypeptide (such as an immunomodulatory polypeptide) in the ICP22 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral UL41 locus (e.g., a recombinant virus comprising a polynucleotide encoding a polypeptide (such as an immunomodulatory polypeptide) at the UL41 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral ICP27 locus (e.g., a recombinant virus comprising a polynucleotide encoding a polypeptide (such as an immunomodulatory polypeptide) at the ICP27 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral ICP47 locus (e.g., a recombinant virus comprising a polynucleotide encoding a polypeptide (such as an immunomodulatory polypeptide) at the ICP47 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral UL55 locus (e.g., a recombinant virus comprising a polynucleotide encoding a polypeptide (such as an immunomodulatory polypeptide) at the UL55 locus).In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral tk locus (e.g., a recombinant virus comprising a polynucleotide encoding a polypeptide (such as an immunomodulatory polypeptide) at the tk locus).

[0143] In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or both viral ICP4 loci and one or more polynucleotides of the present disclosure within the viral ICP22 locus (e.g., a recombinant virus comprising a polynucleotide encoding an immunomodulatory polypeptide (such as a human IL-12 polypeptide) at one or both ICP4 loci and a polynucleotide encoding an immunomodulatory polypeptide (e.g., a human GM-CSF polypeptide) at the ICP22 locus). In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or both viral ICP4 loci and one or more polynucleotides of the present disclosure within the viral UL41 locus (e.g., a recombinant virus comprising a polynucleotide encoding an immunomodulatory polypeptide (e.g., a human IL-12 polypeptide) at one or both ICP4 loci and a polynucleotide encoding an immunomodulatory polypeptide (e.g., a human GM-CSF polypeptide) at the UL41 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral ICP22 locus and one or more polynucleotides of the present disclosure within the viral UL41 locus (e.g., a recombinant virus comprising a polynucleotide encoding an immune modulating polypeptide (e.g., a human IL-12 polypeptide) at the ICP22 locus and a polynucleotide encoding an immune modulating polypeptide (e.g., a human GM-CSF polypeptide) at the UL41 locus).In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or both viral ICP4 loci, one or more polynucleotides of the present disclosure within the viral ICP22 locus, and one or more polynucleotides of the present disclosure within the viral UL41 locus (e.g., a recombinant virus comprising a polynucleotide encoding an immunomodulatory polypeptide (e.g., a human IL-12 polypeptide) at one or both ICP4 loci, a polynucleotide encoding an immunomodulatory polypeptide (e.g., a human GM-CSF polypeptide) at the ICP22 locus, and a polynucleotide encoding an immunomodulatory polypeptide (e.g., a human IL-2 polypeptide) at the UL41 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within one or both viral ICP4 loci, one or more polynucleotides of this disclosure within the viral ICP22 locus, one or more polynucleotides of this disclosure within the viral UL41 locus, one or more polynucleotides of this disclosure within the viral ICP27 locus, one or more polynucleotides of this disclosure within the viral ICP47 locus, one or more polynucleotides of this disclosure within the viral tk locus, and / or one or more polynucleotides of this disclosure within the viral UL55 locus.

[0144] In some embodiments, the recombinant herpesvirus genome (e.g., recombinant herpes simplex virus genome) is engineered to reduce or eliminate expression of one or more herpesvirus genes (e.g., one or more toxic herpesvirus genes), such as one or both copies of the HSV ICP0 gene, the HSV ICP4 gene, the HSV ICP22 gene, the HSV UL41 gene, the HSV ICP27 gene, the HSV ICP47 gene, the HSV tk gene, the HSV UL55 gene, and the like. In some embodiments, the recombinant herpesvirus genome (e.g., recombinant herpes simplex virus genome) is engineered to reduce the cytotoxicity of the recombinant genome (e.g., when introduced into a target cell) compared to the corresponding wild-type herpesvirus genome (e.g., a wild-type herpes simplex virus genome). In some embodiments, the target cell is a human cell (primary cell or a cell line derived therefrom). In some embodiments, the target cell is a respiratory cell (primary cell or a cell line derived therefrom). In some embodiments, the target cell is an airway epithelial cell (primary cell or a cell line derived therefrom). In some embodiments, the target cell is a pulmonary cell (primary cell or a cell line derived therefrom).In some embodiments, the cytotoxicity (e.g., in a target cell) of the recombinant genome (e.g., a recombinant herpes simplex virus genome) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about reduced by 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% (e.g., measuring the relative cytotoxicity of a recombinant ΔICP4 (one or both copies) herpes simplex virus genome compared to a wild-type herpes simplex virus genome in a target cell; measuring the relative cytotoxicity of a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome compared to a wild-type herpes simplex virus genome in a target cell, etc.). In some embodiments, the cytotoxicity (e.g., in a target cell) of a recombinant herpes genome (e.g., a recombinant herpes simplex virus genome) is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about reduced by about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, at least about 1000-fold, or more (e.g., measuring the relative cytotoxicity of a recombinant ΔICP4 (one or both copies) herpes simplex virus genome compared to a wild-type herpes simplex virus genome in a target cell; measuring the relative cytotoxicity of a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome compared to a wild-type herpes simplex virus genome in a target cell, etc.).Methods of measuring cytotoxicity are known to those of skill in the art, including through the use of vital dyes (formazan dyes), protease biomarkers, the MTT assay (or assays using related tetrazolium salts such as XTT, MTS, water soluble tetrazolium salts), measuring ATP content, and the like.

[0145] In some embodiments, the recombinant genome (e.g., recombinant herpes simplex virus genome) is engineered to reduce its effect on the proliferation of a target cell following exposure of the target cell to the recombinant genome compared to the corresponding wild-type genome (e.g., wild-type herpes simplex virus genome). In some embodiments, the target cell is a human cell (primary cell or a cell line derived therefrom). In some embodiments, the target cell is a respiratory cell (primary cell or a cell line derived therefrom). In some embodiments, the target cell is an airway epithelial cell (primary cell or a cell line derived therefrom). In some embodiments, the target cell is a pulmonary cell (primary cell or a cell line derived therefrom). In some embodiments, proliferation of target cells following exposure to a recombinant genome is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 102%, at least about 104%, at least about 105%, at least about 106%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 122%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, at least about 139%, at least about 140 at least about 90%, at least about 95%, or at least about 99% faster (e.g., measuring the relative cell proliferation following exposure to a recombinant ΔICP4 (one or both copies) herpes simplex virus genome compared to the cell proliferation following exposure to a wild-type herpes simplex virus genome in a target cell; measuring the relative cell proliferation following exposure to a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome compared to the cell proliferation following exposure to a wild-type herpes simplex virus genome in a target cell, etc.).In some embodiments, the proliferation of target cells following exposure to a recombinant genome is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500 times, at least about 750 times, or at least about 1000 times faster (e.g., measuring the relative cell proliferation after exposure to a recombinant ΔICP4 (one or both copies) herpes simplex virus genome compared to the cell proliferation after exposure to a wild-type herpes simplex virus genome in a target cell; measuring the relative cell proliferation after exposure to a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome compared to the cell proliferation after exposure to a wild-type herpes simplex virus genome in a target cell, etc.). Methods of measuring cell proliferation, including, for example, by using a Ki67 cell proliferation assay, a BrdU cell proliferation assay, and the like, are known to those of skill in the art.

[0146] A vector (e.g., a herpes virus vector) can include one or more polynucleotides of the present disclosure in a form suitable for expression of the polynucleotide in a host cell. The vector can include one or more regulatory sequences operably linked to the polynucleotide to be expressed (e.g., as described above).

[0147] In some aspects, the disclosure relates to one or more heterologous polynucleotides (eg, bacterial artificial chromosomes (BACs)) that comprise any of the recombinant nucleic acids described herein.

[0148] In some embodiments, the recombinant nucleic acid (e.g., recombinant herpes simplex virus genome) of the present disclosure comprises one or more of the polynucleotides described herein, inserted in any orientation into the recombinant nucleic acid. When the recombinant nucleic acid comprises two or more (e.g., two or more, three or more, etc.) polynucleotides described herein, the polynucleotides can be inserted in the same or opposite orientation to each other. Without wishing to be bound by theory, incorporating two polynucleotides (e.g., two transgenes) in antisense orientation into the recombinant nucleic acid (e.g., vector) can help avoid read-through and ensure proper expression of each polynucleotide.

[0149] In some embodiments, the recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a collagen alpha-1 (VII) chain polypeptide (COL7). In some embodiments, the recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a lysyl hydroxylase 3 polypeptide (LH3). In some embodiments, the recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a keratin type I cytoskeletal 17 polypeptide (KRT17). In some embodiments, the recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a transglutaminase (TGM) polypeptide (e.g., a human transglutaminase polypeptide, such as a human TGM1 polypeptide and / or a human TGM5 polypeptide). In some embodiments, the recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a cosmetic protein (e.g., a collagen protein, fibronectin, elastin, lumican, vitronectin / vitronectin receptor, laminin, neuromodulators, fibrillin, additional skin extracellular matrix proteins, etc.). In some embodiments, the recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding an antibody (e.g., a full-length antibody, an antibody fragment, etc.). In some embodiments, a recombinant nucleic acid of the disclosure does not include a polynucleotide encoding a Kazal-type serine protease inhibitor (SPINK) polypeptide (e.g., a human SPINK polypeptide, such as a SPINK5 polypeptide). In some embodiments, a recombinant nucleic acid of the disclosure does not include a polynucleotide encoding a filaggrin or filaggrin 2 polypeptide (e.g., a human filaggrin or filaggrin 2 polypeptide). In some embodiments, a recombinant nucleic acid of the disclosure does not include a polynucleotide encoding a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide (e.g., a human CFTR polypeptide). In some embodiments, a recombinant nucleic acid of the disclosure does not include a polynucleotide encoding an ichthyosis-associated polypeptide (e.g., an ATP-binding cassette subfamily A member 12 polypeptide, a 1-acylglycerol-3-phosphate O-acyltransferase ABHD5 polypeptide, an aldehyde dehydrogenase family 3 member A2 polypeptide,Arachidonic acid 12-lipoxygenase type 12R polypeptide, hydroperoxide isomerase ALOXE3 polypeptide, AP-1 complex subunit sigma-1A polypeptide, arylsulfatase E polypeptide, caspase-14 polypeptide, corneodesmosin polypeptide, ceramide synthase 3 polypeptide, carbohydrate sulfotransferase 8 polypeptide, claudin-1 polypeptide, cystatin-A polypeptide, cytochrome P450 4F22 polypeptide, 3-beta-hydroxysteroid-delta(8),delta(7)-isomerase polypeptide, very long chain fatty acid elongation protein 4 polypeptide, filaggrin polypeptide, filaggrin 2 polypeptide, gap junction beta-2 polypeptide, gap junction beta-3 polypeptide, gap junction beta-4 polypeptide, gap junction beta-6 polypeptide, 3-ketodihydrosphingosine reductase polypeptide, keratin, type II cytoskeleton 1 polypeptide, keratin, type II cytoskeleton 2 epithelial polypeptide, keratin, type I cytoskeleton 9 polypeptide, keratin, type I cytoskeleton 10 polypeptide, lipase member N polypeptide, loricrin polypeptide, membrane-bound transcription factor site-2 protease polypeptide, magnesium transporter NIPA4 polypeptide, sterol-4-alpha-carboxylic acid 3-dehydrogenase, decarboxime In some embodiments, the polypeptide does not include a polynucleotide encoding a polypeptide encoding a phosphoglycerate polypeptide, a peroxisomal targeting signal 2 receptor polypeptide, a D-3-phosphoglycerate dehydrogenase polypeptide, a phytanoyl-CoA dioxygenase, a peroxisomal polypeptide, a patatin-like phospholipase domain-containing protein 1 polypeptide, a proteasome maturation protein polypeptide, a phosphoserine aminotransferase polypeptide, a short-chain dehydrogenase / reductase family 9C member 7 polypeptide, a serpin B8 polypeptide, a long-chain fatty acid transport protein 4 polypeptide, a synaptosomal associated protein 29 polypeptide, a tumorigenicity suppressor 14 protein polypeptide, a steryl-sulfatase polypeptide, a vacuolar protein sorting-associated protein 33B polypeptide, and a CAAX prenyl protease 1 homolog polypeptide.The recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a collagen alpha-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeletal 17 polypeptide, and / or any chimeric polypeptide thereof. In some embodiments, the recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a collagen alpha-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeletal 17 polypeptide, a transglutaminase (TGM) polypeptide, a filaggrin polypeptide, a cosmetic protein, an antibody, a SPINK polypeptide, a CFTR polypeptide, an ichthyosis-associated polypeptide, an alpha-1-antitrypsin polypeptide, a sodium-dependent phosphate transport protein 2B polypeptide, a dynein heavy chain 5 axonemal polypeptide, a dynein heavy chain 11 axonemal polypeptide, a coiled-coil domain-containing protein 39 polypeptide, a dynein intermediate chain 1 axonemal polypeptide, a coiled-coil domain-containing protein 40 polypeptide, a coiled-coil domain-containing protein 103 polypeptide, a sperm associated antigen 1 polypeptide, a zinc finger MYND domain-containing protein 10 polypeptide, an armadillo repeat-containing protein 4 polypeptide, a coiled-coil domain-containing protein 151 polypeptide, dynein intermediate chain 2 axonemal polypeptide, radial spokehead 1 homolog polypeptide, coiled-coil domain-containing protein 114 polypeptide, radial spokehead protein 4 homolog A polypeptide, dynein assembly factor 1 axonemal polypeptide, dynein assembly factor 2 axonemal polypeptide, leucine-rich repeat-containing protein 6 polypeptide, pulmonary surfactant-associated protein B polypeptide, pulmonary surfactant-associated protein C polypeptide, homeobox protein Nkx-2.1 polypeptide, ATP-binding cassette subfamily A member 3 polypeptide, cytokine receptor common subunit beta polypeptide, granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide, bone morphogenetic protein receptor type 2 polypeptide, sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide, serine / threonine-protein kinase receptor R3 polypeptide, endoglin polypeptide,The invention does not include a polynucleotide encoding a mother's against decapentaplegic homolog 9 polypeptide, a caveolin-1 polypeptide, a potassium channel subfamily K member 3 polypeptide, an eIF-2-alpha kinase GCN2 polypeptide, a pulmonary surfactant associated protein A2 polypeptide, a telomerase reverse transcriptase polypeptide, a dyskerin polypeptide, a telomere elongation regulator helicase 1 polypeptide, a poly(A)-specific ribonuclease PARN polypeptide, a TERF1-interacting nuclear factor 2 polypeptide, a H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide, a mucin-5B polypeptide, a desmoplakin polypeptide, a CST complex subunit STN1 polypeptide, a dipeptidyl peptidase 9 polypeptide, and / or any chimeric polypeptide thereof.

[0150] IV. Viruses Certain aspects of the present disclosure relate to a virus comprising any of the polynucleotides and / or recombinant nucleic acids described herein. In some embodiments, the virus is capable of infecting one or more target cells of a subject (e.g., a human). In some embodiments, the virus is suitable for delivering the polynucleotides and / or recombinant nucleic acids to one or more target cells of a subject (e.g., a human). In some embodiments, the one or more target cells are human cells. In some embodiments, the one or more target cells are one or more airway epithelial cells. In some embodiments, the one or more target cells are one or more cells of the respiratory tract (e.g., airway epithelial cells (e.g., goblet cells, ciliated cells, Clara cells, neuroendocrine cells, basal cells, intermediate or parabasal cells, serous cells, brush cells, oncocytes, non-ciliated columnar cells, and / or metaplastic cells); alveolar cells (e.g., type 1 pneumocytes, type 2 pneumocytes, and / or cuboidal non-ciliated cells); salivary gland cells in the bronchi (e.g., serous cells, mucus cells, and / or duct cells), etc.). In some embodiments, the one or more target cells are one or more cells of the lung.

[0151] Any suitable virus known in the art may be used, including, for example, adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, papilloma virus, herpes virus (e.g., herpes simplex virus), vaccinia virus, and / or any hybrid or derivative virus thereof. In some embodiments, the virus is attenuated. In some embodiments, the virus is replication-competent. In some embodiments, the virus is replication-deficient. In some embodiments, the virus is not oncolytic. In some embodiments, the virus is modified to alter its tissue tropism compared to the tissue tropism of the corresponding unmodified wild-type virus. In some embodiments, the virus has reduced cytotoxicity (e.g., in target cells) compared to the corresponding wild-type virus. Methods for producing viruses containing recombinant nucleic acids are well known to those skilled in the art.

[0152] In some embodiments, the virus is a member of the Herpesviridae DNA virus, including, for example, herpes simplex virus, varicella zoster virus, human cytomegalovirus, herpes virus 6A, herpes virus 6B, herpes virus 7, Epstein-Barr virus, and Kaposi's sarcoma-associated herpes virus. In some embodiments, the herpes virus is attenuated. In some embodiments, the herpes virus is replication-deficient. In some embodiments, the herpes virus is replication-competent. In some embodiments, the herpes virus is engineered to reduce or eliminate expression of one or more herpes virus genes (e.g., one or more toxic herpes virus genes). In some embodiments, the herpes virus has reduced cytotoxicity compared to the corresponding wild-type herpes virus. In some embodiments, the herpes virus is not oncolytic.

[0153] In some embodiments, the virus is a herpes simplex virus. Herpes simplex viruses comprising recombinant nucleic acids are described in, for example, U.S. Pat. No. 10,174,341, U.S. Pat. No. 2019 / 0276845, U.S. Pat. No. 9,877,990, U.S. Pat. No. 10,155,016, U.S. Pat. No. 10,441,614, U.S. Pat. No. 11,185,564, U.S. Pat. No. 2020 / 0093874, U.S. Pat. No. 10,525,090, U.S. Pat. No. 2020 / 0197456, U.S. Pat. No. 2021 / 0261649, U.S. Pat. No. 2021 / 0395775, U.S. Pat. No. 10,829,529, U.S. Pat. No. 2021 / 0087245, U.S. Pat. No. 2021 / 0189427, U.S. Pat. No. 10,786,438, U.S. Pat. 2021 / 0045988, and / or WO2021 / 046131, all of which are incorporated herein by reference in their entirety. In some embodiments, the herpes simplex virus is attenuated. In some embodiments, the herpes simplex virus is replication-deficient. In some embodiments, the herpes simplex virus is replication-competent. In some embodiments, the herpes simplex virus is engineered to reduce or eliminate expression of one or more herpes simplex virus genes (e.g., one or more toxic herpes simplex virus genes). In some embodiments, the herpes simplex virus has reduced cytotoxicity compared to the corresponding wild-type herpes simplex virus. In some embodiments, the herpes simplex virus is not oncolytic. In some embodiments, the herpes simplex virus is HSV-1, HSV-2, or any derivative thereof. In some embodiments, the herpes simplex virus is an HSV-1 virus. In some embodiments, the HSV-1 is attenuated. In some embodiments, the HSV-1 is replication-deficient. In some embodiments, the HSV-1 is replication-competent. In some embodiments, the HSV-1 is engineered to reduce or eliminate expression of one or more HSV-1 genes (e.g., one or more toxic HSV-1 genes). In some embodiments, the HSV-1 has reduced cytotoxicity compared to the corresponding wild-type HSV-1. In some embodiments, the HSV-1 is not oncolytic.

[0154] In some embodiments, the herpes simplex virus is modified to change its tissue tropism compared to that of the unmodified wild-type herpes simplex virus. In some embodiments, the herpes simplex virus comprises a modified envelope. In some embodiments, the modified envelope comprises one or more (e.g., one or more, two or more, three or more, four or more, etc.) mutant herpes simplex virus glycoproteins. Examples of herpes simplex virus glycoproteins can include, but are not limited to, glycoproteins gB, gC, gD, gH, and gL. In some embodiments, the modified envelope changes the tissue tropism of the herpes simplex virus compared to that of the wild-type herpes simplex virus.

[0155] In some embodiments, the transduction efficiency (in vitro and / or in vivo) of a virus of the present disclosure (e.g., a herpes virus, such as a herpes simplex virus) for one or more target cells (e.g., one or more cells of the respiratory tract) is at least about 25%. For example, the transduction efficiency of the virus for one or more target cells may be at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 99.5%, or more. In some embodiments, the virus is a herpes simplex virus and the transduction efficiency of the virus for one or more target cells (e.g., one or more cells of the respiratory tract) is about 85% to about 100%. In some embodiments, the virus is a herpes simplex virus, and the viral transduction efficiency for one or more target cells (e.g., one or more cells of the respiratory tract) is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%. Methods for measuring viral transduction efficiency in vitro or in vivo are well known to those skilled in the art, including, for example, qPCR analysis, deep sequencing, Western blotting, fluorescence analysis (e.g., fluorescent in situ hybridization (FISH), fluorescent reporter gene expression, immunofluorescence, FACS), and the like.

[0156] In some embodiments, provided herein are recombinant viruses, which may or may not be pseudotyped, that produce one or more therapeutic polypeptides for the treatment of cancer, including solid tumors (e.g., advanced solid tumors) and hematological malignancies. In some embodiments, the recombinant viruses are not oncolytic. In some embodiments, the one or more therapeutic polypeptides produced by the recombinant viruses described herein mediate or enhance anti-tumor effects, such as by effector cell-mediated lysis of tumor cells. The present disclosure further provides therapeutic compositions comprising the recombinant viruses and methods of use in the treatment of solid tumors and hematological malignancies.

[0157] In some embodiments, the therapeutic polypeptide is an immunomodulatory polypeptide. In some embodiments, the immunomodulatory polypeptide modulates the activity of one or more cell types, such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), dendritic cells, T cells, macrophages, neutrophils, and / or NK cells.

[0158] V. Pharmaceutical Compositions and Formulations Certain aspects of the present disclosure relate to pharmaceutical compositions or formulations comprising any of the recombinant nucleic acids described herein (e.g., recombinant herpes virus genomes) and / or viruses described herein (e.g., herpes viruses comprising a recombinant genome) (e.g., herpes simplex viruses comprising a recombinant herpes simplex virus genome), and a pharma- ceutically acceptable excipient or carrier.

[0159] In some embodiments, the pharmaceutical composition or formulation comprises any one or more of the viruses (e.g., herpes viruses) described herein. In some embodiments, the pharmaceutical composition or formulation comprises about 10 4 ~about 10 12 For example, the pharmaceutical composition or formulation may contain about 10 plaque forming units (PFU) / mL of virus. 4 ~about 10 12 , about 10 5 ~about 10 12 , about 106 ~about 10 12 , about 10 7 ~about 10 12 , about 10 8 ~about 10 12 , about 10 9 ~about 10 12 , about 10 10 ~about 10 12 , about 10 11 ~about 10 12 , about 10 4 ~about 10 11 , about 10 5 ~about 10 11 , about 10 6 ~about 10 11 , about 10 7 ~about 10 11 , about 10 8 ~about 10 11 , about 10 9 ~about 10 11 , about 10 10 ~about 10 11 , about 10 4 ~about 10 10 , about 10 5 ~about 10 10 , about 10 6 ~about 10 10 , about 10 7 ~about 10 10 , about 10 8 ~about 10 10 , about 10 9 ~about 10 10 , about 10 4 ~about 10 9 , about 10 5 ~about 10 9 , about 10 6 ~about 10 9 , about 10 7 ~about 10 9 , about 10 8 ~about 10 9 , about 10 4 ~about 10 8 , about 10 5 ~about 10 8 , about 10 6 ~ approx. 108, approx. 10 7 ~about 10 8 , about 10 4 ~about 10 7 , about 10 5 ~about 10 7, about 10 6 ~about 10 7 , about 10 4 ~about 10 6 , about 10 5 ~about 10 6 , or about 10 4 ~about 10 5 In some embodiments, the pharmaceutical composition or formulation may contain about 10 PFU / mL of virus. 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , or about 10 12 Contains PFU / mL of virus.

[0160] Pharmaceutical compositions and formulations can be prepared by mixing an active ingredient (such as a recombinant nucleic acid and / or a virus) having a desired degree of purity with one or more pharma- ceutically acceptable carriers or excipients. Pharmaceutically acceptable carriers or excipients are generally non-toxic to recipients at the dosages and concentrations employed and may contain any of the following: buffers (such as phosphates, citrates, acetates, and other organic acids); antioxidants (such as ascorbic acid and methionine); preservatives (such as octadecyldimethylbenzylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine); low molecular weight (less than about 10 residues) polypeptides; proteins (serum albumin, arginine, arginine, lysine, argin ... The carriers may include, but are not limited to, polyols (such as glycerol, e.g., formulations containing 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc., of glycerol); hydrophilic polymers (such as polyvinylpyrrolidone); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (such as sodium); metal complexes (such as Zn-protein complexes); and / or non-ionic surfactants (such as polyethylene glycol (PEG)). A thorough description of pharma- ceutically acceptable carriers is available from REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991).

[0161] In some embodiments, the pharmaceutical composition or formulation comprises one or more lipid (e.g., cationic lipid) carriers. In some embodiments, the pharmaceutical composition or formulation comprises one or more nanoparticle carriers. Nanoparticles are submicron (less than about 1000 nm) sized drug delivery vehicles that can carry encapsulated drugs (synthetic small molecules, proteins, peptides, cells, viruses, and nucleic acid-based biotherapeutics) for rapid or controlled release. A variety of molecules (e.g., proteins, peptides, recombinant nucleic acids, etc.) can be efficiently encapsulated in nanoparticles using methods well known in the art. In some embodiments, a molecule "encapsulated" in a nanoparticle can refer to a molecule (such as a virus) that is contained within the nanoparticle or attached and / or bound to the surface of the nanoparticle, or any combination thereof. Nanoparticles for use in the compositions or formulations described herein can be any type of biocompatible nanoparticle known in the art, including, for example, nanoparticles comprising poly(lactic acid), poly(glycolic acid), PLGA, PLA, PGA, and any combination thereof (see, e.g., Vauthier et al. Adv Drug Del Rev. (2003) 55: 519-48; US 2007 / 0148074; US 2007 / 0092575; US 2006 / 0246139; U.S. Pat. No. 5,753,234; U.S. Pat. No. 7,081,483; and US 2008 / 0260851, all of which are incorporated by reference herein in their entireties).

[0162] In some embodiments, the pharma- ceutically acceptable carrier or excipient may be adapted or suitable for any route of administration known in the art, including, for example, intravenous, intramuscular, subcutaneous, dermal, oral, intranasal, intratracheal, sublingual, buccal, topical, transdermal, intradermal, intraperitoneal, intraorbital, intravitreal, subretinal, transmucosal, intraarticular, implant, inhalation, intrathecal, intraventricular, and / or intranasal administration. In some embodiments, the pharma- ceutically acceptable carrier or excipient is adapted or suitable for oral, intranasal, intratracheal, and / or inhalation administration. In some embodiments, the pharma- ceutically acceptable carrier or excipient is adapted or suitable for intranasal and / or inhalation administration. In some embodiments, the pharma- ceutically acceptable carrier or excipient is adapted or suitable for inhalation administration.

[0163] In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for any route of administration known in the art, including, for example, intravenous, intramuscular, subcutaneous, dermal, oral, intranasal, intratracheal, sublingual, buccal, topical, transdermal, intradermal, intraperitoneal, intraorbital, intravitreal, subretinal, transmucosal, intraarticular, implant, inhalation, intrathecal, intraventricular, or intranasal administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for oral, intranasal, intratracheal, and / or inhalation administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for intranasal and / or inhalation administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for inhalation administration.

[0164] In some embodiments, the pharmaceutical composition or formulation further comprises one or more additional components. Examples of additional components include binders (such as pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, maize starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.). The polynucleotides may include, but are not limited to, agents that can stabilize polynucleotides or prevent their degradation, such as, but not limited to, cellulose acetate, cellulose esters ... In some embodiments, the pharmaceutical composition or formulation comprises glycerol (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, etc.). In some embodiments, the pharmaceutical composition or formulation comprises a phosphate buffer and glycerol.

[0165] Pharmaceutical compositions and formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0166] In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to deliver one or more polynucleotides encoding a polypeptide (e.g., an immunomodulatory polypeptide, e.g., an IL-2 or IL-12 polypeptide) into one or more cells of a subject (e.g., one or more cells of the respiratory tract of a subject). In some embodiments, the subject is suffering from cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat carcinoma. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat lymphoma. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat blastoma. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat sarcomas. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat neuroendocrine tumors. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat mesotheliomas. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat schwannomas. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat meningiomas. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat adenocarcinomas.In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat melanoma. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat leukemia. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat lymphoid malignancies.

[0167] In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to deliver one or more polynucleotides encoding a polypeptide (e.g., an immune-modulating polypeptide, such as an IL-2 or IL-12 polypeptide) into one or more cells of a subject (e.g., one or more cells of the respiratory tract of a subject). In some embodiments, the subject is suffering from cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat solid tumors. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat hematological cancers. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat bladder cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat brain cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat breast cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat colon cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat gastric cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat glioma. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat head cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat leukemia.In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat liver cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat lung cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat lymphoma. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat myeloma. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat cervical cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat ovarian cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat melanoma. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat pancreatic cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat renal cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat salivary gland cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat skin cancer (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, atypical nevi, and / or Merkel cell carcinoma). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat gastric cancer.In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat thymic epithelial carcinoma. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat thyroid cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat osteosarcoma.

[0168] In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to deliver one or more polynucleotides encoding a polypeptide (e.g., an immunomodulatory polypeptide, such as an IL-2 or IL-12 polypeptide) into one or more cells of a subject (e.g., one or more cells of the respiratory tract of a subject). In some embodiments, the subject suffers from lung cancer. Lung cancer is often divided into the broad classification of small cell lung cancer (SCLC), also called oat cell carcinoma, and non-small cell lung cancer (NSCLC). NSCLC is further divided into three main types: squamous cell carcinoma (SCC), adenocarcinoma, and large cell carcinoma. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat small cell lung cancer. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat lung adenocarcinoma. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat lung squamous cell carcinoma.In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat non-small cell lung cancer.

[0169] VI. Method Certain aspects of the present disclosure relate to methods of delivering an immunomodulatory polypeptide to one or more cells of a subject (e.g., one or more cells of the respiratory tract, e.g., airway epithelial cells (goblet cells, ciliated cells, Clara cells, neuroendocrine cells, basal cells, intermediate or parabasal cells, serous cells, brush cells, oncocytes, non-ciliated columnar cells, and / or metaplastic cells); alveolar cells (type 1 pneumocytes, type 2 pneumocytes, and / or cuboidal non-ciliated cells); salivary gland cells in the bronchi (serous cells, mucus cells, and / or duct cells), etc.), comprising administering to the subject a pharmaceutical composition comprising any of the viruses described herein (e.g., herpes simplex virus, e.g., HSV-1), including any of the recombinant nucleic acids described herein (e.g., recombinant herpes simplex virus genomes, e.g., recombinant HSV-1 genomes) that comprise one or more polynucleotides encoding an immunomodulatory polypeptide, and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, bucally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, intratumorally, topically, or via inhalation. In some embodiments, the pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the pharmaceutical composition is administered to the subject intranasally or via inhalation. In some embodiments, the pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the herpes virus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpes virus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer. In some embodiments, the herpes virus (e.g., herpes simplex virus) is replication competent. In some embodiments, the herpes virus (e.g., herpes simplex virus) is conditionally replication incompetent. In some embodiments, the herpes virus (e.g., herpes simplex virus) does not replicate in cancerous cells.In some embodiments, the herpes virus (e.g., herpes simplex virus) is replication-deficient. In some embodiments, the herpes virus (e.g., herpes simplex virus) is not oncolytic.

[0170] In some embodiments, the subject is a human. In some embodiments, the subject suffers from cancer. In some embodiments, the cancer is acute myeloid leukemia (LAML or AML), acute lymphoblastic leukemia (ALL), adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), brain stem glioma, brain low-grade glioma (LGG), brain tumor, breast cancer (BRCA), bronchial tumor, Burkitt's lymphoma, cancer of unknown primary, carcinoid tumor, carcinoma of unknown primary, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, cervical squamous tumor, Skin cancer, Cervical adenocarcinoma (CESC), Childhood cancer, Bile duct cancer (CHOL), Chordoma, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Chronic myeloproliferative disorder, Colon cancer (adenocarcinoma) (COAD), Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Endocrine islet cell tumor, Endometrial cancer, Ependymoblastoma, Ependymoma, Esophageal cancer (ESCA), Esthesioneuroblastoma, Ewing's sarcoma, Extracranial germ cell tumor, Extragonadal germ cell tumor, Extrahepatic bile duct cancer, Gallbladder cancer, Gastric (gastric) stric / stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioblastoma multiforme (GBM), hairy cell leukemia, head and neck cancer (HNSD), cardiac cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, liver cancer, lymphoid neoplasms Diffuse large B-cell lymphoma [D LBCL), malignant fibrous histiocytoma bone cancer, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, Merkel cell skin cancer, mesothelioma (MESO), metastatic squamous neck cancer of unknown primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative neoplasm, nasal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer cancer), oral cavity cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumors, ovarian low malignant potential tumors, pancreatic cancer, papilloma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pheochromocytoma and paraganglioma (PCPG), moderately differentiated pineal parenchymal tumor, pineoblastoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma,Primary central nervous system (CNS) lymphoma, primary hepatocellular carcinoma, prostate cancer including prostate adenocarcinoma (PRAD), rectal cancer, renal cell (kidney) cancer, renal cell carcinoma, respiratory tract cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (SARC), Sézary syndrome, cutaneous melanoma (SKCM), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell neck cancer, stomach / gastric cancer , supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer testicular germ cell tumor (TGCT), throat cancer, thymic cancer, thymoma (THYM), thyroid cancer (THCA), transitional cell carcinoma, renal pelvis and ureter transitional cell carcinoma, trophoblastic tumor, ureteral cancer, urethral cancer, uterine cancer, uterine cancer, uveal melanoma (UVM), vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and / or Wilms' tumor. In some embodiments, the cancer is a virus-associated cancer. In some embodiments, the cancer is a human papillomavirus (HPV)-associated cancer (e.g., HPV-associated cancer of the back of the throat, cervix, anus, vulva, penis, and / or vagina). In some embodiments, the cancer is not a skin cancer (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, atypical nevi, and / or Merkel cell carcinoma). In some embodiments, the cancer is not a melanoma. In some embodiments, the subject suffers from one or more of carcinoma, lymphoma, blastoma, sarcoma, neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, leukemia, and lymphoid malignancies. In some embodiments, the subject suffers from one or more of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, renal cancer, salivary gland cancer, skin cancer, stomach cancer, thymic epithelial cancer, and thyroid cancer. In some embodiments, the subject suffers from one or more of small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma. In some embodiments, the subject suffers from osteosarcoma.

[0171] In some embodiments, administration of a recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation to a subject increases the level of an immune modulating polypeptide (transcript or protein level) in one or more contacted or treated cells of the subject by at least about two-fold compared to the endogenous level of the polypeptide in one or more corresponding untreated cells in the subject. For example, administration of a recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation may increase the level of an immunomodulatory polypeptide (transcript or protein level) in one or more contacted or treated cells of a subject by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, at least about 1000-fold, or more, compared to the endogenous level of the polypeptide in one or more corresponding untreated cells in the subject. In some embodiments, the one or more contacted or treated cells are one or more cells of the respiratory tract (e.g., one or more cells of the airway epithelium). Methods for measuring transcript or protein levels from a sample are well known to those of skill in the art, including, for example, qPCR, Western blot, mass spectrometry, and the like.

[0172] Another aspect of the present disclosure relates to a method of providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of cancer in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is a human. In some embodiments, the subject is suffering from cancer. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (LAML or AML), acute lymphoblastic leukemia (ALL), adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), brain stem glioma, brain low-grade glioma (LGG), brain tumor, breast cancer (BRCA), bronchial tumor, Burkitt's lymphoma, carcinoma of unknown primary origin, carcinoid tumor, carcinoma of unknown primary origin, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, cervical squamous cell carcinoma, cervical adenocarcinoma. CESC, childhood cancer, bile duct cancer (CHOL), chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer (adenocarcinoma) (COAD), colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine islet cell tumor, endometrial cancer, ependymomas, ependymoma, esophageal cancer (ESCA), esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric / stomach cancer, digestive Intestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioblastoma multiforme (GBM), hairy cell leukemia, head and neck cancer (HNSD), cardiac cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, liver cancer, lymphoid neoplasms (diffuse large B-cell lymphoma [DLBCL]), malignant fibrous histiocytoma (malignant fibrous histiocytoma) bone cancer, medulloblastoma, medulloepithelioma, black Chromoma, Merkel cell carcinoma, Merkel cell skin cancer, Mesothelioma (MESO), Metastatic squamous cell neck cancer of unknown primary site, Oral cancer, Multiple endocrine neoplasia syndrome, Multiple myeloma, Multiple myeloma / plasma cell neoplasm, Mycosis fungoides, Myelodysplastic syndrome, Myeloproliferative neoplasm, Nasal cancer, Nasopharyngeal cancer, Neuroblastoma, Non-Hodgkin's lymphoma, Non-melanoma skin cancer, Non-small cell lung cancer, Oral cancer, Oral cancer, Ovarian cancer, Ovarian germ cell tumor, Osteosarcoma, Other brain and spinal tumors, Ovarian cancer, Ovarian epithelial cancer, Ovarian germ cell tumor,Ovarian low malignant potential tumor, pancreatic cancer, papilloma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pheochromocytoma and paraganglioma (PCPG), moderately differentiated pineal parenchymal tumor, pineoblastoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, primary hepatocellular carcinoma, prostate cancer including prostate adenocarcinoma (PRAD), rectal cancer, renal cell (kidney) cancer, renal cell carcinoma, respiratory tract cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (SARC), Sézary syndrome, cutaneous melanoma (SKCM), small The cancer is selected from lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical cancer, stomach / gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer / testicular germ cell tumor (TGCT), throat cancer, thymic cancer, thymoma (THYM), thyroid cancer (THCA), transitional cell carcinoma, renal pelvis / ureter transitional cell carcinoma, trophoblastic tumor, ureteral cancer, urethral cancer, uterine cancer, uterine cancer, uveal melanoma (UVM), vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and / or Wilms' tumor. In some embodiments, the cancer is a virus-associated cancer. In some embodiments, the cancer is a human papillomavirus (HPV)-associated cancer (e.g., HPV-associated cancer of the back of the throat, cervix, anus, vulva, penis, and / or vagina). In some embodiments, the cancer is not a skin cancer (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, atypical nevi, and / or Merkel cell carcinoma). In some embodiments, the cancer is not a melanoma. In some embodiments, the subject does not suffer from one or more of carcinoma, lymphoma, blastoma, sarcoma, neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, leukemia, and lymphoid malignancies. In some embodiments, the subject suffers from one or more of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, renal cancer, salivary gland cancer, gastric cancer, thymic epithelial cancer, and thyroid cancer. In some embodiments, the subject has one or more of small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma. In some embodiments, the subject has osteosarcoma.

[0173] The recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation described herein may be administered by any suitable method or route known in the art, including, but not limited to, oral, intranasal, intratracheal, sublingual, buccal, topical, rectal, inhalation, transdermal, subcutaneous, intradermal, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravaginal, intravitreal, intraorbital, subretinal, intraarticular, periarticular, topical, epicutaneous, or any combination thereof. Thus, the present disclosure encompasses a method of delivering any of the recombinant nucleic acid, virus, medicament, or pharmaceutical composition or formulation described herein to an individual (e.g., an individual with cancer). In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation described herein is administered orally, intranasally, intratracheally, and / or via inhalation. In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation described herein is administered intranasally or via inhalation. In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation described herein is administered via inhalation.In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation described herein is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device.In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation described herein is administered using a nebulizer.In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0174] Methods of delivering drugs to the airways and / or lungs via oral, intranasal, intratracheal, and / or inhalation routes of administration are generally known to those of skill in the art (see, e.g., Gardenhire et al. A Guide to Aerosol Delivery Devices for Respiratory Therapists, 4 thEdition, American Association for Respiratory care, 2017; Patil et al. Pulmonary Drug Delivery Strategies: A Concise, Systematic Review, Lung India. 2012. 29(1):44-9; Marx et al. Intranasal Drug Administration - An Attractive Delivery Route for Some Drugs, 2015).

[0175] In some embodiments, the recombinant nucleic acid, virus, medicament, and / or composition or formulation is delivered to the lung by inhalation of an aerosolized formulation. In some embodiments, the recombinant nucleic acid, virus, medicament, and / or composition or formulation is delivered to the lung by inhalation of an aerosolized formulation after resection of a lung tumor. Inhalation can occur via the subject's nose and / or mouth. Exemplary devices for delivering recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation to the lung can include, but are not limited to, dry powder inhalers, pressurized metered dose inhalers, soft mist inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, vibrating mesh nebulizers), colliding jets, extruded jets, surface wave microfluidic nebulization, capillary aerosol generation, electrohydrodynamic aerosol devices, and the like (see, e.g., Carvalho and McConville. The function and performance of aqueous devices for inhalation therapy. (2016) Journal of Pharmacy and Pharmacology).

[0176] Liquid formulations can be administered to the lungs of a subject using, for example, a pressurized metered dose inhaler (pMDI). A pMDI generally includes at least two components: a canister in which the liquid formulation is contained under pressure in combination with one or more propellants, and a receptacle used to contain and actuate the canister. The canister may contain a single or multiple doses of the formulation. The canister may include a valve, typically a metered valve, from which the contents of the canister may be released. Aerosolized drugs are dispensed from a pMDI by applying force to the canister to force it into the receptacle, thereby opening the valve and transferring drug particles from the valve through the receptacle outlet. Upon release from the canister, the liquid formulation is atomized to form an aerosol. A pMDI typically employs one or more propellants to pressurize the contents of the canister and to expel the liquid formulation from the receptacle outlet to form an aerosol. Any suitable propellant may be employed and may take a variety of forms including, for example, compressed gas or liquefied gas.

[0177] Liquid formulations can be administered to the lungs of a subject, for example, using a nebulizer. A nebulizer is a liquid aerosol generator that converts liquid formulations into a mist or a cloud of droplets, often with an aerodynamic mass median diameter of less than about 5 microns in diameter, that can be inhaled into the lower respiratory tract. When the aerosol cloud is inhaled, the droplets carry the active agent to the nose, upper respiratory tract, and / or deep lungs. Any type of nebulizer known in the art can be used to administer the formulation to a patient, including, but not limited to, pneumatic (jet) nebulizers, electromechanical nebulizers (e.g., ultrasonic nebulizers, vibrating mesh nebulizers), and the like. Pneumatic (jet) nebulizers use a pressurized gas supply as a driving force for the atomization of liquid formulations. Compressed gas is delivered through a nozzle or jet to create a low pressure field that entrains the surrounding liquid formulation and shears it into a thin film or filament. This film or filament is unstable and breaks down into droplets, which are carried into inspiration by the compressed gas flow. A baffle inserted into the droplet plume intercepts larger droplets and returns them to the bulk liquid reservoir. Electromechanical nebulizers use electrically generated mechanical force to atomize liquid formulations. Electrical driving force can be applied, for example, by vibrating the liquid formulation at ultrasonic frequencies or by forcing the bulk liquid through small holes in a thin film. The force creates a thin liquid film or filament stream that breaks into droplets to form a slow-moving aerosol stream that can be entrained into the inspiratory air stream. In some embodiments, the nebulizer is a vibrating mesh nebulizer. Examples of vibrating mesh nebulizers include, for example, Phillips InnoSpire, Aerogen Solo, PARI eFlow, etc.

[0178] Liquid formulations can be administered to the lungs of a subject, for example, using an electrohydrodynamic (EHD) aerosol device, which uses electrical energy to aerosolize a liquid drug solution or suspension.

[0179] Dry powder formulations can be administered to the lungs of a subject using, for example, a dry powder inhaler (DPI). DPIs typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which the subject can then inhale. In DPIs, the dose to be administered is stored in the form of a non-pressurized dry powder, and upon actuation of the inhaler, the powder particles are inhaled by the subject. In some cases, compressed gas can be used to dispense the powder, similar to pMDIs. In some cases, DPIs can be breath-activated (aerosols are created in exact response to inspiration). Typically, dry powder inhalers administer doses of less than tens of milligrams per inhalation to avoid inducing coughs. Examples of DPIs include, for example, Turbohaler® inhaler (AstraZeneca), Clickhaler® inhaler (Innovata), Diskus® inhaler (Glaxo), EasyHaler® (Orion), Exubera® inhaler (Pfizer), and the like.

[0180] In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation is administered to the subject once. In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition is administered to the subject at least twice (e.g., at least twice, at least three times, at least four times, at least five times, at least ten times, etc.). In some embodiments, at least about 1 hour (e.g., at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 15 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, at least about 120 days, etc.) elapses between administrations (e.g., between a first and second administration, between a second and third administration, etc.). In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation is administered to a subject 1, 2, 3, 4, 5 or more times per day. In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation is administered to a subject 1, 2, 3, 4, 5 or more times per month.

[0181] VII. Host cells Certain aspects of the present disclosure pertain to one or more host cells containing any of the recombinant nucleic acids described herein. For example, prokaryotic cells including eubacteria, e.g., gram-negative or gram-positive organisms, e.g., Enterobacteriaceae, e.g., Escherichia (e.g., E. coli), Enterobacter, Erminia, Klebsiella, Proteus, Salmonella (e.g., S. typhimurium), Serratia (e.g., S. marcescans), and Shigella, as well as Bacillus, e.g., B. subtilis and B. licheniformis; fungal cells (e.g., S. cerevisiae, S. aeruginosa, S. cerevisiae ... cerevisiae); insect cells (e.g., S2 cells); and SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli cells (TM4), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC Any suitable host cell (prokaryotic or eukaryotic) known in the art may be used, including mammalian cells, including human hepatoma cells, including CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (Hep G2), Chinese hamster ovary (CHO) cells, including DHFR" CHO cells, and myeloma cell lines, such as NS0 and Sp2 / 0.In some embodiments, the host cell is a human or non-human primate cell. In some embodiments, the host cell is a cell from a cell line. Examples of suitable host cells or cell lines may include, but are not limited to, 293, HeLa, SH-Sy5y, Hep G2, CACO-2, A549, L929, 3T3, K562, CHO-K1, MDCK, HUVEC, Vero, N20, COS-7, PSN1, VCaP, CHO cells, and others.

[0182] In some embodiments, the recombinant nucleic acid is a herpes simplex virus vector. In some embodiments, the recombinant nucleic acid is a herpes simplex virus amplicon. In some embodiments, the recombinant nucleic acid is an HSV-1 amplicon or an HSV-1 hybrid amplicon. In some embodiments, a host cell containing a helper virus is contacted with the HSV-1 amplicon or the HSV-1 hybrid amplicon described herein, resulting in the production of a virus that contains one or more recombinant nucleic acids described herein. In some embodiments, the virus is collected from the supernatant of the contacted host cell. Methods for generating a virus by contacting a host cell containing a helper virus with the HSV-1 amplicon or the HSV-1 hybrid amplicon are known in the art.

[0183] In some embodiments, the host cell is a complementing host cell. In some embodiments, the complementing host cell expresses one or more genes that are inactivated in any of the viral vectors described herein. In some embodiments, the complementing host cell is contacted with a recombinant herpesvirus genome (e.g., a recombinant herpes simplex virus genome) described herein. In some embodiments, contacting the complementing host cell with the recombinant herpesvirus genome results in the production of a herpesvirus that comprises one or more recombinant nucleic acids described herein. In some embodiments, the virus is collected from the supernatant of the contacted host cell. Methods for producing a virus by contacting a complementing host cell with a recombinant herpes simplex virus are generally described in U.S. Pat. No. 10,174,341, U.S. Pat. No. 2019 / 0276845, U.S. Pat. No. 9,877,990, U.S. Pat. No. 10,155,016, U.S. Pat. No. 10,441,614, U.S. Pat. No. 11,185,564, U.S. Pat. No. 2020 / 0093874, U.S. Pat. No. 10,525,090, U.S. Pat. No. 2020 / 0197456, U.S. Pat. No. 2021 / 0261649, U.S. Pat. No. 2021 / 0395775, U.S. Pat. No. 10,829,529, U.S. Pat. No. 2021 / 0087245, U.S. Pat. No. 2021 / 0189427, U.S. Pat. No. 10,786,438, U.S. Pat. No. 2021 / 0189427, U.S. Pat. No. 2021 / 0199428, U.S. Pat. No. 2021 / 0261649, U.S. Pat. No. 2021 / 0395775, U.S. Pat. No. 2021 / 0261649 ... 2021 / 0045988, and / or WO2021 / 046131, all of which are incorporated by reference in their entireties.

[0184] VIII. Articles of Manufacture or Kits Certain aspects of the present disclosure relate to articles of manufacture or kits that include any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the articles of manufacture or kits include a package insert that includes instructions for administering the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation.

[0185] Suitable containers for recombinant nucleic acid, virus, medicine, and / or pharmaceutical composition or formulation may include, for example, bottles, vials, bags, tubes, and syringes. Containers may be formed from a variety of materials, for example, glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or Hastelloy). In some embodiments, the container includes a label on or associated with the container, where the label displays instructions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, bulking agents, inhalers, nebulizers, intranasal administration devices, package inserts, and the like.

[0186] IX. Enumerated Aspects Embodiment 1: A recombinant herpesvirus genome comprising one or more polynucleotides encoding an immunomodulatory polypeptide.

[0187] Embodiment 2: A replication-competent recombinant herpesvirus genome according to embodiment 1.

[0188] Embodiment 3: A recombinant herpesvirus genome according to any of embodiments 1 or 2, which is replication-deficient.

[0189] Embodiment 4: The recombinant herpesvirus genome of any of embodiments 1 to 3, wherein the recombinant herpesvirus genome is selected from the group consisting of a recombinant herpes simplex virus genome, a recombinant varicella zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, a recombinant Epstein-Barr virus genome, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any derivatives thereof.

[0190] Embodiment 5: The recombinant herpesvirus genome of any of embodiments 1 to 4, which is a recombinant herpes simplex virus genome.

[0191] Embodiment 6: The recombinant herpesvirus genome of any of embodiments 1 to 5, wherein the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof.

[0192] Embodiment 7: The recombinant herpesvirus genome of any of embodiments 1 to 6, wherein the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome.

[0193] Embodiment 8: The recombinant herpesvirus genome of any of embodiments 1 to 7, wherein the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 2 (HSV-2) genome.

[0194] Embodiment 9: The recombinant herpesvirus genome of any of embodiments 1 to 8, wherein the recombinant herpes simplex virus genome has been engineered to reduce or eliminate expression of one or more toxic herpes simplex virus genes.

[0195] Embodiment 10: The recombinant herpesvirus genome of any of embodiments 1 to 9, wherein the recombinant herpes simplex virus genome has been engineered to reduce or eliminate expression of one or more toxic herpes simplex virus genes.

[0196] Embodiment 11: The recombinant herpesvirus genome of any of embodiments 1 to 10, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation.

[0197] Embodiment 12: The recombinant herpesvirus genome of any of embodiments 1 to 11, wherein the inactivating mutation is present in a herpes simplex virus gene.

[0198] Embodiment 13: The recombinant herpesvirus genome of any of embodiments 1 to 12, wherein the inactivating mutation is a deletion of a coding sequence of a herpes simplex virus gene.

[0199] Embodiment 14: The recombinant herpesvirus genome of any of embodiments 1 to 13, wherein the herpes simplex virus gene is selected from the group consisting of infected cell protein (ICP) 0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41, and UL55.

[0200] Embodiment 15: The recombinant herpesvirus genome of any of embodiments 1 to 14, wherein the herpes simplex virus gene is ICP4.

[0201] Embodiment 16: The recombinant herpesvirus genome of any of embodiments 1 to 15, wherein the herpes simplex virus gene is ICP22.

[0202] Embodiment 17: The recombinant herpesvirus genome of any of embodiments 1 to 16, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP4 gene.

[0203] Embodiment 18: The recombinant herpesvirus genome of any of embodiments 1 to 17, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP4 gene.

[0204] Embodiment 19: The recombinant herpesvirus genome of any of embodiments 1 to 18, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene.

[0205] Embodiment 20: The recombinant herpesvirus genome of any of embodiments 1 to 19, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP0 gene.

[0206] Embodiment 21: The recombinant herpesvirus genome of any of embodiments 1 to 20, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in one copy of the ICP0 gene.

[0207] Embodiment 22: The recombinant herpesvirus genome of any of embodiments 1 to 21, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in both copies of the ICP0 gene.

[0208] Embodiment 23: The recombinant herpesvirus genome of any of embodiments 1 to 22, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene.

[0209] Embodiment 24: The recombinant herpesvirus genome of any of embodiments 1 to 23, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene.

[0210] Embodiment 25: The recombinant herpesvirus genome of any of embodiments 1 to 24, wherein the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP47 gene.

[0211] Embodiment 26: The recombinant herpesvirus genome of any of embodiments 1 to 25, wherein the recombinant herpes simplex virus does not contain an inactivating mutation in one or both copies of the ICP34.5 gene.

[0212] Embodiment 27: The recombinant herpesvirus genome of any of embodiments 1 to 26, wherein the recombinant herpes simplex virus does not contain an inactivating mutation in one copy of the ICP34.5 gene.

[0213] Embodiment 28: The recombinant herpesvirus genome of any of embodiments 1 to 27, wherein the recombinant herpes simplex virus does not contain an inactivating mutation in both copies of the ICP34.5 gene.

[0214] Embodiment 29: A recombinant herpesvirus genome of any of embodiments 1 to 28, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide within one or both ICP4 viral loci.

[0215] Embodiment 30: A recombinant herpesvirus genome according to any of embodiments 1 to 29, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide within one of the ICP4 viral loci.

[0216] Embodiment 31: A recombinant herpesvirus genome of any of embodiments 1 to 30, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide within both ICP4 viral loci.

[0217] Embodiment 32: A recombinant herpesvirus genome according to any of embodiments 1 to 31, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide within the ICP22 viral locus.

[0218] Embodiment 33: A recombinant herpesvirus genome according to any of embodiments 1 to 32, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide within the UL41 viral locus.

[0219] Embodiment 34: A recombinant herpesvirus genome of any of embodiments 1 to 33, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide within one or both ICP0 viral loci.

[0220] Embodiment 35: A recombinant herpesvirus genome according to any of embodiments 1 to 34, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide within one ICP0 viral locus.

[0221] Embodiment 36: A recombinant herpesvirus genome according to any of embodiments 1 to 35, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding immunomodulatory polypeptides within both ICP0 viral loci.

[0222] Embodiment 37: A recombinant herpesvirus genome according to any of embodiments 1 to 36, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide within the ICP27 viral locus.

[0223] Embodiment 38: A recombinant herpesvirus genome of any of embodiments 1 to 37, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an immunomodulatory polypeptide within the UL55 viral locus.

[0224] Embodiment 39: A recombinant herpesvirus genome of any of embodiments 1 to 38, wherein the immunomodulatory polypeptide is a human immunomodulatory polypeptide.

[0225] Embodiment 40: A recombinant herpesvirus genome of any of embodiments 1 to 39, wherein the immunomodulatory polypeptide is a cytokine or chemokine.

[0226] Embodiment 41: The recombinant herpesvirus genome of any of embodiments 1 to 40, wherein the cytokine is a proinflammatory cytokine.

[0227] Embodiment 42: The recombinant herpesvirus genome of any of embodiments 1 to 42, wherein the cytokine is selected from the group consisting of interleukin (IL)-1, IL-2, IL-7, IL-12, IL-13, IL-15, IL-17, IL-18, IL-28, IL-32, IL-33, IL-34, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), granulocyte colony stimulating factor (G-CSF), and granulocyte macrophage colony stimulating factor (GM-CSF).

[0228] Embodiment 43: The recombinant herpesvirus genome of any of embodiments 1 to 42, wherein the cytokine is IL-2.

[0229] Embodiment 44: The recombinant herpesvirus genome of any of embodiments 1 to 43, wherein the cytokine is IL-12.

[0230] Embodiment 45: The recombinant herpesvirus genome of any of embodiments 1 to 44, wherein the cytokine is G-CSF.

[0231] Embodiment 46: A recombinant herpesvirus genome of any of embodiments 1 to 45, wherein the cytokine is GM-CSF.

[0232] Embodiment 47: The recombinant herpesvirus genome of any of embodiments 1 to 46, wherein the cytokine is not GM-CSF.

[0233] Embodiment 48: The recombinant herpesvirus genome of any of embodiments 1 to 47, wherein the IL-2 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3.

[0234] Embodiment 49: The recombinant herpesvirus genome of any of embodiments 1 to 48, wherein the IL-12 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:85.

[0235] Embodiment 50: A recombinant herpesvirus genome of any of embodiments 1 to 49, wherein the chemokine is a pro-inflammatory chemokine.

[0236] Embodiment 51: The recombinant herpesvirus genome of any of embodiments 1 to 50, wherein the chemokine is selected from the group consisting of chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, CXCL8, CXCL9, CXCL11, CXCL16, CC motif chemokine ligand 2 (CCL2), CCL3, CCL4, CCL5, and CCL11.

[0237] Embodiment 52: The recombinant herpesvirus genome of any of embodiments 1 to 51, wherein the immunomodulatory polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30.

[0238] Embodiment 53: The recombinant herpesvirus genome of any of embodiments 1 to 52, wherein the immunomodulatory polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 19.

[0239] Embodiment 54: The recombinant herpesvirus genome of any of embodiments 1 to 53, wherein the immunomodulatory polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-30.

[0240] Embodiment 55: A recombinant herpesvirus genome according to any of embodiments 1 to 54, which has reduced cytotoxicity when introduced into a target cell compared to a corresponding wild-type herpesvirus genome.

[0241] Embodiment 56: The recombinant herpesvirus genome of any of embodiments 1 to 55, wherein the target cell is a human cell.

[0242] Embodiment 57: The recombinant herpesvirus genome of any of embodiments 1 to 56, wherein the target cell is a respiratory cell.

[0243] Embodiment 58: The recombinant herpesvirus genome of any of embodiments 1 to 57, wherein the target cell is an airway epithelial cell.

[0244] Embodiment 59: A herpesvirus comprising a recombinant herpesvirus genome according to any one of embodiments 1 to 58.

[0245] Embodiment 60: A herpesvirus of embodiment 59, which is replication-competent.

[0246] Embodiment 61: A herpesvirus of embodiment 59 or 60, which is replication-deficient.

[0247] Embodiment 62: A herpesvirus of any of embodiments 59 to 61, which is not oncolytic.

[0248] Embodiment 63: A herpesvirus of any of embodiments 59 to 62, having reduced cytotoxicity compared to a corresponding wild-type herpesvirus.

[0249] Embodiment 64: The herpesvirus of any of embodiments 59 to 63, which is selected from the group consisting of herpes simplex virus, varicella-zoster virus, human cytomegalovirus, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus.

[0250] Embodiment 65: A herpesvirus of any of embodiments 59 to 64, which is a herpes simplex virus.

[0251] Embodiment 66: The herpesvirus of any of embodiments 59 to 65, wherein the herpes simplex virus is herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), or any derivative thereof.

[0252] Embodiment 67: The herpesvirus of any of embodiments 59 to 66, wherein the herpes simplex virus is herpes simplex virus type 1 (HSV-1).

[0253] Embodiment 68: The herpesvirus of any of embodiments 59 to 67, wherein the herpes simplex virus is herpes simplex virus type 2 (HSV-2).

[0254] Embodiment 69: A herpes virus of any of embodiments 59 to 68, wherein the herpes simplex virus is not oncolytic.

[0255] Embodiment 70: A pharmaceutical composition comprising a recombinant herpesvirus genome according to any of embodiments 1 to 58 or a herpesvirus according to any of embodiments 59 to 69, and a pharma- ceutically acceptable excipient.

[0256] Embodiment 71: The pharmaceutical composition of embodiment 70, which is suitable for topical, transdermal, subcutaneous, intradermal, oral, intranasal, intratracheal, sublingual, buccal, rectal, intravaginal, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, topical, or epicutaneous administration.

[0257] Embodiment 72: A pharmaceutical composition according to embodiment 70 or 71, which is suitable for oral, intranasal, intratracheal, or inhalation administration.

[0258] Embodiment 73: A pharmaceutical composition according to any of embodiments 70 to 72, which is suitable for intranasal or inhaled administration.

[0259] Embodiment 74: A pharmaceutical composition according to any of embodiments 70 to 73, which is suitable for inhaled administration.

[0260] Embodiment 75: The pharmaceutical composition of any of embodiments 70 to 74, which is suitable for intranasal administration.

[0261] Embodiment 76: The pharmaceutical composition of any of embodiments 70 to 75, which is suitable for use in a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, an electrohydrodynamic aerosol device, or any combination thereof.

[0262] Embodiment 77: The pharmaceutical composition of any of embodiments 70 to 76, which is suitable for use in a nebulizer.

[0263] Embodiment 78: The pharmaceutical composition of any of embodiments 70 to 77, wherein the nebulizer is a vibrating mesh nebulizer.

[0264] Embodiment 79: A herpesvirus according to any of embodiments 59 to 69 or a pharmaceutical composition according to any of embodiments 70 to 78 for use as a medicament.

[0265] Embodiment 80: A herpesvirus according to any of embodiments 59 to 69 or a pharmaceutical composition according to any of embodiments 70 to 78 for use in therapy.

[0266] Embodiment 81: Use of a herpesvirus according to any of embodiments 59 to 69 or a pharmaceutical composition according to any of embodiments 70 to 78 in the manufacture of a medicament for treating cancer.

[0267] Aspect 82: The cancer is acute myeloid leukemia, acute lymphoblastic leukemia, adrenal cortical carcinoma, bladder urothelial carcinoma, brain stem glioma, brain low-grade glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoma of unknown primary site, carcinoid tumor, carcinoma of unknown primary site, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, cervical squamous cell carcinoma, cervical adenocarcinoma, childhood cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine islet cell tumor, endometrial cancer. , ependymomas, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal cell tumors, gastrointestinal stromal tumors, gestational trophoblastic tumors, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, cardiac cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumors, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, liver cancer, lymphoid neoplasms diffuse large B-cell lymphoma, malignant fibrous histiocytoma bone cancer, medulloblastoma, medulloepithelioma , melanoma, Merkel cell carcinoma, Merkel cell skin cancer, mesothelioma, metastatic squamous cell neck cancer of unknown primary site, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative neoplasm, nasal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal tumors, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papilloma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, brown cell Cystoma and paraganglioma, Moderately differentiated pineal parenchymal tumor, Pineoblastoma, Pituitary tumor, Plasma cell neoplasm / multiple myeloma, Pleuropulmonary blastoma, Primary central nervous system lymphoma, Primary hepatocellular carcinoma, Prostate cancer including prostate adenocarcinoma, Rectal cancer, Renal cell carcinoma, Renal cell carcinoma, Respiratory cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Sezary syndrome, Cutaneous melanoma, Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Squamous cell carcinoma, Squamous cervical cancer, Gastric cancer, Supratentorial primitive neuroectodermal tumor, T-cell lymphoma, Testicular cancer Testicular germ cell tumor, Throat cancer, Thymic carcinoma, Thymoma, Thyroid cancer,The use of embodiment 81, wherein the tumor is selected from the group consisting of transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureteral cancer, urethral cancer, uterine cancer, uterine cancer, uveal melanoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0268] Embodiment 83: A method for expressing, enhancing, increasing, enhancing and / or replenishing the level of an immune modulating polypeptide in one or more cells of a subject, comprising the step of administering to the subject an effective amount of a herpesvirus of any of embodiments 59 to 69 or a pharmaceutical composition of any of embodiments 70 to 78.

[0269] Embodiment 84: The method of embodiment 83, wherein the one or more cells are one or more respiratory, airway epithelial, and / or pulmonary cells.

[0270] Embodiment 85: The method of any of embodiments 83 or 84, wherein the one or more cells are one or more cells of the respiratory tract.

[0271] Embodiment 86: The method of any of embodiments 83 to 85, wherein the one or more cells are one or more cells of the respiratory epithelium.

[0272] Embodiment 87: The method of any of embodiments 83 to 86, wherein the one or more cells are one or more cells of the lung.

[0273] Embodiment 88: A method for providing prophylactic, palliative, or therapeutic alleviation of one or more signs or symptoms of cancer, comprising the step of administering to a subject an effective amount of a herpesvirus of any of embodiments 59-69 or a pharmaceutical composition of any of embodiments 70-78.

[0274] Embodiment 89: A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a herpesvirus of any of embodiments 59 to 69 or a pharmaceutical composition of any of embodiments 70 to 78.

[0275] Embodiment 90: The method of embodiment 88 or 89, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, leukemia, and lymphoid malignancies.

[0276] Embodiment 91: The method of any of embodiments 88 to 90, wherein the cancer is selected from the group consisting of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, renal cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, and thyroid cancer.

[0277] Embodiment 92: The method of any one of embodiments 88 to 91, wherein the cancer is small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma.

[0278] Embodiment 93: The method of any one of embodiments 88 to 92, wherein the cancer is small cell lung cancer.

[0279] Embodiment 94: The method of any one of embodiments 88 to 93, wherein the cancer is non-small cell lung cancer.

[0280] Embodiment 95: The method of any one of embodiments 88 to 94, wherein the cancer is lung adenocarcinoma.

[0281] Embodiment 96: The method of any one of embodiments 88 to 95, wherein the cancer is lung squamous cell carcinoma.

[0282] Embodiment 97: The method of any one of embodiments 88 to 96, wherein the cancer is osteosarcoma.

[0283] Embodiment 98: The method of any of embodiments 88 to 97, wherein the subject is a human.

[0284] Embodiment 99: The method of any of embodiments 88 to 98, wherein the herpes virus or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, buccally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, intratumorally, topically, or via inhalation.

[0285] Embodiment 100: The method of any of embodiments 88 to 99, wherein the herpes virus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation.

[0286] Embodiment 101: The method of any of embodiments 88 to 100, wherein the herpes virus or pharmaceutical composition is administered to the subject intranasally or via inhalation.

[0287] Embodiment 102: The method of any of embodiments 88 to 101, wherein the herpes virus or pharmaceutical composition is administered intranasally to the subject.

[0288] Embodiment 103: The method of any of embodiments 88 to 102, wherein the herpes virus or pharmaceutical composition is administered to the subject via inhalation.

[0289] Embodiment 104: The method of any of embodiments 88 to 103, wherein the herpes virus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device.

[0290] Embodiment 105: The method of any of embodiments 88 to 104, wherein the herpes virus or pharmaceutical composition is administered using a nebulizer.

[0291] Embodiment 106: The method of any of embodiments 88 to 105, wherein the nebulizer is a vibrating mesh nebulizer.

[0292] The specification is considered to be sufficient to enable one skilled in the art to practice the disclosure. Various modifications of the disclosure, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. EXAMPLES

[0293] The present disclosure will be more fully understood by referring to the following examples. However, this should not be interpreted as limiting the scope of the present disclosure. It will be understood that the examples and embodiments described herein are for illustrative purposes only, and in light of them, various modifications or changes will be suggested to those skilled in the art, and these modifications or changes will be included within the spirit and scope of the present application and the scope of the appended claims.

[0294] Example 1: Modified herpes simplex virus vectors encoding immunomodulatory polypeptides To generate a modified herpes simplex virus genome vector capable of expressing an immunomodulatory polypeptide in a target mammalian cell (such as a cell of the respiratory tract), the herpes simplex virus genome (FIG. 1A) is first modified to inactivate one or more herpes simplex virus genes. Such modifications can reduce the toxicity of the genome in mammalian cells. These modified / attenuated recombinant virus constructs are then mutant to have one or more polynucleotides encoding the desired immunomodulatory polypeptide.These variants include a recombinant ΔICP4 modified HSV-1 genome comprising an expression cassette comprising a coding sequence for an immunomodulatory polypeptide (e.g., SEQ ID NO:3) under the control of a heterologous promoter integrated into each ICP4 locus ( FIG. 1B ); a recombinant ΔICP4 / ΔUL41 modified HSV-1 genome comprising an expression cassette comprising a coding sequence for an immunomodulatory polypeptide (e.g., SEQ ID NO:3) under the control of a heterologous promoter integrated into each ICP4 locus ( FIG. 1C ); a recombinant ΔICP4 / ΔUL41 modified HSV-1 genome comprising an expression cassette comprising a coding sequence for an immunomodulatory polypeptide (e.g., SEQ ID NO:3) under the control of a heterologous promoter integrated into the UL41 locus ( FIG. 1D ); a recombinant ΔICP4 / ΔUL41 modified HSV-1 genome comprising an expression cassette comprising a coding sequence for an immunomodulatory polypeptide (e.g., SEQ ID NO:3) under the control of a heterologous promoter integrated into each ICP4 locus ( FIG. 1E ); a recombinant ΔICP4 / ΔICP22 modified HSV-1 genome comprising an expression cassette comprising a coding sequence for an immunomodulatory polypeptide (e.g., SEQ ID NO:3) under control of a heterologous promoter integrated into the ICP22 locus (FIG. 1F); a recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome comprising an expression cassette comprising a coding sequence for an immunomodulatory polypeptide (e.g., SEQ ID NO:3) under control of a heterologous promoter integrated into each ICP4 locus (FIG. 1G); a recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome comprising an expression cassette comprising a coding sequence for an immunomodulatory polypeptide (e.g., SEQ ID NO:3) under control of a heterologous promoter integrated into the UL41 locus (FIG. 1H); and a recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome comprising an expression cassette comprising a coding sequence for an immunomodulatory polypeptide (e.g., SEQ ID NO:3) under control of a heterologous promoter integrated into the ICP22 locus (FIG. 1H). NO:3) and the recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome (Fig. 1I ).

[0295] These modified herpes simplex virus genome vectors are transfected into engineered cells that are modified to express one or more herpes simplex virus genes.These engineered cells secrete the replication-defective herpes simplex virus that the modified genome is packaged in into the supernatant of cell culture.Then, the supernatant is collected, concentrated, and sterile filtered.

[0296] The modified herpes simplex virus genome vectors described herein can express any one or more of the exemplary immunomodulatory polypeptides in Table 1 below, in any suitable combination.

[0297] Table 1. Representative immunomodulatory polypeptides TIFF2024513826000002.tif214153

[0298] Example 2: Construction of modified herpes simplex virus vectors encoding human IL-12, IL-2, and GM-CSF The following examples describe the engineering of recombinant herpes simplex virus type 1 (HSV-1) that successfully encoded human IL-12 (see, e.g., SEQ ID NO:35 and 36; HSV-IL12), IL-2 (see, e.g., SEQ ID NO:33; HSV-IL2) or GM-CSF (see, e.g., SEQ ID NO:49; HSV-GMCSF) and expressed full-length human IL-12 (see, e.g., SEQ ID NO:5 and 6), IL-2 (see, e.g., SEQ ID NO:3) or GM-CSF (see, e.g., SEQ ID NO:19) proteins.

[0299] Recombinant HSV-1 was engineered to incorporate human IL-12, IL-2 or GM-CSF expression cassettes containing heterologous promoters and polyA sequences (see Example 1). Viral plaques that were suspected to contain human IL-12, IL-2 or GM-CSF cassettes were selected and screened by infection in complementation cell lines to test human IL-12, IL-2 and GM-CSF protein expression by Western blot analysis (data not shown). High-expressing clones, designated HSV-IL12, HSV-IL2 and HSV-GMCSF, were then selected for further in vitro analysis.

[0300] Complementing cells were mock infected with vehicle control or infected with HSV-IL12, HSV-IL2, or HSV-GMCSF at a multiplicity of infection (MOI) of 1 in serum-free cell culture medium. At 24 or 48 h postinfection, cell pellets were harvested, lysed in RIPA buffer containing protease inhibitors, and protein content was quantified by BCA assay. 30–40 μg of each sample was loaded and run on a 4–20% acrylamide gel, and expression of HSV-encoded human proteins was assessed by Western blot analysis (Figures 2A–2B). Recombinant human IL-12 or IL-2 was loaded on the gel as a positive control. No human IL-12 or IL-2 was detected in uninfected control cells, whereas robust expression of human IL-12 (Figure 2A) and IL-2 (Figure 2B) was observed in cells after infection with HSV-IL12 and HSV-IL2, respectively.

[0301] In addition, because IL-12, IL-2, and GM-CSF are naturally secreted proteins, cell culture supernatants were harvested and examined by ELISA for the presence of human proteins.Consistent with the Western blot data, human IL-12 (2.01μg / ml and 2.58μg / ml), IL-2 (0.531μg / ml, 0.850μg / ml, and 1.200μg / ml), and GM-CSF (643.427ng / ml, 513.56ng / ml, and 200.167ng / ml) were detected in the supernatants of cells infected with HSV-IL12, HSV-IL2, and HSV-GMCSF at the MOIs tested, respectively, suggesting that the full-length human proteins were properly processed / secreted after expression from the recombinant vectors.

[0302] In summary, the data presented in this example showed that the recombinant HSV-1 vectors HSV-IL12, HSV-IL2, and HSV-GMCSF can efficiently infect multiple cell types and express the human transgenes encoded therein. Furthermore, the data showed that the exogenous human proteins were then (appropriately) secreted from the infected cells. Without wishing to be bound by theory, this data is believed to further support the use of engineered herpes simplex viruses as novel, targeted, and broadly applicable gene therapy vectors for the treatment of various cancers (e.g., osteosarcoma).

[0303] Example 3: In vitro human and mouse IL-12 bioactivity assays The objective of this study was, in part, to determine whether recombinant human and / or mouse IL-12 proteins produced from recombinant herpes simplex virus type 1 (HSV-1) are as biologically active as commercially available recombinant IL-12 proteins.

[0304] Human embryonic kidney 293 (HEK293) cells were infected with HSV-IL12 at an MOI of 1 for 48 h. IL-12 concentrations in the supernatants from HEK293 infection were measured by ELISA. Human PBMCs and mouse splenocytes were cultured in complete RPMI or DMEM, respectively, and maintained at 37 °C with 5% CO2 in a humidified environment. Commercially available human or mouse αCD3 / αCD28 coated beads were washed and added to PBMC or splenocyte cultures, respectively, in a volume of 2 μL per well. As shown in Figures 3A-3B, serial dilutions of supernatants from HEK293 infection with human or mouse HSV-IL12 were added to PBMC or splenocyte cultures together with αCD3 / αCD28 coated beads. As a comparison, commercially available recombinant IL-12 protein was added to some cultures at similar concentrations. 24 h after stimulation, PBMC or mouse splenocyte culture supernatants were harvested and assayed for IFNγ production by ELISA.

[0305] As shown in Figures 3A-3B, addition of αCD3 / αCD28-coated beads resulted in the release of IFNγ from both human PBMCs (Figure 3A) and mouse splenocytes (Figure 3B). In comparison, addition of viral supernatants from both human HSV-IL12 and mouse HSV-IL12 to αCD3 / αCD28-stimulated human PBMCs (Figure 3A) and mouse splenocytes (Figure 2B) induced higher levels of IFNγ secretion compared to αCD3 / αCD28 stimulation alone. Furthermore, HSV-IL12-dependent IFNγ release was comparable to that of recombinant IL-12 (Figures 3A-3B).

[0306] Taken together, these results suggested that the recombinant human and mouse IL-12 proteins produced from recombinant herpes simplex viruses were as biologically active as commercially available recombinant IL-12 proteins.

[0307] Example 4: Intratracheal administration and in vivo evaluation of HSV-IL12 in healthy mice In recent years, immunotherapy or targeting of the immune system has shown great potential in reducing tumor burden in cancer treatment. Specifically, administration of recombinant interleukin (IL)-12 protein, a potent proinflammatory cytokine, has been evaluated. IL-12 is a heterodimeric cytokine composed of p35 and p40 subunits linked by three disulfide bridges. As a proinflammatory cytokine, IL-12 is primarily produced by activated antigen-presenting cells (e.g., macrophages and dendritic cells) to stimulate increased interferon gamma (IFNγ) production by T cells and natural killer (NK) cells. IFNγ can then initiate tumor cell apoptosis, upregulate tumor cell antigen presentation, and induce macrophage tumoricidal activity, among other effects. Research over the past decades has demonstrated that IL-12 is a potent stimulator of antitumor immune responses, supporting its use as a cancer therapy. However, previous clinical trials have shown that systemic IL-12 treatment in cancer patients produces high levels of toxicity.

[0308] The inventors have utilized non-replicating HSV-1 based gene therapy vectors to develop modified vectors encoding the il12a and il12b genes of the p35 and p40 subunits (HSV-IL12), respectively. Infection with HSV-IL12 resulted in the expression of IL-12 p70 heterodimers, with the subunits covalently linked by G4S linkers. In vitro studies comparing virally produced and recombinant IL-12 proteins showed similar efficacy in inducing IFNγ expression during activation of mouse splenic T cells (see Example 3 above).

[0309] Having confirmed transgene bioactivity through these studies, the next step was to test HSV-IL12 in vivo. This study assessed IL-12 expression along with toxicity following intratracheal (it) administration of a single dose of HSV-IL12 in young (8-10 week old) BALB / c animals. Three different doses of HSV-IL12 were evaluated for IL-12 expression by nucleic acid and protein analysis in lung tissue, bronchoalveolar lavage fluid (BALF), and serum. Toxicity was measured by loss of total body weight over the course of the study (24 and 48 hours post-administration). In addition, a group of animals received recombinant IL-12 protein systemically (subcutaneously; sc) as a means of comparing local HSV-IL12 delivery with previously tested routes of administration.

[0310] All procedures performed were in compliance with applicable animal welfare laws and approved by the local Institutional Animal Care and Use Committee (IACUC). Healthy, 8-week-old, female BALB / c mice were used in this study.

[0311] Mice were sedated with an intraperitoneal mixture of Telazol and Dexdomitor. Sedation was determined by using the toe pinch reflex test. Eye ointment (Puralube Vet) was applied to the eyes to prevent dryness. For dosing, animals were placed on a metal tray on a support table.

[0312] Intratracheal (it) administration was performed by thawing the viral vector on wet ice, diluting in vehicle to achieve the appropriate titer, and administering in a dose of 50 μL. The mandible and tongue were gently moved apart with forceps to expose the epiglottis and larynx. Approximately 4.5 cm of catheter tubing was inserted halfway into the trachea and a 23 G needle connected to a syringe was attached to the tip of the catheter to administer the dose. Negative control mice were injected it with vehicle only. After administration, animals were observed while recovering from anesthesia.

[0313] In addition, a group of animals was injected subcutaneously (sc) with recombinant IL-12 protein (0.5 mg diluted in 1x PBS) as a systemic route of administration. This dose was chosen because it had been demonstrated to be toxic in mice. For administration, awake mice were grasped by the scruff of the neck and the dose was administered at the back of the neck using a 1 mL syringe and a 27G needle.

[0314] At the indicated time points after it or sc administration of the above treatments, animals were euthanized by CO2 asphyxiation, and cardiac puncture was performed to collect blood into serum separator tubes (BD Biosciences). Tubes were centrifuged to pellet red blood cells, and serum was removed and flash frozen in Eppendorf tubes. Animals were then perfused with 40 mL of cold 1x PBS at a rate of 20 mL per minute. Bronchoalveolar lavage (BAL) was performed using 2 mL of 1x PBS. Recovery was recorded for each animal. BAL samples were centrifuged to remove cells, and BAL fluid (BALF) was collected for assay. 100x protease inhibitor (Fisher Scientific) was added to all BALF samples at a final concentration of 1x. The left and right lungs were then excised and flash frozen in liquid nitrogen for protein and nucleic acid analysis.

[0315] The resuspended BAL cell pellet was centrifuged at 2000 rpm for 5 min and the supernatant was removed and discarded. Residual red blood cells were lysed using 1x red blood cell lysis buffer (Sigma) (incubated at room temperature for 2 min). The lysis reaction was stopped by adding 500 μL of 1x PBS and the cells were centrifuged as above. The supernatant was removed and the cells were resuspended in 100-500 μL of 1x PBS depending on the size of the cell pellet. An aliquot of each sample was diluted in trypan blue (Fisher Scientific) and cell count and viability were assessed using a hemocytometer.

[0316] Snap-frozen lungs were stored at -80°C until processing. On the day of processing, frozen lungs were excised using a razor blade, immediately weighed, and returned to dry ice. Two biopsies were excised, one for RNA / DNA work and one to generate homogenates for ELISA. Biopsies for nucleic acid analysis were immediately resuspended in 350 μL of RLT buffer prepared with fresh DTT according to the manufacturer's (Qiagen) protocol. Biopsies for protein analysis were resuspended in 300 μL of Pierce's TPER Reagent (Fisher Scientific) supplemented with 1× protease inhibitor (Fisher Scientific). 5 mm metal beads were added to each tube, and samples were homogenized by Tissue Lyser (Qiagen) at 25 Hz for 3 min. Nucleic acids were extracted immediately and protein homogenates were treated as follows: samples were centrifuged at 10,000×g for 5 min to pellet debris; supernatants were collected and aliquoted into Eppendorf tubes. Homogenates were stored at −20° C. until ELISA and BCA assays.

[0317] DNA and RNA extraction was performed using Qiagen AllPrep DNA / RNA extraction kits according to the manufacturer's protocol. Both DNA and RNA samples were eluted in distilled, deionized RNase-free water. Residual genomic DNA was removed from RNA samples using the TURBO DNA kit (Invitrogen) according to the manufacturer's instructions. All nucleic acids were quantified spectrophotometrically on a Nanodrop (BioTek). Absolute quantification of il12 DNA genomic and RNA transcripts was performed by TaqMan real-time PCR analysis using custom transgene-specific primer / probe pairs. A 20x il12 primer / probe mix containing 45 nmol each of forward and reverse primers and 12.5 nmol of probe was diluted 1x in each reaction to achieve final primer and probe concentrations. RNA was reverse transcribed into cDNA using MultiScribe Reverse Transcriptase (Applied Biosystems) prior to qRT-PCR analysis.

[0318] Taqman® Fast Advanced Master Mix (Applied Biosystems) was used for DNA quantification (qPCR) and RNA quantification (qRT-PCR). 50 ng of DNA or cDNA was used for qPCR and qRT-PCR assays, respectively. All samples were run in duplicate and copy numbers were determined using a standard curve derived from a dilution series of gBlock standards containing known copy numbers of the il12 transgene.

[0319] Prior to ELISA, the protein concentration of lung homogenates was measured by BCA assay (Pierce). For ELISA, all reagents used were obtained from R&D Systems. Lung homogenates, BALF, and serum were then assayed on ELISA plates coated overnight with αIL-12 capture antibody according to the manufacturer's instructions. After blocking the plates with 1× reagent diluent for 1 h at room temperature, samples and standards were diluted accordingly in reagent diluent and assayed in duplicate (50 μL per well). Samples were incubated on the plates with shaking for 2 h at room temperature. After incubation, the plates were washed three times with 1× wash buffer, with blotting performed between each wash.

[0320] Biotinylated detection antibody was diluted in reagent diluent to the working concentration specified in the protocol and 100 μL was added to each well. Samples were incubated for 2 hours at room temperature with shaking. After incubation, plates were washed as above and 40× streptavidin was diluted to 1× in reagent diluent. 100 μL was added to each well and plates were incubated for 20 minutes in the dark at room temperature. Plates were washed again and then developed with TMB substrate for 20 minutes in the dark. The reaction was quenched with 2N H2SO4. Plates were read on a SpectraMax spectrophotometer using SoftMax Pro software. All ODs were background subtracted based on a plate blank and concentrations were determined using a 4-point calibration curve.

[0321] For lung homogenates, IL-12 concentrations measured by ELISA were normalized to total protein concentrations obtained from the BCA assay and calculated as pg of IL-12 per μg of protein.

[0322] Example 4a: Single dose of HSV-IL12 The objectives of this study were (1) to evaluate the kinetics of HSV-IL12-mediated expression of murine IL-12 locally and systemically at the transcript and protein levels upon intratracheal administration to healthy BALB / c mice; (2) to evaluate the toxicity of HSV-IL12 at various doses; and (3) to compare toxicity and IL-12 localization between intratracheally administered HSV-IL12 and systemically administered recombinant IL-12 protein.

[0323] An in vivo study was performed to evaluate HSV-IL12-mediated expression of murine IL-12 in healthy immunocompetent mice upon it delivery of the viral vector. A total of 28 BALB / c mice were used in this study. Table 2 provides an overview of the experimental design. At the indicated time points following it or sc administration of active or control test articles, animals were sacrificed and lungs were flash frozen for downstream analysis.

[0324] Table 2: Study design and test article (TA) administration TIFF2024513826000003.tif77155hr—hours; it—intratracheal administration; sc—subcutaneous administration

[0325] All animals were weighed prior to dosing and also prior to sacrifice to assess test article toxicity. Data showed that animals in the high dose group (9.5E8 pfu) showed dramatic weight loss (approximately 16%) 48 hours after treatment, suggesting toxicity to the virus, IL-12, or both (Figure 4A). Animals in other groups showed no change in weight after treatment, indicating no toxic effects from treatment.

[0326] qPCR analysis of lung tissue after sacrifice showed that il12 genome demonstrated a direct relationship with dose at both 24 and 48 hours after treatment (Figure 4B). In addition, DNA levels were relatively stable between the two time points within each dose group. Although il12 genomes differed between treatment groups, il12 transcript levels were comparable regardless of dose (Figure 4C). These data suggest that transcript levels may plateau at a certain level of genome, in which case the presence of more genomes does not result in increased transcripts. This indicates that the highest levels of transgene expression (undiluted, high dose) are achievable even at a 1:25 dilution of virus (low dose). rIL-12 sc administration did not result in appreciable levels of il12 genome or transcripts, as would be expected when IL-12 protein rather than DNA and / or RNA was administered (Figures 4B-4C). Also, as expected, vehicle control recipients demonstrated il12 genome / transcript levels close to the detection limit of the assay (Figures 4B-4C). The high dose group demonstrated >0.08 μg / mL IL-12 protein in serum (Figure 4D), which was most likely the trigger for toxicity (Figure 4A). However, this was dramatically reduced in the mid and low dose groups (ng / mL range) (Figure 4D). IL-12 was also detected in the BALF of the mid dose group (Figure 4E). Furthermore, there was significantly more IL-12 in the BALF of the HSV-IL12 treated group compared to the recombinant IL-12 treated group, suggesting efficient local delivery of HSV-IL12.

[0327] In addition to serum and BALF, lung biopsies were homogenized for ELISA analysis of IL-12. After normalization for total protein, results showed that there was no significant difference in IL-12 protein concentration between vehicle and sc recombinant protein (Figure 4F). In addition, homogenates from the mid- and low-dose groups contained detectable levels of IL-12 protein.

[0328] This study evaluated (1) the kinetics of HSV-IL12-mediated expression of murine IL-12 upon intratracheal administration to healthy BALB / c mice; (2) the toxicity of HSV-IL12 at various doses; and (3) the tolerability of HSV-IL12 compared to systemically administered recombinant IL-12 protein. Measurement of mouse body weight at sacrifice indicated that the high dose (9.5E8 pfu) was not well tolerated in this study, as animals lost nearly 20% of their body weight 48 hours after administration (Figure 4A). However, further investigations revealed that high levels of recombinant IL-12 protein, purified along with HSV-IL12 and administered it, potentially contributed to toxicity. No significant weight loss was observed in any of the other treatment groups, indicating tolerability. Although we hypothesized that the sc administered rIL-12 dose would be somewhat toxic, animals in this study received only one dose. This is in contrast to previous studies in which toxicity was observed after successive daily administrations.

[0329] qRT-PCR data showed that il12 transcript levels were comparable between the treatment groups at 24 and 48 hours after treatment (Figure 4C), although il12 genome decreased with test substance administration (Figure 4B). These results would indicate that il12 transcription may be more efficient at lower test substance doses and should be further investigated. It would also suggest that lower doses of virus could be administered and still result in high levels of transgene expression. This would be advantageous in a patient situation where HSV-IL12 is administered by nebulization.

[0330] IL-12 protein levels in serum (Figure 4D) and BALF (Figure 4E) were found to trend somewhat with the dose of HSV-IL12 administered. IL-12 protein concentrations were highly variable in lung homogenates (Figure 4F), indicating that further studies are needed to understand protein expression upon HSV-IL12 treatment.

[0331] Example 4b: Weekly administration of HSV-IL12 The purpose of this study was, in part, to evaluate HSV-IL12-mediated expression of murine IL-12 and its toxicity in healthy, immunocompetent mice. Administration of vehicle and intratracheally administered HSV-IL12, along with sc rIL-12, was performed weekly for three consecutive weeks. A total of 22 BALB / c mice were used in this study. Table 3 provides an overview of the experimental design.

[0332] Table 3: Study design and test article (TA) administration TIFF2024513826000004.tif60160i.t.—intratracheal; sc—subcutaneous

[0333] Animal weights were recorded before each dose and before terminal sacrifice. Terminal sacrifice was performed 24 hours after the last dose (day 15). BALF and associated cells, serum, and lungs were collected for downstream analysis.

[0334] Prior to each weekly dose administration and final sacrifice, all animals were weighed to assess test article toxicity. After the second weekly dose administration, all animals in the highest HSV-IL12 dose group (Dose #1) died, indicating strong toxicity at this dose (Figure 4G). Instead, animals in all other groups maintained relatively the same body weight through the second dose. However, from the second to third dose, animals in the Dose #2 group (3.8E7 pfu) lost an average of 10% of their body weight, suggesting toxicity across multiple doses. Also, one animal from this group died after the second dose. Animals in the Dose #3 group did not show any significant signs of toxicity (body weights were similar to vehicle and rIL-12 recipient groups), demonstrating that 7.6E6 pfu weekly was the best tolerated dose in this model.

[0335] qPCR analysis of lung tissue showed that the difference in il12 genome between HSV-IL12 dose groups was not statistically significant (Figure 4H). A similar trend was observed for il12 transcripts in that animals in the dose #2 group had slightly increased transcript copy numbers (Figure 4I). However, this difference was not statistically significant from the total copy numbers in dose #3 animals. Taken together, these results suggest that although there was a 5-fold decrease in pfu administered between animals in the dose #2 (3.8E7 pfu) and dose #3 (7.6E6 pfu) groups, it did not result in higher il12 genome or transcript copies, suggesting efficient transgene expression even at lower viral doses.

[0336] Furthermore, as shown in Figures 4J-4K, IL-12 protein levels in BALF and lung homogenates showed a direct relationship to dosing. In addition to serum and BALF, lung biopsies were homogenized for ELISA analysis of IL-12. After normalizing for total protein, results showed that there was no significant difference in IL-12 protein concentration between vehicle and sc recombinant protein (Figures 4L-4M). In addition, homogenates from the mid- and low-dose groups contained detectable levels of IL-12 protein.

[0337] Taken together, these data demonstrated that intratracheal administration of low-dose (7.6E6 pfu) HSV-IL12 administered once weekly for three consecutive weeks was well tolerated in healthy mice.

[0338] Example 5: Intratracheal administration and in vivo evaluation of HSV-IL2 in healthy mice Interleukin-2 (IL-2) is a monomeric cytokine produced primarily by activated T cells and NK cells. For these cells, IL-2 often signals in a paracrine manner to stimulate cell growth and division. For T cells in particular, post-activation IL-2 stimulation is required for optimal clonal expansion and upregulation of anti-apoptotic signals. Research supports the use of IL-2 as an immunotherapeutic agent in cancer. However, one significant drawback of IL-2 monotherapy is its toxicity. In addition, IL-2 has a very short serum half-life (minutes) and therefore must be administered systemically at very high doses (>600,000 IU / kg) and frequently (multiple times per day) to be effective. At these doses and administration frequencies, IL-2 treatment can result in, for example but not limited to, vascular leak syndrome, hypotension, and cardiac toxicity.

[0339] Example 5a: Single dose of HSV-IL2 The purpose of this study was, in part, to evaluate transgene expression in lung and serum 24 hours after intratracheal administration of HSV-IL2 compared to recombinant protein administration. Young (8-10 week old) BALB / c mice were administered a single dose of HSV-IL2 intratracheally (it). Three different doses of HSV-IL2 were evaluated for IL-2 expression by nucleic acid and protein analysis in lung tissue, bronchoalveolar lavage fluid (BALF), and serum. In addition, a group of animals was administered recombinant IL-2 protein as a means of comparing local HSV-IL2 delivery with previously tested routes of administration. All procedures have been described previously (see, e.g., Example 4 above). Table 4 provides an overview of the experimental design.

[0340] Table 4: Study design and test article (TA) administration TIFF2024513826000005.tif72160IT - Intratracheal administration; IV - Intravenous administration

[0341] Results of this study suggest that no differences in body weight were observed between groups (Figure 5A). qPCR analysis of lung tissue after sacrifice showed that il2 genome demonstrated a direct relationship with dose at 24 hours after treatment (Figure 5B). Although il2 genomes differed between treatment groups, il2 transcript levels were comparable regardless of dose (Figure 5C). These results were similar to those observed with a single dose of HSV-IL12 in healthy mice (Figures 4B-4C) and demonstrated vector consistency independent of the inserted transgene. With regard to protein levels, IL-2 was detected in serum (Figure 5D), but levels in lungs measured in BALF (Figure 5E) were more than 10-fold higher, indicating more significant expression in target tissues with limited systemic exposure. Although IL-2 protein levels in lung homogenates showed some variability between animals, levels were detectable and relatively higher compared to animals treated with recombinant protein (Figure 5F). Furthermore, 24 h after IV injection of recombinant IL-2, IL-2 protein levels in lung homogenates were undetectable, likely due to its short half-life. These data suggest prolonged effector exposure from HSV-IL2 compared to recombinant protein therapy.

[0342] Taken together, these data demonstrated that infection with HSV-IL2 resulted in the expression of full-length functional murine IL-2, and moreover, it administration of HSV-IL2 resulted in high levels of IL-2 protein in the target organ (lung) with limited systemic exposure (as measured in serum).

[0343] Example 5b: HSV-IL2 Pharmacokinetics The objective of this study was, in part, to quantitatively measure the kinetics of vector transduction and IL-2 expression in lung tissue and fluids harvested from healthy mice following intratracheal (it) administration of a single dose of HSV-IL2.

[0344] All procedures have been described previously (see, e.g., Example 4 above). Table 5 provides an overview of the experimental design.

[0345] Table 5. Study design and test article (TA) administration TIFF2024513826000006.tif80160i.t.:tracheal administration;hpit:time after intratracheal administration

[0346] Results from this study are shown in Figures 5G-5I and demonstrate the pharmacokinetics of IL-2 expression in serum (Figure 5G), BALF (Figure 5H), and lysates (Figure 5I) following a single dose intratracheal administration of HSV-IL2. Overall, these results show that IL-2 protein levels peaked 24 hours after it administration of HSV-IL2 and declined 24-48 hours post-administration, which could be due to reduced transgene translation, the short half-life of IL-2, or a combination thereof. However, IL2 protein exposure in these animals was significantly prolonged when expressed from a vector compared to recombinant protein therapy.

[0347] Example 6: Intratracheal administration and in vivo evaluation of HSV-GMCSF in healthy mice Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a monomeric cytokine that plays a key role in the activation and migration of myeloid cells to sites of inflammation. In cancer therapy, GM-CSF has been shown to effectively activate antitumor immune responses, especially in cases of chemotherapy resistance, in addition to enhancing neutrophil recovery after chemotherapy. Clinical trials testing inhaled GM-CSF therapy for OS lung metastases determined that multiple daily administrations in patients were feasible and had limited toxicity.

[0348] Example 6a: Single dose of HSV-GMCSF The objectives of this study were, in part, to evaluate transgene expression of GM-CSF compared with recombinant protein at 24 and 48 hours after intratracheal administration of HSV-GMCSF and to determine the optimal dose for limited toxicity and robust GM-CSF expression.

[0349] Young (8-10 weeks old) BALB / c animals were administered a single dose of HSV-GMCSF intratracheally (it). Three different doses of HSV-GMCSF were evaluated for GM-CSF expression by nucleic acid and protein analysis in lung tissue, bronchoalveolar lavage fluid (BALF), and serum. In addition, a group of animals was administered recombinant GM-CSF protein as a means of comparing local HSV-GMCSF delivery with previously tested routes of administration.

[0350] All procedures have been described previously (see, e.g., Example 4 above). Table 6 provides an overview of the experimental design.

[0351] Table 6: Study design and test article (TA) administration TIFF2024513826000007.tif126160hr - hours; IT - intratracheal administration

[0352] Results of this study showed that animals in the high (4.88E8 pfu) and medium (9.75E7 pfu) dose groups demonstrated weight loss 48 hours after treatment (Figure 6A), suggesting toxicity. Animals in the other groups did not show any change in weight after treatment, indicating no toxic effects due to treatment. qPCR analysis of lung tissue after sacrifice showed that the gmcsf genome demonstrated a direct relationship with dose both 24 and 48 hours after treatment (Figure 6B). Although the gmcsf genome differed between treatment groups, gmcsf transcript levels were comparable regardless of dose (Figure 6C). Additionally, low levels of GM-CSF were detected in the serum of animals treated with HSV-GMCSF (Figure 6D); however, these levels were approximately 40-fold lower than those in BALF (Figure 6E), again demonstrating limited systemic exposure. Lung homogenate analysis revealed detectable GM-CSF levels at both 24 and 48 hours post-administration, with a decrease observed between the two time points (FIG. 6F).

[0353] Taken together, these data demonstrated that infection with HSV-GMCSF resulted in the expression of full-length murine GM-CSF.

[0354] Example 6b: Weekly administration of HSV-GMCSF The purpose of this study was, in part, to evaluate the toxicity of weekly intratracheal administration of HSV-GMCSF compared to recombinant GM-CSF protein over the course of three weeks. All procedures have been described previously (see, e.g., Example 4 above). Table 7 provides an overview of the experimental design.

[0355] Animal weights were recorded before each dose and terminal sacrifice. Terminal sacrifice was performed 24 hours after the last dose (day 15). BALF and associated cells, serum, and lungs were collected for downstream analysis.

[0356] Table 7. Study design and test article (TA) administration TIFF2024513826000008.tif96160IT - Intratracheal

[0357] As shown in Figure 6G, animals at dose #1 or dose #2 remained at relatively the same weight throughout the course of the study, indicating that both dose #1 and dose #2 were well tolerated. qPCR analysis of lung tissue showed that the differences in gmcsf genome and transcriptome between HSV-GMCSF-treated groups were not statistically significant (Figure 6H-6I). Taken together, these results suggest efficient transgene expression at both doses.

[0358] When GM-CSF protein levels were assessed, there was some systemic exposure to GM-CSF with HSV-GMCSF treatment (serum levels; Fig. 6J), but it was again determined to be minimal compared to local expression / exposure (BALF; Fig. 6K).

[0359] Taken together, these data suggested that HSV-GMCSF was well tolerated following weekly intratracheal administration at the doses indicated.

[0360] Example 7: Establishment of an in vivo mouse model of osteosarcoma Osteosarcoma (OS) is the most common type of bone cancer diagnosed in the clinic, occurring primarily in children and young adults. In terms of incidence, 3.4 cases per million are diagnosed each year, making OS a relatively rare malignancy. However, the incidence rate is 5.6 cases per million in children (<15 years of age), making it the third most common cancer in adolescents.

[0361] OS originates from spindle-shaped malignant stromal cells that can generate bone-like tissue. It is usually subdivided into three groups based on the characteristics of the predominant cell type of the tumor: osteoblastic, chondroblastic, or fibroblastic. Eighty percent of cases originate from the metaphysis (location of the growth plate) of long bones, including the proximal tibia, proximal humerus, and distal femur. Although rare, cases have been noted in the spine and pelvis.

[0362] The primary site of OS metastasis is the lung, with an estimated 20% of OS patients having metastatic disease at the time of diagnosis. Unfortunately, the presence of lung metastases dramatically impacts a patient's 5-year survival rate, decreasing it from 70% in non-metastatic patients to 30%. The standard treatment for OS lung metastases is lung resection; however, clinical trials are investigating the use of other monotherapies and combinatorial therapies.

[0363] Several preclinical animal models have been developed to test osteosarcoma tumor progression and treatment efficacy, including the K7M2 tumor BALB / c mouse model. K7M2 cells are a derivative of a spontaneous BALB / c mouse osteosarcoma; however, they differ from their parental lineage in that inoculation leads to lethal lung metastases in >90% of inoculated animals. Also, being a syngeneic tumor, its inoculation into immunocompetent BALB / c animals allows for the study of tumor growth and regression in the presence of a fully intact immune system. Taken together, these features make it a suitable model for the preliminary study of human osteosarcoma lung metastases and drugs under development for this indication.

[0364] The goal of this study was to establish the K7M2 BALB / c model of osteosarcoma lung metastasis after intravenous administration of K7M2 cells as previously described, and to elucidate the kinetics of tumor metastasis. All procedures have been previously described (see, for example, Example 4 above). Table 8 provides an overview of the experimental design.

[0365] Table 8. Study design and test article (TA) administration TIFF2024513826000009.tif75160

[0366] As shown in Figures 7A-7C, body weight (Figure 7A) remained relatively unchanged between groups throughout the study period, but lung weight increased in tumor recipient mice both 3 (Figure 7B) and 6 weeks (Figure 7C) after inoculation with K7M2 cells. Furthermore, histological analysis of lungs from K7M2 recipients 6 weeks after inoculation revealed tumor burden indicative of tumor metastasis in over 90% of blood vessels, as well as partial thrombus formation (Figures 7D-7G).

[0367] Taken together, these data established a K7M2 BALB / c model of osteosarcoma lung metastasis following intravenous administration of K7M2 cells, which showed increased lung weight and positive tumor formation in the lung.

[0368] Example 8: HSV-IL12 efficacy in an in vivo mouse model of osteosarcoma The purpose of this study was, in part, to evaluate the efficacy of intratracheal administration of HSV-IL12 in suppressing established metastatic osteosarcoma lung tumor growth. All procedures have been described previously (see, e.g., Example 4 above). Table 9 provides an overview of the experimental design.

[0369] Table 9: Test plan TIFF2024513826000010.tif119160IT - Intratracheal; IV - Intravenous

[0370] As shown in Figures 8A-8B, body weight (Figure 8A) remained relatively unchanged between groups throughout the study, but lung weights of mice increased 6 weeks after inoculation with K7M2 cells (Figure 8B). Notably, intratracheal administration of HSV-IL12 attenuated the K7M2-mediated increase in lung weight (Figure 8B). In addition, all five animals treated with HSV-IL12 survived to the scheduled time of sacrifice, whereas two animals treated with vehicle died early in the study. HSV-IL12 appeared to have efficacy comparable to recombinant protein therapy.

[0371] Taken together, these data may suggest that HSV-IL12 was effective in inhibiting established metastatic osteosarcoma lung tumor growth.

[0372] Example 9: Intratracheal administration and in vivo evaluation of weekly combinatorial HSV-IL12+HSV-GMCSF in healthy mice The purpose of this study was, in part, to evaluate the toxicity of weekly intratracheally administered combinatorial cytokine-expressing (IL-12 and GM-CSF) non-replicating HSV-1 vectors in healthy mice. All procedures have been previously described (see, e.g., Example 4 above). Table 10 provides an overview of the experimental design.

[0373] (Table 10) Test plan TIFF2024513826000011.tif94160i.t.:Intratracheal administration

[0374] Results of this study suggested that animals in the high dose group showed somewhat dramatic weight loss after each weekly dose administered on days 0 and 7 (Figure 9A). This was somewhat surprising considering that high doses of HSV-GMCSF and HSV-IL12, respectively, were not toxic after weekly administration when administered individually. Instead, animals in the mid-dose group showed no change in weight after treatment, indicating no toxic effects from the indicated doses. Protein analysis of IL-12 and GM-CSF expression after combinatorial treatment revealed high levels of both cytokines in BALF (Figures 9B-9C) and lung homogenates (Figures 9D-9E) of animals treated with high doses of both viruses. There was a decrease in the concentration of both cytokines in lung specimens from animals treated with the mid-dose, respectively, compared to the high-dose group (Figures 9B-9E), indicating a dose effect on transgene expression. Finally, counting of cellular infiltrates in BALF indicated that high-dose treatment led to greater immune cell influx into tissues (Figure 9F), potentially contributing to the toxicity exemplified by weight loss in these animals (Figure 9G).

[0375] Taken together, these data suggested that weekly dosing with intratracheally administered HSV-IL12+HSV-GMCSF nonreplicating HSV-1 vectors resulted in expression of full-length murine IL-12 and GM-CSF, which was well tolerated in healthy animals at doses of 7.46E6 and 3.9E6, respectively.

[0376] Example 10: Combinatorial HSV-IL12 and HSV-GMCSF efficacy in an in vivo mouse model of osteosarcoma The purpose of this study was, in part, to evaluate the efficacy of intratracheal administration of combina...

Claims

1. A recombinant herpesvirus genome comprising one or more polynucleotides encoding an immunomodulatory polypeptide.

2. 2. The recombinant herpesvirus genome of claim 1, which is a recombinant herpes simplex virus genome.

3. The recombinant herpesvirus genome of claim 2, wherein the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof.

4. A recombinant herpes virus genome as described in claim 2 or 3, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in a herpes simplex virus gene selected from the group consisting of infected cell protein (ICP) 0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41, and UL55.

5. 5. The recombinant herpesvirus genome of claim 4, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP4 gene.

6. 5. The recombinant herpesvirus genome of claim 4, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene.

7. 4. The recombinant herpesvirus genome of claim 2 or 3, wherein the recombinant herpes simplex virus genome does not contain an inactivating mutation in one or both copies of the ICP34.5 gene.

8. 4. The recombinant herpesvirus genome of claim 2 or 3, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding immunomodulatory polypeptides within one or both of the ICP4 viral loci.

9. 4. The recombinant herpesvirus genome of claim 2 or 3, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding immune-modulating polypeptides within the ICP22 viral locus.

10. The recombinant herpesvirus genome of claim 1, wherein the immunomodulatory polypeptide is a human immunomodulatory polypeptide.

11. The recombinant herpesvirus genome of claim 1, wherein the immunomodulatory polypeptide is a cytokine or chemokine.

12. The recombinant herpesvirus genome of claim 11, wherein the cytokine is selected from the group consisting of interleukin (IL)-1, IL-2, IL-7, IL-12, IL-13, IL-15, IL-17, IL-18, IL-28, IL-32, IL-33, IL-34, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

13. The recombinant herpesvirus genome of claim 12, wherein the IL-2 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:

3.

14. The recombinant herpesvirus genome of claim 12, wherein the IL-12 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, and 85.

15. The recombinant herpesvirus genome of claim 11, wherein the cytokine is not granulocyte-macrophage colony-stimulating factor (GM-CSF).

16. The recombinant herpesvirus genome of claim 11, wherein the chemokine is selected from the group consisting of chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, CXCL8, CXCL9, CXCL11, CXCL16, CC motif chemokine ligand 2 (CCL2), CCL3, CCL4, CCL5, and CCL11.

17. 2. The recombinant herpesvirus genome of claim 1, wherein the immunomodulatory polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30.

18. A herpesvirus comprising the recombinant herpesvirus genome of claim 1.

19. 20. The herpesvirus of claim 18, which is replication-competent.

20. 20. The herpesvirus of claim 18, which is replication-deficient.

21. 20. The herpesvirus of claim 18, which is not oncolytic.

22. 19. The herpesvirus of claim 18, which is a herpes simplex virus.

23. A pharmaceutical composition comprising the herpesvirus of claim 18 and a pharma- ceutically acceptable excipient.

24. 24. The pharmaceutical composition of claim 23, suitable for topical, transdermal, subcutaneous, intradermal, oral, intranasal, intratracheal, sublingual, buccal, rectal, intravaginal, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, intratumoral, intrathecal, intraventricular, topical, or epicutaneous administration.

25. 24. The pharmaceutical composition of claim 23, suitable for use in a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, an electrohydrodynamic aerosol device, or any combination thereof.

26. 26. The pharmaceutical composition of claim 25, wherein the nebulizer is a vibrating mesh nebulizer.

27. Use of the pharmaceutical composition of any one of claims 23 to 26 in the manufacture of a medicament for treating cancer.

28. The cancer is acute myeloid leukemia, acute lymphoblastic leukemia, adrenal cortical carcinoma, bladder urothelial carcinoma, brain stem glioma, brain low-grade glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma of unknown primary site, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, cervical squamous cell carcinoma, cervical adenocarcinoma, childhood cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine islet cell tumor, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor, gestational trophoblastic tumor, glioblastoma multiforme glioma, hairy cell leukemia, head and neck cancer, cardiac cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, liver cancer, lymphoid neoplasm diffuse large B-cell lymphoma, malignant fibrous tissue cystic bone cancer, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, Merkel cell skin cancer, mesothelioma, metastatic squamous neck cancer of unknown primary site, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative neoplasm, nasal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer cancer), oral cavity cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumors, ovarian low malignant potential tumors, pancreatic cancer, papilloma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pheochromocytoma and paraganglioma, moderately differentiated pineal parenchymal tumors, pineoblastoma, pituitary tumors, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, primary hepatocellular carcinoma, prostate cancer including prostate adenocarcinoma, rectal cancer, kidney cancer, renal cell carcinoma, renal cell carcinoma, respiratory tract cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, cutaneous melanoma, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell neck cancer, stomach cancer28. The use of claim 27, wherein the cancer is selected from the group consisting of supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer, testicular germ cell tumor, throat cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma, renal pelvis and ureteral transitional cell carcinoma, trophoblastic tumor, ureteral cancer, urethral cancer, uterine cancer, uterine cancer, uveal melanoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

29. A pharmaceutical composition described in any one of claims 23 to 26 for providing preventive, palliative, or therapeutic relief of one or more signs or symptoms of cancer in a subject.

30. 30. The pharmaceutical composition of claim 29, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, neuroendocrine tumors, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, leukemia, and lymphoid malignancies.

31. 30. The pharmaceutical composition of claim 29, wherein the cancer is selected from the group consisting of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, renal cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, and thyroid cancer.

32. 32. The pharmaceutical composition of claim 31, wherein the cancer is small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma.

33. 33. The pharmaceutical composition of claim 32, wherein the cancer is non-small cell lung cancer.

34. 30. The pharmaceutical composition of claim 29, wherein the cancer is osteosarcoma.

35. 30. The pharmaceutical composition of claim 29, wherein the subject is a human.

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