Compositions and methods for viral vectors

Abstract

The present invention relates generally to replication-deficient HSV-1 vectors, and more particularly, to replication-deficient HSV-1 vectors and their use to deliver one or more genes encoding transgenic proteins that stimulate immune destruction of tumors.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 331,903, filed April 18, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] The present disclosure relates generally to HSV-1 vectors, and more specifically, the present invention relates to replication-deficient HSV-1 vectors and their use to deliver one or more genes encoding transgenic proteins that stimulate immune destruction of tumors. [Background technology]

[0003] As cancers have evolved mechanisms to evade the immune system, advances in immuno-oncology have provided new therapies to treat cancers that have eluded successful treatment. However, despite significant progress to date in treating various cancers with immune-stimulating therapies such as checkpoint inhibitors and CAR T cells, a significant number of patients remain unresponsive to immunotherapy. Furthermore, solid tumors can be challenging to treat with immuno-oncological approaches due to the complex tumor microenvironment (TME), which contains epithelial, endothelial, mesenchymal, stromal, cancer, and immune cells. In certain solid tumors, cancer cells can affect the activity or inactivity of immune cells in the TME.

[0004] Viral vectors have been developed to deliver therapeutic agents to cells. However, many of these vectors have poor persistence in target cells, preventing sufficient expression of therapeutic agents. In addition, many of the existing vectors cause non-specific inflammation and tissue damage, making delivery unsafe. As a result, despite the progress made to date, there remains a need for methods and compositions that can stimulate immune activity against tumor cells and are effective in treating tumors in a given subject, either alone or in combination with other cancer therapeutic agents. Summary of the Invention

[0005] The present disclosure is based, in part, on the discovery that HSV-1 vectors containing modifications (e.g., gene deletions) that prevent expression of one or more functional infected cell polypeptide 4 (ICP4) and infected cell polypeptide 47 (ICP47) proteins have reduced replication capacity, are oncolytic, have extended payload expression duration, are cytotoxic to proliferating cells (e.g., cancer cells), and exhibit enhanced immunogenicity. As a result, such vectors have been discovered to be useful for delivering therapeutic payloads (e.g., one or more therapeutic polypeptides) useful for reducing tumor size, for example, to treat cancer in a subject in need thereof. The vectors can induce delayed oncolysis and allow sustained expression of therapeutic payloads (e.g., one or more therapeutic polypeptides). Additionally, the vectors exhibit increased immunogenicity (e.g., by increasing expression of human leukocyte antigen (HLA) on the surface of proliferating cells (e.g., cancer cells)), increasing immune activity against tumor cells. These characteristics were unexpected given the unpredictable nature of replication capacity, oncolytic activity, and persistence in tissues among viruses, particularly among various lineages of viruses.

[0006] The present disclosure is also based, in part, on the discovery that certain combinations of therapeutic polypeptides can be used to disrupt one or more pathways (e.g., parallel pathways) that affect the ability of a recipient's immune cells to kill cancer cells, thereby shrinking tumors. For example, certain therapeutic polypeptides that target the tumor stroma, support the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment, induce tertiary lymphoid structures (TLS) in the tumor bed, and / or promote phagocytic autoimmune surveillance may be expressed from a viral vector, e.g., a viral vector lacking functional ICP4 and ICP47 proteins, to enhance the killing ability of immune cells against tumor cells, e.g., to treat cancer in a subject in need thereof. Vectors can be designed to contain certain combinations of therapeutic polypeptides that are effective to treat a given tumor type, such as selecting one or more therapeutic polypeptides to reduce inhibitory features of a particular tumor microenvironment that may prevent the immune system from effectively attacking the tumor.

[0007] Thus, in one aspect, the present disclosure relates to a vector comprising a Herpes Simplex Virus (HSV) genome, wherein the vector comprises a modification that prevents expression of one or more of a functional ICP4 protein and an ICP47 protein. In certain embodiments, the functional ICP4 protein is characterized by the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, and the functional ICP47 protein is characterized by the amino acid sequence of SEQ ID NO:5. In certain embodiments, the HSV genome is an HSV-1 genome, e.g., the McKrae strain genome. In certain embodiments, the vector, when administered to a subject, results in one or more of: (a) delayed tumor lysis; (b) increased immunogenicity; and (c) increased immune activation.

[0008] In certain embodiments, the vector comprises a nucleic acid sequence encoding one or more therapeutic polypeptides. In certain embodiments, the therapeutic polypeptides may have one or more of the following attributes: (a) targeting the tumor stroma; (b) supporting the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment; (c) induce tertiary lymphoid structures (TLS) in the tumor bed; and / or (d) Promoting phagocytic autoimmunosurveillance.

[0009] In certain embodiments, the therapeutic polypeptide targets the tumor stroma by degrading extracellular matrix proteins. Examples of such therapeutic polypeptides include, but are not limited to, hyaluronidase (SEQ ID NO: 6), MMP-9 (SEQ ID NO: 7), or inhibitors of lysyl oxidase. In certain embodiments, the therapeutic polypeptide targets the tumor stroma by activating local endothelium to increase T cell infiltration. Examples of such therapeutic polypeptides include, but are not limited to, inflammatory cytokines, such as TNF (SEQ ID NO: 11), IL-1β (SEQ ID NO: 8), IL-6 (SEQ ID NO: 9), or IL-18 (SEQ ID NO: 18).

[0010] In certain embodiments, the therapeutic polypeptide supports the survival of T cells in the tumor microenvironment by enhancing the recruitment of T cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, CCL19 (SEQ ID NO: 12) or CCL21 (SEQ ID NO: 13). In certain embodiments, the therapeutic polypeptide supports the survival of T cells in the tumor microenvironment by supporting the function of T cells. Examples of such therapeutic polypeptides include, but are not limited to, T cell trophic factors, such as T cell trophic factors selected from IL-7 (SEQ ID NO: 14), IL-12 (SEQ ID NOs: 15 and 16), IL-15 (SEQ ID NO: 17), IL-18 (SEQ ID NO: 18), and IFNγ (SEQ ID NO: 19).

[0011] In some circumstances, e.g., the indication being treated, the T cell is a CAR T cell. In such an embodiment, the therapeutic polypeptide may comprise a soluble TGFβRII (SEQ ID NO: 20 or SEQ ID NO: 21). Alternatively, the therapeutic polypeptide may comprise an antigen recognized by the CAR T cell, e.g., mesothelin. Alternatively, or in addition, the therapeutic polypeptide may comprise a costimulatory molecule, e.g., a costimulatory molecule selected from CD40L (SEQ ID NO: 22) and OX40L (SEQ ID NO: 23).

[0012] In certain embodiments, the therapeutic polypeptide supports the survival of NK cells in the tumor microenvironment by enhancing the recruitment of NK cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, chemokine ligands CCL2 (SEQ ID NO:24), CX3CL1 (SEQ ID NO:25), CXCL16 (SEQ ID NO:26), CCL5 (SEQ ID NO:27), CXCL9 (SEQ ID NO:28), CXCL10 (SEQ ID NO:29), CXCL11 (SEQ ID NO:30). In certain embodiments, the therapeutic polypeptide supports the survival of NK cells in the tumor microenvironment by supporting the function of NK cells. Examples of such therapeutic polypeptides include, but are not limited to, NK cell trophic factors, e.g., NK cell trophic factors selected from IL-2 (SEQ ID NO:31), IL-15 (SEQ ID NO:17), IL-18 (SEQ ID NO:18), and IFNα (SEQ ID NO:32).

[0013] In certain embodiments, the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed. Such therapeutic polypeptides may include CCL19 (SEQ ID NO: 12), lymphotoxin β (SEQ ID NO: 33), CXCL13 (SEQ ID NO: 34), or TNF.

[0014] In certain embodiments, the therapeutic polypeptide promotes phagocytic autoimmune surveillance and may include, but is not limited to, therapeutic polypeptides that disrupt the Sirpα / CD47 axis, such as the Sirpα-IgG fusion transgene (SIRPalpha, SEQ ID NO: 37).

[0015] In certain embodiments, the nucleic acid further comprises a therapeutic polypeptide that supports anti-tumor macrophage polarization. In certain embodiments, the therapeutic polypeptide that supports anti-tumor macrophage polarization comprises TNF, IL-1 (SEQ ID NO: 35), IL-12 (SEQ ID NOs: 15 and 16), IL-17 (SEQ ID NO: 36), or IFNγ (SEQ ID NO: 19).

[0016] In another aspect, the disclosure relates to a method of expressing a polypeptide in a subject, comprising administering to the subject a vector comprising a variant of a Herpes Simplex Virus (HSV) strain that comprises a modification in its genome such that it does not express functional ICP4 and ICP47 proteins. In a particular embodiment, the functional ICP4 protein is characterized by the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, and the functional ICP47 protein is characterized by the amino acid sequence of SEQ ID NO:5. In a particular embodiment, the HSV strain is an HSV-1 strain, e.g., McKrae strain. In a particular embodiment, the vector, when administered to a subject, results in one or more of: (a) delayed tumor lysis; (b) increased immunogenicity; and (c) increased immune activation.

[0017] In certain embodiments, the HSV strain comprises a nucleic acid encoding a therapeutic polypeptide. In certain embodiments, the therapeutic polypeptide may have one or more of the following attributes: (a) targeting the tumor stroma; (b) supporting the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment; (c) induce tertiary lymphoid structures (TLS) in the tumor bed; and / or (d) Promoting phagocytic autoimmunosurveillance.

[0018] In certain embodiments, the therapeutic polypeptide targets the stroma of tumors by degrading extracellular matrix proteins. Examples of such therapeutic polypeptides include, but are not limited to, hyaluronidase, MMP-9, or lysyl oxidase inhibitors. In certain embodiments, the therapeutic polypeptide targets the stroma of tumors by activating local endothelium to increase T cell infiltration. Examples of such therapeutic polypeptides include, but are not limited to, inflammatory cytokines, such as TNF, IL-1β, IL-6, or IL-18.

[0019] In certain embodiments, the therapeutic polypeptide supports the survival of T cells in the tumor microenvironment by enhancing the recruitment of T cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, CCL19 or CCL21. In certain embodiments, the therapeutic polypeptide supports the survival of T cells in the tumor microenvironment by supporting T cell function. Examples of such therapeutic polypeptides include, but are not limited to, T cell trophic factors, such as T cell trophic factors selected from IL-7, IL-12, IL-15, IL-18, and IFNγ.

[0020] In some circumstances, such as the indication being treated, the T cell is a CAR T cell. In such an embodiment, the therapeutic polypeptide may comprise a soluble TGFβRII. Alternatively, the therapeutic polypeptide may comprise an antigen recognized by the CAR T cell, such as mesothelin. Alternatively, or in addition, the therapeutic polypeptide may comprise a costimulatory molecule, such as a costimulatory molecule selected from CD40L and OX40L.

[0021] In certain embodiments, the therapeutic polypeptide supports survival of NK cells in the tumor microenvironment by enhancing recruitment of NK cells to tumor sites. Examples of such therapeutic polypeptides include, but are not limited to, chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11. In certain embodiments, the therapeutic polypeptide supports survival of NK cells in the tumor microenvironment by supporting NK cell function. Examples of such therapeutic polypeptides include, but are not limited to, NK cell trophic factors, e.g., NK cell trophic factors selected from IL-2, IL-15, IL-18, and IFNα.

[0022] In certain embodiments, the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed. Such therapeutic polypeptides may include CCL19, lymphotoxin beta, CXCL13, or TNF.

[0023] In certain embodiments, the therapeutic polypeptide promotes phagocytic autoimmunosurveillance and may include, but is not limited to, therapeutic polypeptides that disrupt the Sirpα / CD47 axis, such as a Sirpα-IgG fusion transgene.

[0024] In certain embodiments, the nucleic acid further comprises a therapeutic polypeptide that supports anti-tumor macrophage polarization. In certain embodiments, the therapeutic polypeptide that supports anti-tumor macrophage polarization comprises TNF, IL-1, IL-12, IL-17, or IFNγ.

[0025] In another aspect, the disclosure relates to a method for preparing a vector comprising a mutant Herpes Simplex Virus (HSV) genome, the vector comprising a modification such that the mutant does not express functional ICP4 and ICP47 proteins, the vector expressing at least one therapeutic polypeptide. The method comprises culturing a cell transfected with (a) a first nucleic acid molecule comprising (i) a portion of the HSV genome, the portion of the HSV genome not encoding functional ICP4 and ICP47 proteins, and (ii) a sequence encoding a marker element, the sequence encoding the marker element being flanked by a first region of homology (HR1) and a second region of homology (HR2); and (b) a second nucleic acid molecule comprising a sequence encoding a therapeutic polypeptide, the sequence encoding the therapeutic polypeptide being flanked by a first region of homology (HR1_) and a second region of homology (HR2_). HR1 is homologous to HR1_ and HR2 is homologous to HR2_, such that the sequence encoding the therapeutic polypeptide is integrated into the first nucleic acid molecule via homologous recombination. In certain embodiments, the cells are ICP4 and / or ICP47 complementing cells. In certain embodiments, the method further comprises purifying viral plaques that do not express the marker element.

[0026] In certain embodiments, the HSV genome is an HSV-1 genome, such as the McKrae strain genome.

[0027] In certain embodiments, a therapeutic polypeptide comprises one or more of the following attributes: (a) targeting the tumor stroma; (b) supporting the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment; (c) induce tertiary lymphoid structures (TLS) in the tumor bed; and / or (d) Promoting phagocytic autoimmunosurveillance.

[0028] In certain embodiments, the therapeutic polypeptide targets the stroma of tumors by degrading extracellular matrix proteins. Examples of such therapeutic polypeptides include, but are not limited to, hyaluronidase, MMP-9, or lysyl oxidase inhibitors. In certain embodiments, the therapeutic polypeptide targets the stroma of tumors by activating local endothelium to increase T cell infiltration. Examples of such therapeutic polypeptides include, but are not limited to, inflammatory cytokines, such as TNF, IL-1β, IL-6, or IL-18.

[0029] In certain embodiments, the therapeutic polypeptide supports the survival of T cells in the tumor microenvironment by enhancing the recruitment of T cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, CCL19 or CCL21. In certain embodiments, the therapeutic polypeptide supports the survival of T cells in the tumor microenvironment by supporting T cell function. Examples of such therapeutic polypeptides include, but are not limited to, T cell trophic factors, such as T cell trophic factors selected from IL-7, IL-12, IL-15, IL-18, and IFNγ.

[0030] In some circumstances, such as the indication being treated, the T cell is a CAR T cell. In such an embodiment, the therapeutic polypeptide may comprise a soluble TGFβRII. Alternatively, the therapeutic polypeptide may comprise an antigen recognized by the CAR T cell, such as mesothelin. Alternatively, or in addition, the therapeutic polypeptide may comprise a costimulatory molecule, such as a costimulatory molecule selected from CD40L and OX40L.

[0031] In certain embodiments, the therapeutic polypeptide supports the survival of NK cells in the tumor microenvironment by enhancing the recruitment of NK cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11. The therapeutic polypeptide can support the survival of NK cells in the tumor microenvironment by supporting the function of NK cells. Examples of such therapeutic polypeptides include, but are not limited to, NK cell trophic factors, e.g., NK cell trophic factors selected from IL-2, IL-15, IL-18, and IFNα.

[0032] In certain embodiments, the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed. Such therapeutic polypeptides may include CCL19, lymphotoxin beta, CXCL13, or TNF.

[0033] In certain embodiments, the therapeutic polypeptide promotes phagocytic autoimmunosurveillance and may include, but is not limited to, therapeutic polypeptides that disrupt the Sirpα / CD47 axis, such as a Sirpα-IgG fusion transgene.

[0034] In certain embodiments, the nucleic acid further comprises a therapeutic polypeptide that supports anti-tumor macrophage polarization, which may include, but is not limited to, TNF, IL-1, IL-12, IL-17, or IFNγ.

[0035] In another aspect, the disclosure relates to a mutated HSV strain comprising a vector described herein.

[0036] In another aspect, the disclosure relates to a cell transduced with the vectors described herein.

[0037] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a vector described herein and a pharma- ceutically acceptable carrier.

[0038] In another aspect, the disclosure relates to a method of reducing tumor size in a subject in need thereof, comprising administering to the subject a vector comprising a mutated strain of Herpes Simplex Virus (HSV) that contains a modification in its genome such that it does not express functional ICP4 and ICP47 proteins. Additionally, the vector comprises a nucleic acid encoding one or more therapeutic polypeptides that function to reduce tumor size.

[0039] In certain embodiments, the mutants are unable to express functional ICP4 and ICP47 proteins, characterized by the amino acid sequences of SEQ ID NOs: 3 and 4, and SEQ ID NO: 5, respectively. In certain embodiments, the HSV strain is an HSV-1 strain, e.g., a McKrae strain.

[0040] In certain embodiments, a therapeutic polypeptide comprises one or more of the following attributes: (a) targeting the tumor stroma; (b) supporting the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment; (c) induce tertiary lymphoid structures (TLS) in the tumor bed; and / or (d) Promoting phagocytic autoimmunosurveillance.

[0041] In certain embodiments, the therapeutic polypeptide targets the stroma of tumors by degrading extracellular matrix proteins. Examples of such therapeutic polypeptides include, but are not limited to, hyaluronidase, MMP-9, or lysyl oxidase inhibitors. In certain embodiments, the therapeutic polypeptide targets the stroma of tumors by activating local endothelium to increase T cell infiltration. Examples of such therapeutic polypeptides include, but are not limited to, inflammatory cytokines, such as TNF, IL-1β, IL-6, or IL-18.

[0042] In certain embodiments, the therapeutic polypeptide supports the survival of T cells in the tumor microenvironment by enhancing the recruitment of T cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, CCL19 or CCL21. In certain embodiments, the therapeutic polypeptide supports the survival of T cells in the tumor microenvironment by supporting T cell function. Examples of such therapeutic polypeptides include, but are not limited to, T cell trophic factors, such as T cell trophic factors selected from IL-7, IL-12, IL-15, IL-18, and IFNγ.

[0043] In some circumstances, such as the indication being treated, the T cell is a CAR T cell. In such an embodiment, the therapeutic polypeptide may comprise a soluble TGFβRII. Alternatively, the therapeutic polypeptide may comprise an antigen recognized by the CAR T cell, such as mesothelin. Alternatively, or in addition, the therapeutic polypeptide may comprise a costimulatory molecule, such as a costimulatory molecule selected from CD40L and OX40L.

[0044] In certain embodiments, the therapeutic polypeptide supports survival of NK cells in the tumor microenvironment by enhancing recruitment of NK cells to tumor sites. Examples of such therapeutic polypeptides include, but are not limited to, chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11. In certain embodiments, the therapeutic polypeptide supports survival of NK cells in the tumor microenvironment by supporting NK cell function. Examples of such therapeutic polypeptides include, but are not limited to, NK cell trophic factors, e.g., NK cell trophic factors selected from IL-2, IL-15, IL-18, and IFNα.

[0045] In certain embodiments, the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed. Such therapeutic polypeptides may include CCL19, lymphotoxin beta, CXCL13, or TNF.

[0046] In certain embodiments, the therapeutic polypeptide promotes phagocytic autoimmunosurveillance and may include, but is not limited to, therapeutic polypeptides that disrupt the Sirpα / CD47 axis, such as a Sirpα-IgG fusion transgene.

[0047] In certain embodiments, the nucleic acid further comprises a therapeutic polypeptide that supports anti-tumor macrophage polarization. In certain embodiments, the therapeutic polypeptide that supports anti-tumor macrophage polarization comprises TNF, IL-1, IL-12, IL-17, or IFNγ.

[0048] In another aspect, the present disclosure relates to a vector comprising a herpes simplex virus (HSV) genome, where the vector comprises a modification that prevents expression of one or more functional ICP4 and ICP47 proteins, and where the vector encodes one or more therapeutic polypeptides that support survival of NK cells in the tumor microenvironment (TME).

[0049] In certain embodiments, the NK cells are natural NK cells or CAR NK cells. In certain embodiments, the one or more therapeutic polypeptides comprise an NK cell-recruiting factor. In certain embodiments, the NK cell-recruiting factor is a chemokine ligand. In certain embodiments, the chemokine ligand is selected from CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, and CXCL11. In certain embodiments, the one or more therapeutic polypeptides comprise one or more NK cell trophic factors selected from IL-2, IL-15, IL-18, and IFNα. In certain embodiments, the one or more therapeutic polypeptides comprise soluble TGFβRII.

[0050] These and other aspects and features of the present invention are set forth in the following detailed description and claims.

[0051] The above and other objects, features and advantages of the present disclosure will become apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings. Like reference elements identify common features in corresponding drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the present disclosure. [Brief description of the drawings]

[0052] [Figure 1A] Schematic diagram of the McKrae strain HSV-1 genome. The wild-type McKrae strain HSV-1 genome is depicted, showing essential genes (encoding UL27 (gB), UL48 (VP16), ICP27, and ICP4) and non-essential genes (encoding ICP0, LAT, UL37, UL38, UL41 (vhs), LAT, ICP0, ICP22, and ICP47). The genome is divided into two parts, the unique long (UL) part and the unique short (US) part, which are connected via a splice region. ICP4 is encoded at two loci and ICP47 at one locus. TR denotes terminal repeat and IR denotes inverted repeat. [Figure 1B] 1 is a schematic representation of the McKrae strain HSV-1 genome. FIG. 2 is a schematic representation of the McKrae strain HSV-1 genome with a deletion of the ICP4 locus. [Figure 1C] 1 is a schematic representation of the McKrae strain HSV-1 genome, FIG. 2 is a schematic representation of the McKrae strain HSV-1 genome with deletions of both the ICP4 and ICP47 loci. [Diagram 2] Exemplary vector designs are shown for delivering multiple payloads (e.g., genes 1-5) to cancer cells. The top diagram shows the general design of a suitable promoter driving expression of the payload cassette. The bottom diagram shows an exemplary design of a payload cassette with the HCMV immediate early promoter (HCMV IEp), payload genes 1-5, and a polyA tail (pA). Tosea asignavirus 2A (T2) and porcine teschovirus-1 2A (P2) show exemplary ribosomal skipping sites. [Figure 3A]1 is a graph showing the viability monitored over 8 days of cancer cells infected with the ICP4-deficient McKrae HSV-1 strain at three MOIs (0, 3, and 10). As shown, infection with the ICP4-deficient virus resulted in delayed oncolysis. [Figure 3B] 1 is a graph showing payload expression monitored over an 8 day period in cancer cells infected with the ICP4-deficient McKrae HSV-1 strain at three MOIs: 0, 3, and 10. As shown, infection with the ICP4-deficient virus resulted in sustained payload expression. [Figure 3C] FIG. 1 is a graph showing the viability of Hs578T cancer cells infected with mutICP4mutICP47 McKrae HSV-1 strain at three MOIs (0, 3, and 10) monitored over a six day period. [Figure 3D] 13 is a graph showing IFNγ payload expression in Hs578T cancer cells infected with mutICP4mutICP47 McKrae HSV-1 strain at an MOI of 10 on days 2, 4, and 6. [Figure 4A] 1 is a graph showing payload (GFP) expression after 24 hours in cells infected with one of two exemplary oncolytic viruses, mutICP4 McKrae HSV-1 and mutICP4mutICP47 McKrae HSV-1, at three MOIs (0.3, 1, and 3). "mutICP4" is a McKrae HSV-1 strain deleted for the gene encoding ICP4. "mutICP4mutICP47" is a McKrae HSV-1 strain deleted for the genes encoding ICP4 and ICP47. These data show that comparable infections were obtained with mutICP4 and mutICP4mutICP47. [Figure 4B]1 is a graph showing GFP and HLA expression after 24 hours in cells infected with one of two exemplary oncolytic viruses, mutICP4 McKrae HSV-1 and mutICP4mutICP47 McKrae HSV-1, at three MOIs (0.3, 1, and 3). These data show that nearly all cells were HLA positive. [Figure 4C] 1 is a flow cytometry graph of HLA expression after 24 hours by flow cytometry in cells infected with one of two exemplary oncolytic viruses, mutICP4 McKrae HSV-1 and mutICP4mutICP47 McKrae HSV-1, at three MOIs (0.3, 1, and 3). These data show that increased cell surface expression of HLA, a component of MHC class I molecules, is observed at all MOIs for viruses deleted for both ICP4 and ICP47 compared to viruses deleted for ICP4 alone. [Figure 4D] FIG. 1 is a graph showing Tap1 expression in Hs578T cells infected with one of two exemplary oncolytic viruses, mutICP4mutICP47 McKrae HSV-1 and mutICP27 McKrae HSV-1, as measured by RNAseq. [Figure 4E] FIG. 1 is a graph showing MHC class I expression in Hs578T cells infected with one of two exemplary oncolytic viruses, mutICP4mutICP47 McKrae HSV-1 and mutICP27 McKrae HSV-1, as measured by flow cytometry. [Figure 4F] mutICP4mutICP47 McKrae Graph showing cell signaling pathways upregulated in cancer cells following HSV-1 infection. [Figure 4G] FIG. 1 is a graph showing cell signaling pathways upregulated in cancer cells following mutICP27 McKrae HSV-1 infection. [Figure 4H]Graph showing expression of TNF superfamily members (TNF, LTB, TNFSF14, PYCARD, and CASP10) in cancer cells following infection with mutICP4mutICP47 McKrae HSV-1 or mutICP27 McKrae HSV-1. [Figure 5A] FIG. 1 is a graph showing the number of CD31+ endothelial cells in mouse salivary glands following infection with mutICP4mutICP47 McKrae HSV-1 viruses carrying payloads of LTB, CXCL13, CCL19, CCL21, and IL-7 in mice. [Figure 5B] 1 is a graph showing the number of PDGFRa+PDPN+ stromal cells in mouse salivary glands following infection with mutICP4mutICP47 McKrae HSV-1 viruses carrying payloads of LTB, CXCL13, CCL19, CCL21, and IL-7 in mice. [Figure 5C] Graph showing expression of TGFβ1 in Hs578T cells following infection with mutICP4mutICP47 (encoding GFP as a marker) as measured by RNAseq. [Figure 6A] FIG. 13 is a graph showing the viability of Hs578T cells after infection with viral backbone alone (mutICP4mutICP47-GFP) or mutICP4mutICP47 vectors encoding human IFNγ, human IL-12, or human IFNγ and human IL-12, or after co-culture with PBMCs and addition of recombinant rhIL-15. [Figure 6B] Graph showing the number of Ki67+ Granzyme B+ CD8+ T cells after 24 hours of co-culture with Hs578T cells and PBMCs following infection with MutICP4mutICP47-IFNγ or MutICP4mutICP47-GFP (backbone). [Figure 6C] Graph showing the number of Ki67+ Granzyme B+ CD8+ T cells after 72 hours of co-culture with Hs578T cells and PBMCs following infection with MutICP4mutICP47-IFNγ or MutICP4mutICP47-GFP (backbone). [Figure 6D] Graph showing the number of Granzyme B+Ki67-NK cells after 24 hours of co-culture with Hs578T cells and PBMCs following infection with MutICP4mutICP47-IFNγ or MutICP4mutICP47-GFP (backbone). [Figure 6E] Graph showing antigen presenting cell numbers (CD11c+CD16+CD14-Ki67+MHCII++) 24 hours after co-culture of Hs578T cells and PBMCs following infection with MutICP4mutICP47-IFNγ or MutICP4mutICP47-GFP (backbone). [Figure 6F] Graph showing viability of Hs578T cells after infection with MutICP4mutICP47-IFNγ or MutICP4mutICP47-GFP (backbone) with or without PBMC co-culture. [Figure 6G] Graph showing expression of multiple T cell and NK cell chemokines (CCL2, CXCL9, CXCL10, CXCL11, CCL5, CX3CL1, and CXCL16) by Hs578T cells after 24 h infection with mutICP4mutICP47-GFP or mutICP4mutICP47-IFNγ as measured by RNAseq. [Figure 7A] FIG. 13 is a graph showing the percentage of CD45+ cells in mouse salivary glands harvested 15 days after administration of mutICP4mutICP-GFP HSV-1 vector or vehicle to mice. [Figure 7B] Photographs taken by a microscope at 40x resolution of cells isolated from mouse salivary glands taken 15 days after administration of the mutICP4mutICP-GFP HSV-1 vector to mice are shown. Cells are stained with CD4+ (T cell marker), B220 (B cell marker), CD11c (dendritic cell marker), and DAPI (nuclear marker). [Figure 7C]Photographs taken by microscope at 10x and 40x resolution from mouse salivary glands taken 15 days after administration of mice with a combination of mutICP4mutICP47 HSV-1 vectors expressing mouse TNF, CCL19, IL-17a, and IL-7. Cells are stained for CD4+ (T cell marker), B220 (B cell marker), CD11c (dendritic cell marker), and DAPI (nuclear marker). [Figure 8] Figure 1 shows the percentage of phagocytosis of labeled Raji cells by macrophages. Raji cells were pretreated with conditioned medium from mutICP4mutICP47-SIRPα-IgG-infected Hs578T cells (MOI 3 or 10), conditioned medium from mutICP4mutICP47-GFP cells, IgG, or anti-CD47 antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present disclosure is based, in part, on the discovery that HSV-1 vectors containing modifications (such as gene deletions or inactivating mutations) that prevent expression of one or more functional ICP4 and ICP47 proteins have reduced replication capacity, are oncolytic, have extended payload expression duration, are cytotoxic to proliferating cells (e.g., cancer cells), and exhibit enhanced immunogenicity. As a result, the vectors have been found to be useful for delivering therapeutic payloads (e.g., therapeutic polypeptides) useful for reducing tumor size, for example, to treat cancer in a subject in need thereof. The vectors can induce delayed oncolysis and allow sustained expression of therapeutic payloads (e.g., therapeutic polypeptides). Additionally, the vectors exhibit increased immunogenicity and increase immune activity against tumor cells.

[0054] The present invention is also based, in part, on the discovery that certain combinations of therapeutic polypeptides can be used to disrupt one or more pathways (e.g., parallel pathways) that affect the ability of a recipient's immune cells to kill cancer cells, thereby shrinking tumors. For example, therapeutic polypeptides that target the tumor stroma, support the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment, induce tertiary lymphoid structures (TLS) in the tumor bed, and / or promote phagocytic autoimmune surveillance may be expressed from a viral vector, e.g., a viral vector lacking functional ICP4 and ICP47 proteins, to enhance the killing ability of immune cells against tumor cells, e.g., to treat cancer in a subject in need thereof. Vectors can be designed to contain a specific combination of therapeutic polypeptides that are effective to treat a given tumor type, such as selecting one or more therapeutic polypeptides to reduce inhibitory features of a particular tumor microenvironment that may prevent the immune system from effectively attacking the tumor.

[0055] I. Definition In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one," (ii) the term "or" may be understood to mean "and / or," (iii) the terms "comprising" and "including," whether listed by themselves or with one or more additional components or steps, may be understood to encompass the listed components or steps, and (iv) when ranges are provided, the endpoints are included.

[0056] As used herein, the term "administration" refers to administration of a composition to a subject or system. Administration to an animal subject (e.g., a human) can be by any suitable route. For example, in some embodiments, administration can be bronchial (including by bronchial infusion), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intratumoral, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including intratracheal infusion), transdermal, intravaginal, and intravitreal. Depending on the circumstances, administration can involve intermittent administration and / or continuous administration (e.g., perfusion) for at least a selected period of time.

[0057] As used herein, the term "agent" refers to any chemical class of compound or entity, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, or combinations thereof. In some embodiments, an agent is or includes a natural product in that it is found in nature and / or obtained from nature. In some embodiments, an agent is or includes one or more artificial entities that are man-made in that it is designed, engineered, and / or produced by the action of the hand of man and / or is not found in nature. Some specific embodiments of agents that may be utilized in accordance with the present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNA, shRNA, DNA / RNA hybrids, antisense oligonucleotides, ribozymes), peptides, peptidomimetics, and the like.

[0058] As used herein, the term "amelioration" refers to the prevention, alleviation or remission of a condition in a subject, or the improvement of a condition. Amelioration includes, but does not require, complete recovery or complete prevention of a disease, disorder, or condition.

[0059] As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans of either sex and at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be genetically modified animals, genetically engineered animals, and / or clones. Depending on the context, the terms "human," "patient," and "subject" are used interchangeably herein.

[0060] The terms "about" and "approximately" may be understood to allow for standard variations as understood by one of ordinary skill in the art. For example, as used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise indicated or implied.

[0061] As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, the two or more agents may be administered simultaneously, in some embodiments, such agents may be administered sequentially, and in some embodiments, such agents are administered in an overlapping dosing regimen.

[0062] As used herein, the term "engineered" refers to an aspect of being manipulated by the hand of man. For example, a polynucleotide is considered to be "engineered" if it is manipulated by the hand of man such that two or more sequences that are not linked together in that order in nature are directly linked to each other in the engineered polynucleotide. For example, in some embodiments of the present disclosure, an engineered polynucleotide includes a regulatory sequence that is found in nature in a working relationship with a first coding sequence, but not in a working relationship with a second coding sequence, and that is linked by the hand of man to be in a working relationship with the second coding sequence. Equivalently, a cell or organism is considered to be "engineered" if its genetic information is manipulated to be altered (e.g., new genetic material not previously present is introduced by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or previously present genetic material is altered or removed, such as by substitution or deletion mutations, or mating protocols). As is common practice and understood by those of skill in the art, the progeny of engineered polynucleotides or cells are typically still referred to as "engineered," even though actual manipulation occurred on the progenitor entity.

[0063] As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0064] As used herein, the term "homologous" refers to the overall relatedness between polymeric molecules, such as between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be "homologous" to one another if their sequences are at least 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymeric molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar.

[0065] As used herein, the term "isolated" refers to substances and / or entities that are (1) separated from at least some of the components with which they were associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99% or more of the other components with which they were originally associated. In some embodiments, an isolated agent has a purity of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99% or greater. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, a substance may be considered "isolated" or "pure" even after it has been combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.), as will be understood by those of skill in the art. In such embodiments, the isolation or purity of a substance is calculated without including such carriers or excipients. By way of example, in some embodiments, a biological polymer, such as a naturally occurring polypeptide or polynucleotide, is considered to be "isolated" if: a) because of its origin or source, it is not associated in its natural state in nature with some or all of the components that accompany it; b) it is substantially free of other polypeptides or nucleic acids of the same species as the species that produces it in nature; or c) it is expressed by or is otherwise associated with components from cells or other expression systems other than the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the one that produces it in nature is considered to be an "isolated" polypeptide.Alternatively, or in addition, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from a) other components with which it is naturally associated, and / or b) other components with which it was associated when originally produced.

[0066] As used herein, the term "marker element" refers to a detectable or selectable agent. In some embodiments, a "marker element" is a detectable or selectable nucleic acid sequence. In some embodiments, a "marker element" is an expression product (e.g., RNA or protein) whose presence or absence is detectable and / or selectable in a cell. In some embodiments, the expression product is or includes an enzyme. In some embodiments, the expression product is a fluorescent substance.

[0067] As used herein, the term "nucleic acid" refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As is clear from the context, in some embodiments, a "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or a nucleoside), and in some embodiments, a "nucleic acid" refers to an oligonucleotide chain that includes individual nucleic acid residues. In some embodiments, a "nucleic acid" is or includes RNA. In some embodiments, a "nucleic acid" is or includes DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, but are considered within the scope of the present disclosure. Alternatively, or in addition, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, or more residues in length. In some embodiments, the nucleic acid is single-stranded, and in some embodiments, the nucleic acid is double-stranded.In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide, hi some embodiments, the nucleic acid has enzymatic activity.

[0068] As used herein, the terms "patient" or "subject" are used interchangeably and refer to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Exemplary subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the subject is a human. In some embodiments, the subject is suffering from or susceptible to one or more disorders or conditions. In some embodiments, the subject exhibits symptoms of one or more disorders or conditions. In some embodiments, the subject has been diagnosed with one or more disorders or conditions. In some embodiments, the subject is undergoing or has undergone a particular treatment to diagnose and / or treat a disease, disorder, or condition.

[0069] As used herein, the term "pharmaceutical composition" refers to an active agent (e.g., a vector) formulated with one or more pharma- ceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical compositions may be formulated for administration in solid or liquid form, including those adapted for: oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), e.g., tablets, boluses, powders, granules, tongue pastes intended for buccal, parenteral, sublingual, and systemic absorption; parenteral administration, e.g., as a sterile solution or suspension, or sustained release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, e.g., as a cream, ointment, or sustained release patch or spray applied to the skin, lungs, or buccal cavity; vaginal or rectal administration, e.g., as a pessary, cream, or foam; sublingual administration; ocular administration; transdermal administration; or administration to nasal, pulmonary, and other mucosal surfaces.

[0070] As used herein, the term "pharmacologically acceptable" applied to a carrier, diluent, or excipient used to formulate a composition as disclosed herein, means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0071] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in the carrying or transport of a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Examples of substances which may function as pharma- ceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; celluloses and their derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffers; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0072] As used herein, the term "prevent" or "prevention," when used in relation to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention is considered complete when the onset of the disease, disorder, or condition has been delayed for a predefined period of time.

[0073] As used herein, the term "treatment" (also the terms "treat" or "treating") refers to any administration of a substance that partially or completely alleviates, improves, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition (e.g., cancer). Such treatment may be treatment of subjects who do not exhibit symptoms of the associated disease, disorder, and / or condition, and / or subjects who exhibit only early symptoms of the disease, disorder, and / or condition. Alternatively, or in addition, such treatment may be treatment of subjects who exhibit one or more established symptoms of the associated disease, disorder, and / or condition. In some embodiments, treatment may be of subjects who have been diagnosed as suffering from the associated disease, disorder, and / or condition. In some embodiments, treatment may be of subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated disease, disorder, and / or condition.

[0074] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, into which a DNA segment may be ligated to a viral genome or a portion thereof. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having an origin of replication, episomal mammalian vectors, herpes simplex virus (HSV) vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are linked in a working relationship. Such vectors are referred to herein as "expression vectors."

[0075] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)).

[0076] The use of clauses is not intended to limit the invention. Each clause may be applicable to any aspect of the invention. In this application, the use of "or" means "and / or" unless otherwise stated or clearly disjunctive from the context.

[0077] Throughout this specification, when compositions are described as having, including, or comprising particular ingredients, or when processes and methods are described as having, including, or comprising particular steps, it is additionally contemplated that there are compositions of the invention that essentially include, or comprise, the recited ingredients, and that there are processes and methods according to the invention that essentially include, or comprise, the recited processing steps.

[0078] In this application, when an element or component is said to be included in and / or selected from a recited list of elements or components, it is to be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group comprising two or more of the recited elements or components.

[0079] Furthermore, it should be understood that elements and / or features of the compositions or methods described herein, whether express or implicit in the specification, can be combined in various ways without departing from the spirit and scope of the invention. For example, when a particular compound is mentioned, that compound can be used in various embodiments of the compositions of the invention and / or in the methods of the invention, unless otherwise understood from the context. In other words, within this application, the embodiments have been described and depicted in a manner that allows a clear and concise application to be written and depicted, but it is intended and understood that the embodiments can be variously combined or separated without departing from the present teachings and invention(s). For example, it will be understood that all features described and depicted herein are applicable to all aspects of the invention described and depicted herein.

[0080] The phrase "at least one" should be understood to include each of the subsequent listed objects individually and the various combinations of two or more of the listed objects, unless otherwise understood from context and usage. Additionally, the phrase "and / or" in the context of three or more listed objects should be understood to have the same meaning, unless otherwise understood from context.

[0081] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, should be understood as generally open-ended and open-ended, e.g., not excluding additional, unrecited elements or steps, unless otherwise stated or understood from context.

[0082] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0083] The use of any and all examples or exemplary language herein, such as "such as" or "including," is intended merely to better describe the invention and does not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0084] II. Viral Vectors and HSV-1 (McKrae) Viral vectors can be used to facilitate the transfer of nucleic acids into cells. HSV-1 vectors can usually accommodate up to 25 kb of foreign DNA sequences. HSV-1 has a double-stranded linear DNA genome of approximately 152 kb and can be maintained episomally in the nucleus of the cell. HSV-1 virions are enveloped and have a diameter of approximately 110 nm.

[0085] At least 17 strains of HSV-1 have been isolated, including, but not limited to, McKrae, 17, F, H129, HF10, MacIntyre, HF, ATCC 2011, and KOS (for review, see Watson et al. (2012) Virology 433(2):528-537).

[0086] A.McKrae The McKrae strain was isolated from a patient with herpes simplex keratitis and subsequently passaged in tissue culture. The partial genomic sequence of McKrae is set forth in SEQ ID NO: 1 (GenBank Accession No.: JQ730035.1).

[0087] Strain differences in HSV-1 peripheral replication and pathogenicity have been observed following injection into animals. McKrae undergoes spontaneous or induced reactivation at a higher frequency than any other known strain and is one of the most pathogenic HSV-1 strains.

[0088] HSV genes affect the viral properties and phenotype. There are at least nine genes and several non-coding sequences that are unique to the McKrae strain. In addition to genes related to virulence and reactivation of latency such as RL1, RSI, and RL2, three UL genes (UL36, UL49A, UL56) and three US genes (US7, US10, and US11) are unique to the McKrae strain. In addition to gene variants, non-coding sequences such as LAT, 'a' sequences, and miRNAs contain variants unique to the McKrae strain.

[0089] One or more of the following gene and non-coding sequences may be considered characteristic of the McKrae strain: In the McKrae strain, RL1 (ICP34.5) has eight repeated extended PAT repeats between residues 159 and 160, whereas other strains have only three to five repeats. The PAT repeats are thought to affect the subcellular localization of the ICP34.5 protein (Mao et al. (2012) J. Biol. Chem. 277(13):11423-31). ICP34.5 is thought to be a neuroviral factor involved in viral replication and anti-host responses.

[0090] The McKrae strain also contains an expanded repeat element of 6 repeats of the internal tandem repeat STPSTTT (SEQ ID NO: 38) located within the coding sequence of US07 (gl). Furthermore, in McKrae, UL36 contains a premature stop codon introduced due to a G nucleotide deletion in the mononucleotide stretch encoding amino acid residue 2453 (nt 72,535), and UL56 (180aa) contains a 1 base pair insertion at nucleotide 115,992 (amino acid 97). The McKrae strain also contains an expanded ORF in US10 due to a 1 bp insertion at nucleotide 143,416, causing a frameshift resulting in the loss of a stop codon in McKrae and a unique C-terminal protein sequence. In McKrae, amino acids at residues 28 and 51 of UL49A differ from other strains. At residues 28 and 51, McKrae has histidine and threonine, whereas 17 has arginine and threonine, and other strains (e.g., KOS) have histidine and alanine. McKrae also has a reduced tandem repeat at the UL-RL junction (181 bp in 17 and KOS versus 49 bp in McKrae) and is missing approximately 330 nucleotides immediately following the UL-RL junction repeat. McKrae also has a unique mutation in the direct repeat 2 (DR2) sequence of the 'a' sequence. Instead of a series of unbroken tandem repeats, the McKrae DR2 repeat is interrupted twice by the same guanine-rich sequence.

[0091] The main variation within the LAT intron between strains is due to differences in the number of repeats of a repeat element (GCACCCCCACTCCCAC) (SEQ ID NO: 39) beginning at nucleotide 119,482 in the McKrae strain, with the McKrae strain containing 13 repeats, whereas the F, H129 and 17 strains contain 9 repeats and the KOS strain contains 15 repeats. There is also variation in the tandem repeats between strains, beginning at base 125,520 in the McKrae strain. The McKrae repeat element contains 12 repeats of CCCCAGCCCTCCCCAG (SEQ ID NO: 40) and 8 repeats of CCCCTCGCCCTCCCCG (SEQ ID NO: 41). The first repeat unit is unique from the other strains in that it contains a GA transition, with the McKrae strain containing 3 more repeats than any other strain. The second repeat element in strain McKrae is folded, missing 188 nucleotides compared with all other strains, and is separated from the upstream repeat by a 105-bp 100% conserved sequence that contains miR-H5.

[0092] McKrae further contains a unique coding sequence for ICP4 that is not found in other known strains (Watson et al., (2012) supra). ICP4 is an immediate early transcription factor and is involved in reactivation. Whereas other strains have an alanine-rich region (AASAPDAADALAAA) (SEQ ID NO: 42) between residues 707 and 720, in McKrae the alanine-rich region is replaced with a serine-rich sequence (GPRRSSSSSGVAA) (SEQ ID NO: 43). The serine-rich substitution block present in McKrae is adjacent to a nuclear localization signal (NLS) (amino acids 728-734). Conformational changes in this region may alter the NLS and thus affect the localization of not only ICP4 but also other viral proteins (e.g., ICP0, ICP8) that are affected by ICP4 localization (Knipe and Smith, Molecular and Cellular Biology, (1986), 6(7), 2371-2381). Thus, this region may affect the viral phenotype in part by altering the localization of the protein to the nucleus.

[0093] B. Modification of HSV-1 Vectors Vectors containing the HSV-1 genome (e.g., from the McKrae strain) may have one or more HSV genes required for replication that are no longer functional. For example, vectors containing the HSV genome may contain modifications that prevent expression of one or more functional ICP0, ICP4, ICP22, ICP27, and ICP47 genes. HSV genes required for replication include immediate early genes, such as ICP4 and ICP47. ICP4 is a viral transcription factor that is expressed soon after infection and maintains the viral cycle of HSV-1. ICP47 of HSV-1 is a cytoplasmic polypeptide of 87 amino acids, 88 residues including the initiator methionine. It binds to the TAP1-TAP2 heterodimer in human cells, but not in mouse cells, and prevents peptide transport by inhibiting the peptide binding site of TAP. As a result, MHC class I molecules cannot be loaded with peptides. The resulting empty class I molecules are retained in the ER, and epitope presentation to CTLs is abrogated in human cells infected with HSV. Mutations that inhibit expression of one or more functional HSV genes (e.g., ICP4 and ICP47) can include mutations (e.g., missense mutations, nonsense mutations, insertions, deletions, etc.) in the coding sequence of the gene or in regulatory sequences that affect expression of the gene (e.g., promoter). ICP47 mutations include, but are not limited to, mutations of A4, D27, K31, R32, R34, or R41 relative to SEQ ID NO:5, and combinations thereof (as described in Mozzie et al. (2022) Mol Biol Evol. 39(7):msac142). Mutants and deletion mutants of ICP4 that disrupt the ability of ICP4 to activate transcription include, but are not limited to, n208, d8-10, nd8-10, ΔSER, d120, n12, ΔSERn7, d3-8, nd3-8, m20, m20n7, m90, m90n7, d143, d143n7, and nd3-10 (as described in Wagner et al. (2012) J Viol. 86(12):6862-74).Deletion of the region between the N-terminal amino acids 30 and 210 of ICP4 is sufficient to eliminate transcriptional activation (as described in Wagner et al. (2013) J Viol. 87(2):1010-1018). In some embodiments, the disclosure provides replication-deficient HSV vectors having modifications in one or more of ICP0, ICP4, ICP22, ICP27, and ICP47.

[0094] The HSV-1 IE promoter contains one or more copies of the consensus TAATGARAT (SEQ ID NO: 44) IE-specific regulatory sequence, where R is a purine. Although these motifs are usually located within a few hundred base pairs of the proximal IE promoter sequence, they are separate functional entities that, together with their flanking sequences, can confer IE-specific control to other proximal promoter elements of different temporal classes. In some embodiments, the replication-defective virus is generated by deleting nucleotides in the IE-specific regulatory sequence that affect expression of one or more of the IE-specific regulatory sequences, e.g., ICP0, ICP4, ICP22, ICP27, and ICP47. In some embodiments, the IE-specific regulatory sequence comprises an internal deletion. In some embodiments, the IE-specific regulatory sequence comprises a terminal deletion. In some embodiments, the IE-specific regulatory sequence is completely deleted.

[0095] In some embodiments, the disclosure provides HSV vectors having non-functional ICP4 and ICP47 genes. In certain embodiments, the ICP4 gene or the regulatory sequence affecting the expression of ICP4 comprises a mutation, and the ICP47 gene or the regulatory sequence affecting the expression of ICP47 comprises a mutation. For example, the ICP4 gene can comprise a mutation, and the ICP47 gene can comprise a mutation. In another example, the regulatory sequence affecting the expression of ICP4 comprises a mutation, and the regulatory sequence affecting the expression of ICP47 comprises a mutation. In one embodiment, the ICP4 gene or the regulatory sequence affecting the expression of ICP4 comprises a deletion (e.g., a complete deletion or a partial deletion sufficient to abolish activity), and the ICP47 gene or the regulatory sequence affecting the expression of ICP47 comprises a deletion (e.g., a complete deletion or a partial deletion sufficient to abolish activity). The ICP4 gene can comprise a deletion, and the ICP47 gene can comprise a deletion. Additionally, combinations of the above are contemplated, e.g., a deletion of ICP4 and a mutant of the regulatory sequence of ICP47.

[0096] In some embodiments, the disclosure provides an HSV vector or strain with a non-functional ICP47 gene. In some embodiments, the disclosure provides an HSV vector or strain with non-functional ICP4 and ICP47 genes. In some embodiments, the disclosure provides an HSV vector or strain that is ICP4 and ICP47 deleted. In some embodiments, the gene encoding ICP4 and the gene encoding ICP47 are deleted completely or partially without disrupting expression of additional immediate-early genes.

[0097] HSV-1 vectors with altered (e.g., mutated) HSV genes can be produced in a cell line that expresses the defective proteins in trans. In some embodiments, the HSV-1 vectors are produced in a mammalian cell line, e.g., a mammalian cell line of the Vero line. In some embodiments, the cell line expresses ICP4. In some embodiments, the cell line expresses ICP47. In some embodiments, the cell line expresses ICP4 and ICP47. In some embodiments, the cell line expresses one or more of ICP0, ICP4, ICP22, ICP27, and ICP47. In some embodiments, the cell line expresses ICP4, ICP22, and ICP47. In some embodiments, the cell line expresses ICP4, ICP22, and UL55. In some embodiments, the cell line expresses ICP4, ICP27, and UL55. In some embodiments, the cell line comprises a nucleic acid molecule having a Simian Virus 40 polyadenylation signal (SV40 pA). Optionally, viral vectors can be produced in Vero 6-5C cells or Vero D cells.

[0098] In certain embodiments, the viral vector of the disclosure is a McKrae HSV-1 viral vector. Wild-type McKrae strain HSV-1 contains two copies of the gene encoding ICP4 and one copy of the gene encoding ICP47. In some embodiments, the disclosure provides an HSV vector having a non-functional ICP4 gene and an ICP47 gene. In certain embodiments, at least one ICP4 gene (i.e., one or both copies of the gene) or a regulatory sequence that affects the expression of ICP4 contains a mutation, and the ICP47 gene or a regulatory sequence that affects the expression of ICP47 contains a mutation. For example, at least one copy of the ICP4 gene (i.e., one or both copies of the ICP4 gene) can contain a mutation and the ICP47 gene can contain a mutation. In another example, a regulatory sequence that affects the expression of at least one ICP4 gene (i.e., one or both copies of the ICP4 gene) contains a mutation and a regulatory sequence that affects the expression of ICP47 contains a mutation. In one embodiment, at least one ICP4 gene (i.e., one or both copies of the ICP4 gene), or a regulatory sequence that affects expression of at least one ICP4 gene (i.e., one or both copies of the ICP4 gene) comprises a deletion, and the ICP47 gene, or a regulatory sequence that affects expression of ICP47, comprises a deletion. At least one ICP4 gene (i.e., one or both copies of the ICP4 gene) can comprise a deletion, and the ICP47 gene can comprise a deletion.

[0099] In some embodiments, the disclosure provides an HSV vector or strain with a non-functional ICP47 gene. In some embodiments, the disclosure provides an HSV vector or strain with a non-functional ICP4 gene and an ICP47 gene. In some embodiments, the disclosure provides an HSV vector or strain with one or both copies of ICP4 and ICP47 deleted. In some embodiments, one or both copies of the gene encoding ICP4 are fully or partially deleted, and the gene encoding ICP47 is fully or partially deleted without disrupting expression of additional immediate early genes.

[0100] C. Application of HSV-1 Vectors The viral vectors described herein (eg, HSV-1 vectors) can exhibit advantageous characteristics for use in the treatment of disease, eg, the treatment of cancer.

[0101] In certain embodiments, the viral vectors described herein exhibit delayed oncolysis (e.g., when administered to a subject). Delayed oncolysis allows for viral persistence in target (e.g., cancer) cells, sustained expression of the payload, exposure of tumor-associated antigens within the payload-primed tumor microenvironment, and limited non-specific inflammation and tissue damage. Delayed oncolysis allows for viral persistence in target cells, sustained expression of the payload, exposure of tumor-associated antigens within the payload-primed tumor microenvironment, and limited non-specific inflammation and tissue damage.

[0102] In certain embodiments, the viral vector exhibits increased immunogenicity (e.g., when administered to a subject). Immunogenicity can be measured, for example, by detecting increased Tap1 gene expression and / or increased MHC class I expression (HLA). As described in the Examples herein, methods for measuring Tap1 gene expression are known in the art, including, for example, RNA seq analysis. Methods for measuring HLA expression are known in the art, including, for example, flow cytometry.

[0103] In certain embodiments, the viral vector (e.g., when administered to a subject) exhibits increased immune activation. Immune activation can be measured, for example, by detecting increased or decreased RNA expression in pathways associated with immune cell activation, including, but not limited to, the IFNα response pathway, the inflammatory response pathway, the myc signaling pathway, and / or the MtorC1 signaling pathway. Genes involved in such pathways are listed on the GSEA website. See, for example, gsea-msigdb.org / gsea / msigdb / collections.jsp. Immune activation can also be detected by detecting increased expression of members of the TNF superfamily, including tumor necrosis factor alpha (TNF), lymphotoxin beta (LTB), APRIL (TNFSF13), and LIGHT (TNFSF14), and / or by detecting changes in expression of immunogenic cell death regulators, indicative of increased pyroptosis and / or apoptosis, such as detecting increased expression of PYCARD and caspase 10 (CASP10).

[0104] III. Payload The viral vector according to the present disclosure comprises a nucleic acid molecule that comprises the payload of the vector. The payload comprises a nucleic acid molecule that encodes one or more polypeptides. It is contemplated that the payload can be comprised of a nucleic acid molecule that comprises a sequence that is complementary to the nucleic acid sequence that encodes the polypeptide. The payload can encode a nucleic acid molecule that has a regulatory function, for example, a small interfering RNA (siRNA) polynucleotide or a microRNA (miRNA) polynucleotide.

[0105] The payload can be a nucleic acid molecule that encodes a protein that is exogenous to the target tissue or subject to which the vector is administered. For example, the payload can be a nucleic acid molecule that encodes a protein that is endogenous to the target tissue or subject to which the vector is administered. In some embodiments, the nucleic acid molecule is codon-optimized.

[0106] A nucleic acid comprising a vector payload can encode, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 therapeutic polypeptides. In certain embodiments, a vector payload can encode 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, or 5-10 therapeutic polypeptides.

[0107] The therapeutic polypeptide encoded on the vector can have one or more of the following functional attributes: (1) Targeting the tumor stroma; (2) supporting the survival of T cells (e.g., CAR T cells) in the tumor microenvironment; (3) induce tertiary lymphoid structures (TLS) in the tumor bed; and / or (4) Promoting phagocytic autoimmunosurveillance.

[0108] One or more therapeutic polypeptides from one or more categories (1-4 above) can be encoded on the same vector to target multiple pathways to increase immune activity against tumors. For example, a vector can encode one, two, or three therapeutic polypeptides from category 1 and one, two, or three therapeutic polypeptides from category 2, 3, or 4. Alternatively, a vector can encode one, two, or three therapeutic polypeptides from category 2 and one, two, or three therapeutic polypeptides from category 3 or 4. Alternatively, a vector can encode one, two, or three therapeutic polypeptides from category 3 and one, two, or three therapeutic polypeptides from category 4. Alternatively, a vector can encode one or two therapeutic polypeptides from category 1 and one or two therapeutic polypeptides from category 2, 3, and / or 4. Alternatively, a vector can encode one or two therapeutic polypeptides from category 2 and one or two therapeutic polypeptides from category 3 and / or 4. Similarly, a vector can encode one or two therapeutic polypeptides from category 3 and one or two therapeutic polypeptides from category 4.

[0109] The therapeutic polypeptides in the payload can be cloned in tandem and driven from the same promoter, with a linker sequence separating the individual polypeptides. In certain embodiments, the linker sequence includes a ribosome skipping site (sequence), such as the 2A peptide. When such a ribosome skipping site is included in a linker located between two polypeptide sequences in an expression construct, translation of the transcribed mRNA will generate the two polypeptides separately, rather than as a fusion protein. The 2A peptide sequences share a core sequence motif of DXEXNPGP, where X is any amino acid (SEQ ID NO: 45). Non-limiting examples of suitable 2A peptide sequences include T2A (EGRGSLLTCGDVEENPGP; SEQ ID NO: 46), P2A (ATNFSLLKQAGDVEENPGP; SEQ ID NO: 181), E2A (QCTNYALLKLAGDVESNPGP; SEQ ID NO: 47), and F2A (VKQTLNFDLKLAGDVESNPGP; SEQ ID NO: 48). Additionally, an optional Gly-Ser-Gly (GSG) tripeptide can be added to the N-terminus of the 2A peptide to increase efficiency.

[0110] A. Targeting the Tumor Stroma The viral vector can encode one or more therapeutic polypeptides that target the tumor stroma and / or activate the local endothelium to increase T cell infiltration into the tumor, thereby reducing tumor size.

[0111] Tumor stroma can be targeted by various mechanisms. For example, the extracellular matrix can be dissolved by proteins involved in extracellular matrix (ECM) degradation, including but not limited to hyaluronidase, MMP-9, and inhibitors of lysyl oxidase. As a result, in certain embodiments, the viral vector encodes one or more therapeutic polypeptides involved in extracellular matrix (ECM) degradation, including but not limited to hyaluronidase, MMP-9, and inhibitors of lysyl oxidase.

[0112] Additionally, cancer-associated fibroblasts can be targeted with proteins that disrupt the TGFβ pathway. Thus, in certain embodiments, the viral vector encodes one or more therapeutic polypeptides that disrupt the TGFβ pathway, including, for example, a polypeptide that includes an ectodomain (e.g., a non-transmembrane domain) of TGFβ. In certain embodiments, the therapeutic polypeptide comprises a fusion protein that includes an ectodomain of TGFβ, an IgG domain (e.g., an Fc domain), and an ectodomain of TGFβRII.

[0113] The local endothelium is activated by expressing inflammatory cytokines, including but not limited to TNF, IL-1β, IL-6, and IL-18, promoting the infiltration of T cells. Expression of enzymes that degrade tumor extracellular matrix simultaneously disrupts physical barriers within the tumor, allowing the infiltration of immune cells. As a result, in certain embodiments, the viral vector encodes one or more inflammatory cytokines, including, for example, TNF, IL-1β, IL-6, and IL-18.

[0114] Tumor stromal targeting and local endothelial activation can be regulated together or separately by the local expression of these therapeutic polypeptides.

[0115] In certain embodiments, one or more genes encoding hyaluronidase, MMP-9, inhibitors of lysyl oxidase, therapeutic polypeptides inhibiting the TGFβ pathway, TNF, IL-1β, IL-6, or IL-18 are cloned into a non-replicating McKrae strain HSV-1 vector. The McKrae strain HSV-1 vector can be packaged into a McKrae strain HSV-1 virus, for example, using a packaging line. In certain embodiments, cancer cells are transduced with the resulting McKrae strain HSV-1 virus.

[0116] In certain embodiments, the effects on the tumor stroma or local endothelium by cancer cells that secrete factors that lyse the tumor stroma or activate the local endothelium for T cell infiltration can be assessed in vitro or in vivo using methods known in the art.

[0117] B. Supporting survival of T cells (e.g., CAR T cells) in the tumor microenvironment In certain embodiments, the viral vector encodes one or more therapeutic polypeptides that support the survival of T cells in the tumor microenvironment (TME). In the embodiments described herein, the T cells can be natural T cells (e.g., T cells present in the subject to which the viral vector is administered) or CAR T cells (e.g., CAR T cells administered to the subject). In certain embodiments, the viral vector is administered together with T cells (e.g., CAR T cells), for example, to treat a tumor.

[0118] T cells in the tumor microenvironment can be supported by various mechanisms. For example, recruitment of T cells, such as natural T cells or CAR T cells, to the tumor site is improved by the presence of T cell recruiting factors, such as the chemokine ligands CCL19 or CCL21, in the tumor microenvironment.

[0119] Local T cells can be supported to generate strong and durable anti-tumor responses. For example, local expression of T cell trophic factors such as IL-7, IL-12, IL-15, IL-18, and IFNγ can elicit strong and durable anti-tumor responses from local or recruited T cells. Thus, in certain embodiments, the viral vector encodes one or more T cell trophic factors, including, for example, IL-7, IL-12, IL-15, IL-18, and IFNγ.

[0120] Furthermore, TGFβ signaling in cells within a tumor can be suppressed by expression of soluble TGFβRII. Thus, in certain embodiments, the therapeutic polypeptide comprises soluble TGFβRII.

[0121] Local expression of genes encoding costimulatory molecules such as CD40L or OX40L can stimulate local or recruited T cells, depending on the target TME. Thus, in certain embodiments, the therapeutic polypeptide comprises CD40L or OX40L.

[0122] T cell help or T cell recruitment may be modulated by these therapeutic polypeptides together or separately.

[0123] In certain embodiments, one or more genes encoding CCL19, CCL21, IL-7, IL-12, IL-15, IL-18, IFNγ, soluble TGFβRII, CD40L, or OX40L are cloned into a non-replicating McKrae strain HSV-1 vector. The McKrae strain HSV-1 vector can be packaged into a McKrae strain HSV-1 virus, for example, using a packaging line. In certain embodiments, cancer cells are transduced with the resulting McKrae strain HSV-1 virus.

[0124] The effect of cancer cells secreting these factors on T cell recruitment or T cell function can be assessed in vitro or in vivo using methods known in the art.

[0125] C. Supporting survival of NK cells (e.g., CAR NK cells) in the tumor microenvironment In certain embodiments, the viral vector encodes one or more therapeutic polypeptides that support the survival of NK cells in the tumor microenvironment (TME). In the embodiments described herein, the NK cells can be natural NK cells (e.g., NK cells present in the subject to which the viral vector is administered) or CAR NK cells (e.g., CAR NK cells administered to the subject). In certain embodiments, the viral vector is administered together with NK cells (e.g., CAR NK cells), for example, to treat a tumor.

[0126] NK cells in the tumor microenvironment can be supported by various mechanisms. For example, recruitment of NK cells, including natural NK cells and CAR NK cells, to tumor sites can be improved by the presence of NK cell recruiting factors, such as the chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, and CXCL11, in the tumor microenvironment.

[0127] Local NK cells can be supported and generate strong and durable anti-tumor responses. For example, local expression of NK cytotrophic factors, such as IL-2, IL-15, IL-18, and IFNα, can elicit strong and durable anti-tumor responses from local or recruited NK cells. Thus, in certain embodiments, the viral vector encodes one or more NK cytotrophic factors, including, for example, IL-2, IL-15, IL-18, and IFNα.

[0128] Furthermore, TGFβ signaling in cells within a tumor can be suppressed by expression of soluble TGFβRII. Thus, in certain embodiments, the therapeutic polypeptide comprises soluble TGFβRII.

[0129] NK cell help or NK cell recruitment can be modulated together or separately by these therapeutic polypeptides.

[0130] In certain embodiments, one or more genes encoding CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11, IL-2, IL-15, IL-18, and IFNα, or soluble TGFβRII are cloned into a non-replicating McKrae strain HSV-1 vector. The McKrae strain HSV-1 vector can be packaged into a McKrae strain HSV-1 virus, for example, using a packaging line. In certain embodiments, cancer cells are transduced with the resulting McKrae strain HSV-1 virus.

[0131] The effect of cancer cells secreting these factors on NK cell recruitment or NK cell function can be assessed in vitro or in vivo using methods known in the art.

[0132] D. Inducing tertiary lymphoid structures in the tumor bed In certain embodiments, the viral vector encodes one or more therapeutic polypeptides and induces tertiary lymphoid structures (TLS) within the tumor bed.

[0133] The formation of TLS in tumors can play a role in antitumor immunity and is associated with the response to immune checkpoint inhibition.Thus, viral vectors encoding therapeutic polypeptides that induce TLS formation can be used according to the methods described herein.In certain embodiments, viral vectors are used in combination with additional solid tumor therapeutic agents, including but not limited to CAR T cell therapy and immune checkpoint inhibitors.

[0134] In certain embodiments, one or more genes encoding therapeutic polypeptides involved in the establishment and maintenance of TLS, including but not limited to CCL19, lymphotoxin beta, CXCL13, and TNF, are cloned into a non-replicating McKrae strain HSV-1 vector. The McKrae strain HSV-1 vector can be packaged into a McKrae strain HSV-1 virus, for example, using a packaging line. In certain embodiments, cancer cells are transduced with the resulting McKrae strain HSV-1 virus.

[0135] The effect of cancer cells secreting these factors on tertiary lymphoid structures (TLS) within the tumor bed can be assessed in vitro or in vivo using methods known in the art.

[0136] E. Promoting phagocytic autoimmune surveillance In certain embodiments, the viral vector encodes one or more therapeutic polypeptides that promote phagocytic autoimmunosurveillance and elimination of tumor cells.

[0137] CD47 is an immunoglobulin that is overexpressed on the surface of many types of cancer cells. CD47 forms a signaling complex with signal regulatory protein alpha (SIRPα) on macrophages, allowing cancer cells to escape macrophage-mediated phagocytosis. Phagocytic autoimmunological surveillance and elimination of tumor cells can be induced by disruption of the Sirpα / CD47 axis, for example, by the presence of a Sirpα-IgG fusion transgene. Disruption of the Sirpα / CD47 axis can promote phagocytic autoimmunological surveillance and elimination of tumor cells by exposing the tumor's "eat" signal. As a result, in certain embodiments, the viral vector encodes a therapeutic polypeptide that disrupts the Sirpα / CD47 axis, thereby promoting phagocytic autoimmunological surveillance and elimination of tumor cells. In certain embodiments, the therapeutic polypeptide is an anti-CD47 antibody. In certain embodiments, the viral vector encodes a Sirpα-IgG fusion polypeptide.

[0138] Additionally, the vector can encode therapeutic polypeptides that support anti-tumor macrophage polarization, including, but not limited to, TNF, IL-1, IL-12, IL-17, and IFNγ.

[0139] In certain embodiments, one or more therapeutic polypeptides involved in the establishment and maintenance of TLS in tissues, including but not limited to Sirpα-IgG fusion transgenes, TNF, IL-1, IL-12, IL-17, and IFNγ, may be cloned into a non-replicating McKrae strain HSV-1 vector.

[0140] The effect on phagocytic autoimmunosurveillance by cancer cells secreting these factors can be assessed in vitro or in vivo using methods known in the art.

[0141] IV. Regulatory elements The nucleic acid of the present disclosure is intended herein to include non-natural regulatory sequences, gene control sequences, promoters, non-coding sequences, introns, or coding sequences.It is further intended herein to include nucleic acids that code for nucleic acid tags or signaling sequences, or protein tags or protein signaling sequences.Typically, the coding region is operably linked to one or more regulatory nucleic acid components.

[0142] The promoter contained in the nucleic acid of the present disclosure can be a tissue or cell type specific promoter, a promoter that is specific to multiple tissues or cell types, an organ specific promoter, a promoter that is specific to multiple organs, a general or ubiquitous promoter, or a nearly general or nearly ubiquitous promoter.Promoters that have stochastic expression, inducible expression, conditional expression, or otherwise discontinuous, variable, or unpredictable expression are also included within the scope of the present disclosure.The promoter of the present disclosure can include any of the above characteristics or other promoter characteristics known in the art.

[0143] Examples of known promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the CMV / human β3 globin promoter, the GFAP promoter, the chicken β-actin (CBA) promoter, the β-glucuronidase (GUSB) promoter, and ubiquitin promoters such as those isolated from human ubiquitin A, human ubiquitin B, and human ubiquitin C.

[0144] In some embodiments, the promoter is a neuron-specific promoter in that it is a promoter that has specific expression in neurons, preferential expression in neurons, or a neuron-specific promoter that typically drives expression of an associated coding sequence in neurons or a subset of neurons, but not in one or more other tissues or cell types. Examples of such promoters include calcitonin gene-related peptide (CGRP), synapsin I (SYN), calcium / calmodulin-dependent protein kinase II, tubulin alpha I, neuron-specific enolase, microtubule-associated protein IB (MAP1B), and platelet-derived growth factor beta chain promoters, and derivatives thereof. In some embodiments, the promoter is a calcitonin gene-related peptide (CGRP) promoter or a derivative thereof.

[0145] Other regulatory elements, such as enhancers and polyadenylation sites, may be further operably linked to the payload. In some embodiments, the enhancer comprises a human cytomegalovirus (HCMV) sequence. In some embodiments, the polyadenylation site comprises a bovine growth hormone (BGH) polyadenylation signal.

[0146] In some embodiments, the promoter is a chimera of one or more promoters or regulatory elements found in nature, hi some embodiments, the viral vector comprises a payload whose expression is driven by the CGRP promoter with an HCMV enhancer sequence.

[0147] V. Vector Preparation The present disclosure particularly relates to McKrae strain viral vectors that are replication-defective. The viral vectors can be made by mutation (e.g., deletion) of one or more immediate-early genes or regulatory sequences that affect their expression. The viral genes can be mutated using recombinant technology methods known in the art. The viral vectors of the present disclosure can be made replication-defective as a result of a homologous recombination event. The replication-defective viral vectors can be made by mutation of the ICP4 gene and mutation of the ICP47 gene. For example, the replication-defective viral vectors are made by deletion of the ICP4 gene and deletion of the ICP47 gene.

[0148] In some embodiments, the viral vectors of the present disclosure are generated by deletion of a locus encoding one or more ICPs (e.g., ICP4 and ICP47) by homologous recombination. In some embodiments, the generation of the viral vectors of the present disclosure includes a step of homologous recombination between a first plasmid and a second plasmid. In some embodiments, the first plasmid includes a nucleic acid sequence that is homologous to a region of the HSV genome adjacent to the nucleic acid region of the HSV genome that is intended to be replaced. In some embodiments, the second plasmid includes an HSV genome or a fragment thereof. In some embodiments, the first plasmid includes a nucleic acid sequence that encodes a gene of interest between the homologous nucleic acid sequences. In some embodiments, the gene of interest may be or include a marker protein detectable by fluorescence, chemiluminescence, or other properties, which may be used to select vectors resulting from successful homologous recombination.

[0149] In some embodiments, the viral vectors of the present disclosure are generated by homologous recombination between a first plasmid containing a nucleic acid sequence homologous to the upstream region of the ICP4 promoter, which contains the viral origin contained in the short inverted conversion junction region of HSV, and a second plasmid containing the HSV McKrae strain genome.

[0150] In some embodiments, the vector is made by first replacing both copies of the ICP4 locus by homologous recombination with plasmid SASB3 and screening for green fluorescent protein (GFP) expressing plaques. In some embodiments, the plasmid is constructed by cloning the Sph I to Afl III (linkered Sal I) fragment (1928 bp) of the HSV-1 KOS strain genome (nucleotides 124485-126413, KT899744, KOS strain) into Sph I / Sal I digested pSP72, and then inserting a 695 bp Bgl II to BamH I fragment (GenBank Accession No: KT899744.1 (SEQ ID NO: 2), KOS strain, nucleotides 131931-132626), which contains the upstream region of the ICP4 promoter including the viral origin contained within the short inverted conversion circuit region, into the Bgl II to BamH I site of the vector plasmid. In some embodiments, the plasmid is constructed by cloning the HCMV-eGFP fragment into the BamHI site of the plasmid as described above. In some embodiments, the plasmid as described above is then recombined into a specific locus of wild-type McKrae virus. In some embodiments, the resulting viral vector is isolated using a stable cell line that expresses one or more genes that are deleted or disrupted in the HSV genome required for replication.

[0151] In some embodiments, the vector is made by first replacing both copies of the ICP4 locus with plasmid SDAXB by homologous recombination and screening for green fluorescent protein (GFP) expressing plaques. In some embodiments, the plasmid is constructed by cloning the Sph I to Afl III fragment (1928 bp) of the HSV-1 KOS strain genome (nucleotides 124346 to 126273 of GenBank Accession No.: KT899744.1 (SEQ ID NO: 2), KOS strain) into Sph I / Afl III digested pSP72 to generate plasmid SDA, and then modifying the Afl III site to a BamHI site (plasmid SDAB). A BamHI to Bgl II DNA PCR fragment containing the upstream region of the ICP4 promoter, including the viral origin (nucleotides 144933 to 145534 of GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), McKrae strain), contained within the short inverted conversion circuit region, was cloned into the BamHI site of plasmid SDAB to generate plasmid SDAXB. In some embodiments, a plasmid is constructed by cloning an HCMV-eGFP fragment into the BamHI site of the plasmid as described above. In some embodiments, a plasmid as described above is then recombined into a specific locus of wild-type McKrae virus. In some embodiments, the resulting vector is isolated using a stable cell line expressing one or more genes deleted or disrupted in the HSV genome required for replication.

[0152] In some embodiments, a vector containing a combination of ICP4 and ICP47 deletions is generated. In some embodiments, McKrae sequence base pairs 143521-144562 and 144640-145534 (GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), McKrae strain) can be synthesized. The SCMV promoter flanked by Pme I sites can be cloned between these flanking sequences. In some embodiments, a marker, e.g., a red fluorescent marker, can be placed in the appropriate orientation relative to the SCMV promoter. In some embodiments, a plasmid as described above is then recombined into a specific locus of McKrae virus with a deletion of the ICP4 locus. In some embodiments, fluorescent clones can be isolated and insertion of the construct into the ICP47 locus can be confirmed by PCR. The resulting recombinant McKrae virus has a deletion of base pairs 144562 to 144640 according to the reference sequence (GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), McKrae strain). In some embodiments, the resulting viral vector is isolated using a stable cell line expressing one or more genes deleted or disrupted in the HSV genome required for replication.

[0153] VI. Vector Characterization The viral vector according to the present disclosure can be characterized by genome sequencing to determine whether the expected vector is successfully created.Any sequencing method known in the art is acceptable for this purpose.Sequencing methods include, for example, nanopore sequencing, single molecule real-time sequencing (SMRT), DNA nanoball (DNB) sequencing, pyrosequencing, and using DNA array.

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

[0155] VII. Applications Viral vectors according to the present disclosure are useful for a wide variety of therapeutic applications. The vectors described herein are useful for delivering one or more payloads to one or more target cells. In certain embodiments, the payloads persist in the target cells for up to 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, or more than 1 year.

[0156] In some embodiments, the target cells are present in tissues that are poorly vascularized and difficult to reach by the systemic circulation. In some embodiments, the target cells are cells susceptible to infection by HSV. In some embodiments, the target cells are susceptible to infection by the McKrae strain of HSV.

[0157] Viral vectors according to the present disclosure are useful for delivering one or more therapeutic polypeptides to cells, for example, cells in a subject. For example, viral vectors comprising a heterologous nucleic acid segment operably linked to a promoter are useful for any disease or clinical condition associated with the reduction or absence of the protein encoded by the heterologous nucleic acid segment, or any disease or clinical condition that can be effectively treated by expressing the encoded protein in a subject. Viral vectors comprising an expression cassette for synthesizing an RNAi agent (e.g., one or more siRNAs or shRNAs) are useful for treating any disease or clinical condition associated with the overexpression of a transcript or its encoded protein in a subject, or any disease or clinical condition that can be effectively treated by causing a reduction of a transcript or its encoded protein in a subject. Viral vectors comprising an expression cassette for synthesizing one or more RNAs that self-hybridize or hybridize with each other to form an RNAi agent that targets a transcript encoding a cytokine can be used to regulate immune system responses (e.g., responses that cause organ transplant rejection, allergy, autoimmune disease, inflammation, etc.). Viral vectors that provide templates for the synthesis of one or more RNAs that self-hybridize or hybridize with each other to form RNAi agents that target transcripts of infectious agents or cellular transcripts whose encoded products are necessary for or contribute to any aspect of the infectious process can be used to treat infectious diseases.

[0158] In one aspect, the present disclosure relates to a method for reducing tumor size in a subject in need thereof. The method comprises administering to the subject a vector comprising a mutant of a Herpes Simplex Virus (HSV) strain, the mutant comprising a modification in its genome such that it does not express functional ICP4 and ICP47 proteins. Additionally, the vector comprises a nucleic acid encoding a therapeutic polypeptide, which functions to reduce tumor size. In certain embodiments, the mutant cannot express functional ICP4 and ICP47 proteins, characterized by the amino acid sequences of SEQ ID NO: 3 and 4, and SEQ ID NO: 5, respectively. In certain embodiments, the HSV strain is an HSV-1 strain, for example, a McKrae strain.

[0159] In certain embodiments, a therapeutic polypeptide comprises one or more of the following functional attributes: (a) targeting the tumor stroma; (b) supporting the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment; (c) induce tertiary lymphoid structures (TLS) in the tumor bed; and / or (d) Promoting phagocytic autoimmunosurveillance.

[0160] Exemplary therapeutic polypeptides are described above in Section III, incorporated herein.

[0161] VIII. Administration Compositions comprising the viral vectors described herein may be formulated for delivery by any available route, including, but not limited to, intratumoral, parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal, and intravaginal. Preferred delivery routes include intratumoral. In some embodiments, the pharmaceutical composition comprises the viral vector in combination with a pharma- ceutically acceptable carrier. As used herein, the term "pharma-ceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the composition. In some embodiments, the viral vector is formulated in glycerol. In some embodiments, the viral vector is formulated in about 10% glycerol in phosphate buffered saline.

[0162] For ease of administration and uniformity of dosage, it is advantageous to formulate the composition in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for subjects to be treated, each unit containing a predetermined amount of viral vector calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier.

[0163] The pharmaceutical composition can be administered at various intervals and for various periods of time, as needed, for example, once a week for about 1-10 weeks, for about 2-8 weeks, for about 3-7 weeks, for about 4, 5, or 6 weeks, etc. One of skill in the art will appreciate that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat a subject. Treatment of a subject with a viral vector can include a single treatment or, in many cases, can include a series of treatments.

[0164] In certain embodiments, the pharmaceutical compositions may be administered to a patient one or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times), e.g., as a result of the improved safety profile exhibited by the vectors described herein.

[0165] In some embodiments, the active agent, i.e., the viral vector of the present disclosure and / or other agents administered with the viral vector of the present disclosure, are prepared with a carrier that protects the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and the like. Methods for preparing such compositions will be apparent to those skilled in the art. In some embodiments, the composition is targeted to a specific cell type or cells infected with a virus.

[0166] IX. Combination Therapy According to the present disclosure, the compositions provided may be administered in combination with one or more other active agents and / or therapeutic modalities, such as known therapeutic agents and / or independently active biologically active agents. In some embodiments, the compositions provided include one or more such other active agents, and in some embodiments, such other active agents are provided as part of a separate composition. In some embodiments, the combination therapy includes co-administration of one or more doses or units of two or more different active agents and / or therapeutic modalities, and in some embodiments, the combination therapy includes simultaneous exposure to two or more different active agents and / or therapeutic modalities, for example, through overlapping dosing regimens.

[0167] In some embodiments, provided compositions comprise or are administered in combination with one or more other active agents useful in the treatment of the associated disease, disorder and / or condition.

[0168] Throughout this specification, when devices, apparatus and systems are described as having, including, or comprising particular components, or processes and methods are described as having, including, or comprising particular steps, it is further contemplated that there are devices, apparatus and systems of the invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the invention that consist essentially of, or consist of, the recited processing steps.

[0169] The practice of this invention will be more fully understood from the foregoing examples, which are presented herein for illustrative purposes only and should not be construed as limiting the invention in any manner. EXAMPLES

[0170] The following examples are given for the purpose of illustrating various embodiments of the present invention and are not intended to limit the present invention in any manner. The examples, together with the methods described herein, are representative of presently preferred embodiments and are exemplary and are not intended to limit the scope of the present invention. Those skilled in the art will be able to make modifications thereof and other uses that are encompassed within the spirit of the invention as defined by the scope of the claims.

[0171] Example 1: Construction of ICP4 and ICP47 modified McKrae strain HSV-1 vectors This example describes the cloning and construction of non-replicating McKrae strain HSV-1 vectors containing modifications of the ICP4 and ICP47 regions.

[0172] Wild-type McKrae strain HSV-1 contains two copies of the gene encoding ICP4 (Figure 1A). First, a plasmid containing the full-length viral genome was modified using homologous recombination by replacing both copies of the two ICP4 loci with the modified locus and screened for green fluorescent protein (GFP)-expressing plaques, for example as described in WO2017 / 165813. Briefly, the plasmid was constructed by cloning an Sph I to Afl III fragment (1928 bp) of the HSV-1 KOS strain genome (nucleotides 124346 to 126273 of GenBank Accession No.: KT899744.1 (SEQ ID NO: 2), KOS strain) into Sph I / Afl III digested pSP72 to generate plasmid SDA, and then modifying the Afl III site to a BamHI site (plasmid SDAB). A BamHI-Bgl II DNA PCR fragment containing the upstream region of the ICP4 promoter, including the viral origin (nucleotides 144933-145534 of GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), McKrae strain), contained within the short inverted conversion circuit region, was cloned into the BamHI site of plasmid SDAB to generate plasmid SDAXB. Additionally, an HCMV-eGFP fragment was cloned into the BamHI site of plasmid SDAXB. This SDAXB plasmid was then recombined into the ICP4 locus of the wild-type McKrae virus genome to generate mutICP4 McKrae HSV-1. The resulting viral vector structure is shown in Figure 1B.

[0173] Next, the ICP47 locus of mutICP4 McKrae HSV-1 was modified. Briefly, a plasmid sequence (GenBank Accession No. JQ730035.1 (SEQ ID NO: 1), McKrae strain) was synthesized that contained base pairs 143521 to 144562 and 144640 to 145534 of the McKrae sequence. Between these flanking sequences, the SCMV promoter was cloned, flanked by Pme I sites. In addition, a red fluorescent marker was placed in the appropriate orientation relative to the SCMV promoter, and the resulting plasmid was used to recombine with mutICP4 McKrae HSV-1 to generate mutICP4mutICP47 McKrae HSV-1. Fluorescent clones were isolated and PCR was performed to confirm insertion into the ICP47 locus. The resulting virus has a deletion of base pairs 144562 to 144640 according to the reference sequence (GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), McKrae strain). The resulting viral vector structure is shown in FIG. 1C.

[0174] After generation of the mutICP4mutICP47 vector, vector stocks were generated by infecting complementing cells and purifying the vector from the supernatant.

[0175] Example 2: Cloning, production, and evaluation of single or multigene payloads This example describes the cloning, production, and evaluation of multigene payload viral vectors.

[0176] Briefly, mutICP4 McKrae HSV-1 or mutICP4mutICP47 McKrae HSV-1 vectors containing a marker element (e.g., GFP) instead of the genes encoding ICP4 and / or ICP47 were further modified by replacing the marker element with one or more genes of interest (GOI), e.g., therapeutic polypeptides, to prepare the constructs used in the following examples. To replace the marker element of mutICP4 McKrae HSV-1, mutICP27, or mutICP4mutICP47 McKrae HSV-1 with a gene of interest (GOI), the GOI flanked by the HCMV immediate early protein gene (IEp) and polyadenylation signal (HCMV-GOI-pA) was cloned into a plasmid and transduced into a stable ICP4-expressing Vero cell line. After cell transduction, plaques not expressing the marker element were isolated and tested for GOI expression by ELISA. In the following examples, the GOIs IFNγ, LTB, CXCL13, CCL19, CCL21, and IL-7 were cloned into vectors and expressed. Exemplary multigene payload viral vectors are shown in FIG.

[0177] Example 3: Evaluation of cancer transformation and payload expression This example describes the initiation of oncolysis and evaluation of payload expression of mutICP4 McKrae HSV-1 expressing murine IL-17a and mutICP4mutICP47-IFNγ McKrae HSV-1 expressing IFNγ in human breast cancer cells.

[0178] mutICP4 McKrae HSV-1 Hs578T human breast cancer cells were infected with increasing doses of mutICP4 McKrae HSV-1 expressing murine IL-17a (multiplicity of infection (MOI) of 0, 3, and 10) followed by cell viability assessment by CellTiter-Glo assay (CellTiter-Glo® Luminescent Cell Viability Assay (Promega Corp, Madison, WI)) (Figure 3A) and expression of murine IL-17a payload (Figure 3B) by ELISA for 8 days.

[0179] These results indicate that mutant ICP4 McKrae HSV-1-mediated changes in cell viability were not observed until several days post-infection, and robust expression of the payload was sustained for up to 8 days.

[0180] mutICP4mutICP47McKrae HSV-1 Hs578T human breast cancer cells were infected with increasing amounts of IFNγ-expressing mutICP4mutICP47-IFNγ McKrae HSV-1 (multiplicity of infection (MOI) of 0, 3, and 10), after which cell viability was assessed by RealTime-Glo™ assay (Promega Corp., Madison, WI) (Figure 3C) and expression of the IFNγ payload was assessed by ELISA for 6 days (Figure 3D).

[0181] These results show that mutICP4mutICP47-IFNγ infection suppressed cell proliferation in a dose-dependent manner (Figure 3C). IFNγ production was highest during the first 2 days postinfection and was detected above baseline by day 6 postinfection. #p<0.0001 (Figure 3D). The results indicate that mutICP4mutICP47-IFNγ-mediated changes in viability were not observed until several days postinfection, and robust expression of the payload was sustained for up to 6 days (Figures 3C and 3D). Delayed oncolysis allows for viral persistence in target cells, sustained expression of the payload, exposure of tumor-associated antigens within the payload-primed tumor microenvironment, and limited nonspecific inflammation and tissue damage. Delayed oncolysis may lead to an improved safety profile and allow the vector to be administered repeatedly.

[0182] Example 4: Evaluation of immunogenicity and immune activation immunogenicity This example describes the evaluation of the immunogenic impact of mutICP4 McKrae HSV-1 and mutICP4mutICP47 McKrae HSV-1 in human breast cancer cells. This example also describes the evaluation of the immunogenic impact of mutICP4mutICP47 McKrae HSV-1 in human breast cancer cells compared to another replication-deficient McKrae HSV-1 with mutated ICP27 (mutICP27). ICP27 is an immediate early gene (like ICP4) that, when disrupted, causes viral replication to malfunction. The mutICP27 vector allows the comparison of the mutICP4mutICP47 replication-deficient vector with another replication-deficient vector with a fully intact ICP47 locus in the same strain. The ICP4 and ICP47 loci are close to each other, making it difficult to disrupt the ICP4 locus without affecting the ICP47 locus, and the ICP4 control used in the example may also disrupt ICP47 function. Therefore, the mutICP27 vector is a good alternative for comparison of the control vector.

[0183] Hs578T human breast cancer cells were infected with increasing amounts of mutICP4 McKrae HSV-1-GFP or mutICP4mutICP47 McKrae HSV-1-GFP at multiplicities of infection (MOI) of 0, 3, and 10. After 24 hours, infection (GFP+ cells) and cell surface MHC class I expression (HLA) were assessed by flow cytometry.

[0184] The mutICP4 and mutICP4mutICP47 McKrae HSV-1 strains showed comparable infection rates at all three MOIs tested (Figure 4A). Results further showed that nearly all cells were HLA positive (Figure 4B), and cell surface expression of HLA was increased in mutICP4mutICP47 McKrae HSV-1 compared to mutICP4 McKrae HSV-1 (Figure 4C).

[0185] These results indicate that mutICP4mutICP47 McKrae HSV-1 induces increased immunogenicity in breast cancer cells compared with mutICP4 McKrae HSV-1.

[0186] Next, Hs578T cells were infected with mutICP4mutICP47 or mutICP27 carrying the respective HSV-1 strains encoding GFP as a marker at 10 PFU per cell. 24 hours after infection, cells were harvested for (1) RNAseq analysis of Tap1, a protein that mediates unidirectional translocation of peptide antigens from the cytosol to the endoplasmic reticulum (ER) for loading onto MHC class I, and (2) flow cytometry analysis of cell surface MHC class I (MHC-I) expression (HLA).

[0187] As shown in Figure 4D, mutICP4mutICP47 infection increased antigen presentation as measured by increased Tap1 gene expression (Figure 4D) and upregulation of cell surface MHC-I (Figure 4E) compared to mutICP27 infection. The results indicate that cell surface expression of MHC-I was not downregulated with mutICP4mutICP47 vector infection, as observed with mutICP27 vector infection, but was increased by approximately 50% above the levels of uninfected cells (Figure 4E; **p<0.01, #p<0.0001). Thus, infection with mutICP4mutICP47 vectors results in increased immunogenicity in breast cancer cells compared to infection with mutICP27 vectors.

[0188] Immune activation-gene set enrichment analysis The cellular response to the viral scaffold can be measured by gene expression analysis as determined by RNAseq. Gene Set Enrichment Analysis (GSEA) is a bioinformatics method that uses this RNAseq data to identify gene-associated pathways that are altered by treatment (see, for example, the GSEA website, gsea-msigdb.org / gsea / msigdb / collections.jsp). By identifying pathways that are significantly altered in response to treatment, GSEA provides insight into the cellular mechanisms underlying the response to the viral scaffold.

[0189] To determine the genes up- or down-regulated by infection with mutICP4mutICP47 or mutICP27, Hs578T cells were infected with mutICP4mutICP47 (with encoded GFP as a marker) or mutICP27 at 10 PFU per cell for 24 hours, and RNA was extracted from the cells for RNAseq analysis. RNAseq data were analyzed using GSEA software and reported as normalized enrichment scores and p-values.

[0190] The results show that compared to mutICP27, the pathways most upregulated by mutICP4mutICP47 were immune activation pathways, including HALLMARK_INTERFERON_ALPHA_RESPONSE and HALLMARK_INFLAMMATORY_RESPONSE (Figure 4F). The pathways most upregulated by mutICP27 infection were pathways involved in cellular response, including HALLMARK_MYC_TARGETS and MTORC1_SIGNALING (Figure 4G). Hallmark gene sets are curated and succinctly summarize well-defined biological cell states or processes that show consistent expression patterns (see, for example, Liberzon et al. (2015) Cell Syst.;1(6):417-425). For example, the HALLMARK_INTERFERON_ALPHA_RESPONSE pathway includes genes such as Tap1, whose expression is upregulated in response to alpha interferon protein. The mutICP4mutICP47 vector induced greater immune activation than the mutICP27 vector.

[0191] Immune activation-TNFSF expressionTNSF expression Members of the TNF superfamily (TNFSF) are key inducers of both autoimmune and adaptive immune responses. These molecules act by directly inducing immunogenic cell death, such as apoptosis and pyroptosis, or by directly stimulating immune cells.

[0192] To examine whether TNFSF is involved in the cellular response of mutICP4mutICP47 or mutICP27, Hs578T cells were infected with mutICP4mutICP47 or mutICP27 with each GFP-encoding HSV-1 strain as a marker at 10 PFU per cell for 24 h, and RNA was extracted from the cells for RNAseq analysis.

[0193] The results show that mutICP4mutICP47 induced higher expression of members of the TNF superfamily, including tumor necrosis factor alpha (TNF), lymphotoxin beta (LTB), APRIL (TNFSF13), and LIGHT (TNFSF14), compared with mutICP27 (Figure 4H). These genes can act as inducers of immunogenic cell death and stimulators of immune responses, respectively. Furthermore, mutICP4mutICP47 upregulated the expression of PYCARD and caspase 10 (CASP10), regulators of pyroptosis and apoptosis, respectively, to a greater extent than mutICP27 infection. mutICP4mutICP47 infection preferentially activated immune responses and immunogenic cell death pathways compared with mutICP27 virus infection.

[0194] Example 5: Multigene payload targeted to the tumor stroma This example describes the design and evaluation of viral vectors carrying a multigene payload to target tumor stroma and activate local endothelium to promote T cell infiltration of tumors and reduce tumor size.

[0195] Tumor stroma can be targeted by various mechanisms. For example, extracellular matrix can be dissolved by proteins involved in extracellular matrix (ECM) degradation, including but not limited to hyaluronidase, MMP-9, and inhibitors of lysyl oxidase. In addition, cancer-associated fibroblasts can be targeted by proteins that interfere with the TGFβ pathway.

[0196] Local endothelium can be activated by inflammatory cytokines, including but not limited to TNF, IL-1β, IL-6, and IL-18, to promote T cell infiltration. It is contemplated that the incorporation of enzymes that degrade tumor extracellular matrix will simultaneously disrupt the physical barrier within the tumor and allow immune cell infiltration.

[0197] Tumor stromal targeting and local endothelial activation may be regulated together or separately by the local expression of these factors.

[0198] As a result, one or more of hyaluronidase, MMP-9, inhibitors of lysyl oxidase, genes inhibiting the TGFβ pathway, TNF, IL-1β, IL-6, or IL-18 can be cloned into a non-replicating McKrae strain HSV-1 vector as described in Example 2 and expressed and purified as described in Example 1. Cancer cells can be transduced with one or more GOIs including McKrae strain HSV-1 virus at different MOIs as described in Example 3 and assessed for oncolysis, payload expression and / or secretion, and immunogenicity as described in Examples 3 and 4.

[0199] The effect on the tumor stroma or local endothelium by cancer cells secreting factors that dissolve the tumor stroma or activate the local endothelium for T cell infiltration can be evaluated in vitro or in vivo. It is contemplated that expression of genes that target the tumor stroma and / or local endothelium will increase T cell infiltration and reduce tumor size.

[0200] Example 6: Multigene payload of mutICP4mutICP47 targeted to tumor stroma This example describes the design and evaluation of mutICP4mutICP47 HSV-1 vectors carrying a multigene payload to target tumor stroma and activate local endothelium to favor T cell infiltration of tumors and reduce tumor size using a salivary gland model (see, e.g., Barone et al. PNAS (2015) 112(35) 11024-11029).

[0201] Briefly, both submandibular salivary glands of wild-type BALB / c mice (8–10 weeks old) were inoculated with 3 × 10 guanosine monophosphate (Guacam) via the excretory duct under anesthesia. 7PFU of mutICP4mutICP47 HSV-1 vector expressing GFP or a combination of mutICP4mutICP47 HSV-1 vectors expressing one each of murine LTB, CXCL13, CCL19, CCL21, or IL-7 (6 × 10 6 PFU, total dose 3 × 10 7 Mice were cannulated with either PFU or the "payload" (prepared as described in Example 2) and salivary glands were then harvested 15 days later for flow cytometric analysis using antibodies against CD31, PDGFRa, and PDPN (a marker of stromal cell activation). The number of positive cells was quantified.

[0202] The results show that the payload-encoding mutICP4mutICP47 virus increased the number of CD31+ endothelial cells and PDGFRa+PDPN+ stromal cells in the salivary glands (Figures 5A-5B). These data indicate that activation of stromal and endothelial cells can be achieved using the combination of mutICP4mutICP47 viruses carrying one of the mouse LTB, CXCL13, CCL19, CCL21, and IL-7 genes in their payloads, respectively.

[0203] Example 7: Evaluation of expressed TGFβ pathway components following mutICP4mutICP47 infection This example describes the assessment of TGFβ pathway component expression following infection with the mutICP4mutICP47-GFP HSV-1 vector.

[0204] The TGF-β pathway is a multifunctional cytokine that regulates stromal, endothelial, and immune cells, and therapies targeting this pathway have been reported in cancer.

[0205] To examine the expression of TGFβ pathway components by mutICP4mutICP47 virus after infection of cancer cells, Hs578T cells were infected with 10 PFU / cell of mutICP4mutICP47-GFP (no payload with encoded GFP as a marker) (uninfected cells as control) for 24 h, and RNA was extracted from the cells for RNAseq analysis. TGFβ1 mRNA expression was quantified.

[0206] The results show that infection with the mutICP4mutICP47 vector reduced TGFβ1 expression by approximately 50% at 24 h postinfection ( Fig. 5C ), thus indicating that infection with the mutICP4mutICP47 viral backbone itself reduces TGFβ1 expression.

[0207] Example 8: Multigene payload for T cell survival in the tumor microenvironment This example describes the design and evaluation of viral vectors carrying a multigene payload to support T cell survival in the tumor microenvironment (TME).

[0208] T cells in the tumor microenvironment can be supported by various mechanisms. For example, the presence of T cell recruiting factors in the tumor microenvironment, such as the chemokine ligands CCL19 or CCL21, can improve recruitment of T cells to the tumor site.

[0209] Local T cells can be supported to generate strong and durable antitumor responses. For example, local expression of T cell trophic factors such as IL-7, IL-12, IL-15, IL-18, and IFNγ can elicit strong and durable antitumor responses from local or recruited T cells. Furthermore, TGFβ signaling in cells within the tumor can be suppressed by expression of soluble TGFβRII. Furthermore, local expression of transgenes, and combinations with other vectors or therapeutic agents, encoding costimulatory molecules such as CD40L or OX40L depending on the target TME can stimulate local or recruited T cells.

[0210] T cell help and T cell recruitment can be regulated by these factors together or separately.

[0211] One or more of CCL19, CCL21, IL-7, IL-12, IL-15, IL-18, IFNγ, soluble TGFβRII, CD40L, or OX40L can be cloned into a non-replicating McKrae strain HSV-1 vector as described in Example 2 and expressed and purified as described in Example 1. Cancer cells can be transduced with one or more GOIs including McKrae strain HSV-1 virus at different MOIs as described in Example 3 and assessed for oncolysis, payload expression and / or secretion, and immunogenicity as described in Examples 3 and 4.

[0212] The effect of cancer cells secreting these factors on T cell recruitment or T cell function can be evaluated in vitro or in vivo. It is contemplated that expression of genes related to T cell recruitment or T cell function will result in recruitment of T cells to the tumor site and support local T cell function, thereby reducing tumor size.

[0213] Example 9: Multigene payload in mutICP4mutICP47 for survival of T and NK cells in the tumor microenvironment This example describes the design and evaluation of mutICP4mutICP47 HSV-1 vectors carrying a multigene payload to support T and NK cell survival in the tumor microenvironment (TME).

[0214] Immune-mediated tumor cell killing can be assessed in ex vivo co-culture models involving peripheral blood mononuclear cells (PBMCs) and target tumor cells. Cytotoxic cells in these models include CD8+ T cells and NK cells present in the PBMCs.

[0215] To examine the cytotoxic activity following treatment with mutICP4mutICP47 HSV-1 vectors with or without a multigene payload in an in vitro breast cancer model, CellTracker Red-labeled Hs578T cells (a human breast cancer cell line) were infected with mutICP4mutICP47 vectors encoding GFP, human IFNγ, human IL-12, or a combination thereof at a total viral dose of 0.5 PFU / cell. After 2 h, CellTracker Violet-labeled human PBMCs were added to the cultures at a ratio of approximately 10 PBMCs per Hs578T cell and co-cultured for 72 h. Recombinant human IL-15 (rh IL-15) was added to mutICP4mutICP47-GFP-infected cells to mimic virus-mediated delivery of human IL-15. A PBMC activator cocktail (ImmunoCult™ Human CD3 / CD28 / CD2 T cell activators + rh IL-2) served as a positive control for maximal tumor cell killing under these conditions. After 72 hours, samples were harvested and viable Hs578T cells were quantified by flow cytometry.

[0216] Results showed that viral-mediated expression of IFNγ or IL-12 in Hs578T:PBMC coculture cells reduced viable Hs578T cell numbers to a greater extent than viral backbone treatment alone (mutICP4mutICP47-GFP) (Figure 6A). Similarly, addition of rh IL-15 to mutICP4mutICP47-GFP-infected cells also increased Hs578T tumor cell killing. The combination of IFNγ- and IL-12-expressing mutICP4mutICP47 vectors further enhanced immune-mediated tumor cell killing in this assay. These data support the use of human IFNγ, IL-12, IL-15, and their combinations encoded in vectors designed to support T cell or NK cell function and immune-mediated tumor cell killing in the tumor microenvironment.

[0217] Example 10: IFNγ payload of mutICP4mutICP47 for survival of T and NK cells in the tumor microenvironment This example describes the evaluation of mutICP4mutICP47 HSV-1 vectors with an IFNγ payload to support survival of T and NK cells in the tumor microenvironment (TME).

[0218] To measure T cell and NK cell activity following treatment with mutICP4mutICP47 HSV-1 vectors with or without IFNγ payload, Hs578T cells were infected with mutICP4mutICP47 encoding GFP or mutICP4mutICP47 encoding human IFNγ at 1 or 3 PFU / cell in OPTI-MEM + 5% FBS for 2 h and then co-cultured with PBMCs for 24 or 72 h. Cells were harvested after 24 or 72 h, stained with fluorescently labeled antibodies, and analyzed by flow cytometry.

[0219] The results show that infection with mutICP4mutICP47 increased the number of Ki67+ granzyme B+ CD8+ T cells after 24 hours, and the effect was further increased after 72 hours of co-culture (Figures 6B and 6C). mutICP4mutICP47-IFNγ showed a stronger effect on T cell activation than mutICP4mutICP47-GFP.

[0220] The results show that mutICP4mutICP47-IFNγ induced more granzyme B+Ki67- NK cells and antigen-presenting cells (CD11c+CD16+CD14-Ki67+MHCII++) after 24 hours than either the uninfected control or mutICP4mutICP47-GFP (Figures 6D and 6E).

[0221] Infection with mutICP4mutICP47-GFP or mutICP4mutICP47-IFNγ reduced the number of viable tumor cells and PBMC-mediated target cell killing (Figure 6F). The effect of mutICP4mutICP47 virus on T cell activation was dose-dependent. These data indicate that infection of cancer cells with the mutICP4mutICP47 vector encoding human IFNγ regulates T and NK cell function, increases antigen-presenting capacity, and activates immune-mediated tumor cell killing.

[0222] Example 11: Evaluation of chemokine expression in cancer cells following mutICP4mutICP47-IFNγ infection This example describes the evaluation of the effect of mutICP4mutICP47 HSV-1 vectors carrying an IFNγ payload on chemokine expression in cancer cells.

[0223] Chemokines in the tumor microenvironment are important factors controlling the recruitment of T cells and NK cells to tumors.

[0224] To examine chemokine expression after infection of cancer cells with mutICP4mutICP47-GFP or mutICP4mutICP47-IFNγ, Hs578T cells were infected with 10 PFU / cell of mutICP4mutICP47-GFP (with encoded GFP as a marker) or mutICP4mutICP47 vector encoding human IFNγ for 24 h and RNA was extracted from cells for RNAseq analysis. Gene numbers are reported for each gene of interest (****p<0.0001).

[0225] The results showed that infection with the mutICP4mutICP47 vector upregulated the expression of multiple T and NK cell chemokines, including CCL2, CXCL9, CXCL10, CXCL11, and CX3CL1, an effect that could be further enhanced by encoding human IFNγ in the vector (Figure 6G). This data indicates that the response to the mutICP4mutICP47 viral backbone itself supports the recruitment of T and NK cells to tumors.

[0226] Example 12: Multigene payload for survival of CAR T cells in the TME This example describes the design and evaluation of viral vectors carrying a multigene payload to support CAR T cell survival in the TME.

[0227] T cells in the tumor microenvironment can be supported by various mechanisms. For example, the presence of T cell recruiting factors in the tumor microenvironment, such as the chemokine ligands CCL19 or CCL21, can improve recruitment of CAR T cells to the tumor site.

[0228] Local CAR T cells can be supported to generate strong and durable antitumor responses. For example, local expression of CAR T cell trophic factors such as IL-7, IL-15, and IL-18 can elicit strong and durable antitumor responses from local or recruited CAR T cells. Furthermore, TGFβ signaling in cells within the tumor can be suppressed by expression of soluble TGFβRII. Furthermore, local expression of transgenes encoding costimulatory molecules such as CD40L or OX40L depending on the target TME, and combination with other vectors or therapeutic agents, can stimulate local CAR T cells or recruited T cells.

[0229] Ectopic or overexpression of a cognate CAR antigen, such as mesothelin, on tumor cells can redirect CAR T cells to target tumor cells that express the antigen.

[0230] CAR T cell help and CAR T cell recruitment can be regulated by these factors together or separately.

[0231] One or more of CCL19, CCL21, IL-7, IL-15, IL-18, soluble TGFβRII, CD40L, or OX40L can be cloned into a non-replicating McKrae strain HSV-1 vector as described in Example 2 and expressed and purified as described in Example 1. Cancer cells can be transduced with one or more GOIs including McKrae strain HSV-1 virus at different MOIs as described in Example 3 and assessed for oncolysis, payload expression and / or secretion, and immunogenicity as described in Examples 3 and 4.

[0232] The effect of cancer cells secreting these factors on CAR T cell recruitment or CAR T cell function can be evaluated in vitro or in vivo. It is contemplated that expression of genes related to CAR T cell recruitment or CAR T cell function will recruit CAR T cells to the tumor site, support the function of local CAR T cells, and reduce tumor size. In addition, expression of ectopic tumor antigens in tumors is expected to recruit CAR T cells with cognate CARs to tumors and reduce tumor size.

[0233] Example 13: Multigene payload in mutICP4mutICP47 HSV-1 vectors to induce tertiary lymphoid structures in tumor beds This example describes the design and evaluation of the mutICP4mutICP47 HSV-1 vector carrying a multigene payload to induce tertiary lymphoid structures (TLS) within the tumor bed.

[0234] Tertiary lymphoid structures (TLS) act as functional niches for the maturation of T and B cell responses to locally present tumor antigens and, although originally described in the context of chronic inflammation, have recently been recognized as elements that help orchestrate robust immune responses to solid tumors. The formation of TLS appears to be associated with improved efficacy of immunotherapy in mice and humans, providing ICI-activated TILs with signals for recruitment and survival within tumors.

[0235] To determine the induction of TLS in the tumor bed by the mutICP4mutICP47 HSV-1 vector, we used a model of vector introduction into the salivary gland, an environment that has proven to be highly permissive for TLS formation. Briefly, both submandibular salivary glands of wild-type BALB / c mice (8–10 weeks old) were transfected with 3 × 10 7 PFU of mutICP4mutICP47 HSV-1 vector expressing GFP, or a combination of mutICP4mutICP47 HSV-1 vectors expressing murine TNF, CCL19, IL-17a, and IL-7 (6 × 10 of each vector, including the vector encoding GFP). 6 PFU, total dose 3 × 10 7 PFU) and then salivary glands were harvested 15 days later for flow cytometry and immunofluorescence staining (Figure 7A). Dendritic cells (CD11c), CD4+ T cells (CD4), B cells (B220), and nuclei (DAPI) were visualized (Figures 7B and 7C). Two regions of interest (A1, A2) are shown at both low (10x) and high (40x) magnification (Figure 7C).

[0236] Results show that the mutICP4mutICP47 viral backbone alone enhanced immune infiltration of salivary glands (measured as CD45+ cells by flow cytometry) (Figure 7A) and formed small immune cell aggregates characterized by dendritic cells and CD4+ T cells (Figures 7B and 7C). Administration of a combination of mutICP4mutICP47 vectors expressing TNF, CCL19, IL-17a, and IL-7, which have reported roles in TLS formation, increased the number, size, and organization of the observed TLS structures, as shown by spatially defined B and T cell regions infiltrated with dendritic cells in immunofluorescence images. This data indicates that the response to the mutICP4mutICP47 viral backbone itself supports the underlying lymphopoiesis process, which is enhanced by the virus-mediated expression of a combination of factors known to regulate TLS formation, including TNF, CCL19, IL-17a, and IL-7. Lymphotoxin β and CXCL13, which are believed to play a role in the establishment and maintenance of TLS, are also believed to be available to support TLS formation.

[0237] Example 14: Multigene payload to promote phagocytic autoimmune surveillance This example describes the design and evaluation of viral vectors carrying multigene payloads to promote phagocytic autoimmune surveillance and elimination of tumor cells.

[0238] When SIRPα on tumor cells binds to CD47 on macrophages, phagocytosis is inhibited. This "don't eat me" signal allows tumor cells to escape immune destruction by macrophages. To block this pathway, a mutICP4mutICP47 vector was constructed that encodes a SIRPα-IgG fusion protein. To determine the activity of this vector in an ex vivo phagocytosis model, macrophages were co-cultured with mutICP4mutICP47 SIRPα-IgG medium. Briefly, CD14+ cells were enriched from human PBMCs using the STEMCELL EasySep™ Human Monocyte Enrichment Kit. The enriched cells were plated in 24-well plates and cultured for 8 days with STEMCELL ImmunoCult™-SF Macrophage Differentiation Medium and 50ng / ml m-CSF. Fresh medium was added on day 4, and 50ng / ml IFNγ was added on day 6 to stimulate the cells to polarize into M1 macrophages. On day 8, CFSE-labeled Raji cells were pretreated (30 min) with conditioned medium from Hs578T cells infected with IgG control or anti-CD47 antibody (positive control), or with mutICP4mutICP47-GFP at 10 PFU / cell, or with mutICP4mutICP47-SIRPα-IgG at 3 or 10 PFU / cell. Macrophages were then added to CFSE-labeled Raji cells and co-cultured for 3 h. Samples were harvested and phagocytosis was measured by flow cytometry. The percentage of phagocytosis was defined as the percentage of CFSE-positive macrophages (CD14+).

[0239] The results showed that conditioned medium from Hs578T cells infected with mutICP4mutICP47-SIRPα-IgG induced a higher percentage of phagocytosis than IgG control, anti-CD47 antibody, or mutICP4mutICP47-GFP, and this effect was dose-dependent (Figure 8). These data indicate that infection of cells with the mutICP4mutICP47 viral vector encoding a SIRPα-IgG fusion protein increases phagocytosis in macrophages.

[0240] Example 15: Multigene payload for survival of NK cells or CAR NK cells in the TME This example describes the design and evaluation of viral vectors carrying a multigene payload to promote survival of NK cells and CAR NK cells in the TME.

[0241] NK cells and CAR NK cells in the tumor microenvironment can be supported by various mechanisms. For example, recruitment of NK cells and CAR NK cells to the tumor site can be improved by the presence of NK cell and CAR NK cell recruitment factors, such as chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11, or CAR antigens, in the tumor microenvironment.

[0242] Local NK cells and CAR NK cells can be supported to elicit strong and sustained anti-tumor responses. For example, local expression of NK cell and CAR NK cell trophic factors, such as IL-2, IL-15, IL-18, and IFNα, can elicit strong and sustained anti-tumor responses from local or recruited NK cells and CAR NK cells. Furthermore, TGFβ signaling in cells within the tumor can be suppressed by expression of soluble TGFβRII.

[0243] NK cell and CAR NK cell help, or NK cell and CAR NK cell recruitment, can be regulated by these factors together or separately.

[0244] One or more of CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11, IL-2, IL-15, IL-18, IFNα, or soluble TGFβRII can be cloned into a non-replicating McKrae strain HSV-1 vector as described in Example 2 and expressed and purified as described in Example 1. Cancer cells can be transduced with one or more GOIs including McKrae strain HSV-1 virus at different MOIs as described in Example 3 and assessed for oncolysis, payload expression and / or secretion, and immunogenicity as described in Examples 3 and 4.

[0245] The effect of cancer cells secreting these factors on NK and CAR NK cell recruitment or NK cell and CAR NK cell function can be assessed in vitro or in vivo. It is contemplated that expression of genes related to NK and CAR NK cell recruitment or NK cell and CAR NK cell function will result in recruitment of NK and CAR NK cells to the tumor site and support local NK and CAR NK cell function, thereby reducing tumor size.

[0246] Incorporation by Reference The entire disclosure of each patent and scientific publication referenced herein is incorporated by reference for all purposes.

[0247] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments should therefore be construed in all respects as illustrative and not limiting of the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than by the above description, and all changes that come within the meaning and range of equivalents of the claims are intended to be embraced therein. [Table 1-1] [Table 1-2]

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Claims

1. A vector comprising a herpes simplex virus (HSV) genome, wherein the vector includes modifications that inhibit the expression of one or more functional ICP4 and ICP47 proteins.

2. The vector according to claim 1, wherein the functional ICP4 and ICP47 proteins are characterized by the amino acid sequences of SEQ ID NOs. 3 and 4 (ICP4) and SEQ ID NOs. 5 (ICP47).

3. The vector according to claim 1, wherein the HSV genome is an HSV-1 genome.

4. The vector according to claim 3, wherein the HSV genome is the McKrae strain genome.

5. When the aforementioned vector is administered to the subject, (a) Delay in tumor lysis, (b) Increased immunogenicity, and (c) Enhancement of immune activation, resulting in one or more of the following: The vector according to claim 3.

6. The vector according to claim 1, wherein the vector comprises nucleic acid sequences encoding one or more therapeutic polypeptides.

7. The aforementioned therapeutic polypeptide (a) Targeting the tumor stroma, (b) Support the survival of T cells or NK cells in the tumor microenvironment, (c) Induce tertiary lymphoid structures (TLS) in the tumor bed, and / or (d) Promote phagocytic autoimmune surveillance, The vector according to claim 6.

8. The vector according to claim 7, wherein the therapeutic polypeptide targets the tumor stroma by degrading extracellular matrix proteins.

9. The vector according to claim 8, wherein the therapeutic polypeptide comprises an inhibitor of hyaluronidase, MMP-9, or lysyl oxidase.

10. The vector according to claim 7, wherein the therapeutic polypeptide targets the tumor stroma by activating local endothelium and increasing T cell or NK cell infiltration.

11. The vector according to claim 10, wherein the therapeutic polypeptide comprises an inflammatory cytokine.

12. The vector according to claim 11, wherein the inflammatory cytokine comprises TNF, IL-1β, IL-6, or IL-18.

13. The vector according to claim 7, wherein the therapeutic polypeptide enhances the recruitment of T cells or NK cells to the tumor site, thereby supporting the survival of T cells or NK cells in the tumor microenvironment.

14. The vector according to claim 13, wherein the therapeutic polypeptide comprises CCL19, CCL21, CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, or CXCL11.

15. The vector according to claim 7, wherein the therapeutic polypeptide supports the survival of T cells or NK cells in the tumor microenvironment by supporting the function of T cells or NK cells.

16. The vector according to claim 15, wherein the therapeutic polypeptide comprises a T cell or NK cell trophic factor or a costimulatory molecule.

17. The vector according to claim 16, wherein the T cell or NK cell trophic factor is selected from IL-2, IL-7, IL-12, IL-15, IL-18, IFNα, and IFNγ, or the costimulatory molecule is selected from CD40L and OX40L.

18. The vector according to claim 7, wherein the T cell is a CAR T cell, or the NK cell is a CAR NK cell.

19. The vector according to claim 7, wherein the therapeutic polypeptide comprises CCL19, lymphotoxin β, CXCL13, or TNF.

20. The vector according to claim 7, wherein the therapeutic polypeptide promotes phagocytic autoimmune surveillance.

21. The vector according to claim 20, wherein the therapeutic polypeptide disrupts the Sirpα / CD47 axis.

22. The vector according to claim 21, wherein the therapeutic polypeptide comprises a Sirpα-IgG fusion transgene.

23. The vector according to claim 7, wherein the therapeutic polypeptide supports antitumor macrophage polarization.

24. The vector according to claim 23, wherein the therapeutic polypeptide that supports the polarization of antitumor macrophages comprises TNF, IL-1, IL-12, IL-17, or IFNγ.

25. A vector according to any one of claims 1 to 24, wherein a target is administered the vector, thereby expressing a polypeptide in the target.

26. A method for preparing a vector according to claim 1, wherein the vector expresses a therapeutic polypeptide, and the method is (a) A first nucleic acid molecule, (i) Consists of a portion of the HSV genome that does not encode functional ICP4 and ICP47 proteins, (ii) A first nucleic acid molecule comprising a sequence encoding a marker element, wherein the sequence encoding the marker element is flanked by a first homology region (HR1) and a second homology region (HR2), and (b) A second nucleic acid molecule comprising a sequence encoding a therapeutic polypeptide, wherein the sequence encoding the therapeutic polypeptide is flanked by a first homology region (HR1_) and a second homology region (HR2_), such that HR1 is homologous to HR1_ and HR2 is homologous to HR2_, so that the sequence encoding the therapeutic polypeptide is incorporated into the first nucleic acid molecule via homologous recombination. A method comprising culturing cells transfected with [a specific agent].

27. A mutant HSV strain comprising the vector described in any one of claims 1 to 24.

28. Cells transduced with the vector described in any one of claims 1 to 24.

29. A pharmaceutical composition comprising a vector according to any one of claims 1 to 24 and a pharmaceutically acceptable carrier.

30. A composition comprising the vector according to claim 1 for reducing tumor size in a subject requiring such reduction, wherein the vector comprises a nucleic acid encoding a therapeutic polypeptide that functions to reduce tumor size.

31. The vector according to claim 1, wherein the vector encodes one or more therapeutic polypeptides that support the survival of NK cells in the tumor microenvironment (TME).

32. A composition comprising the vector according to claim 1 for use in treatment.

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