Nucleic Acid Vectors and Methods of Use

Abstract

The present invention includes nucleic acid vectors (e.g., circular DNA vectors) and compositions thereof. Also provided herein are methods of administering nucleic acid vectors (e.g., circular DNA vectors) and compositions thereof, for example in combination with pulsed electric field therapy. Also provided are methods and compositions for treating cancer (e.g., cancer characterized by the presence of solid tumors) by administering a nucleic acid vector carrying a heterologous gene to modulate the tumor microenvironment.

Description

[Technical field]

[0001] In general, the invention features compositions and methods including nucleic acid vectors (eg, circular DNA vectors), for example, for the treatment of cancer. [Background technology]

[0002] Cancer occurs when normal physiological responses to stimuli (e.g., responding to injury or damage, initiating self-repair, or increasing innate and acquired defenses) become abnormal or excessive, leading to further disease or pathology (e.g., tumor growth and / or metastasis causing tissue damage and overall body failure). The pathogenesis of cancer has been extensively studied, leading to numerous hypotheses suggesting a wide variety of therapeutic approaches with various success rates. Nevertheless, cancer remains one of the most deadly threats to human health. According to the World Health Organization, cancer is the second leading cause of death worldwide, accounting for approximately one in six deaths, and the American Cancer Society estimates that cancer causes more than 1500 deaths per day in the United States alone.

[0003] Gene therapy holds promise as an effective treatment for cancer, but its full potential has yet to be realized, and the field is therefore in need of improved gene therapies that can confer robust and durable antitumor effects. Summary of the Invention

[0004] The present invention provides compositions and methods for treating cancer (e.g., cancer characterized by the presence of a solid tumor) by administering a nucleic acid vector (e.g., a non-viral nucleic acid vector, e.g., a circular DNA vector) carrying a heterologous gene to modulate the tumor microenvironment. In some embodiments, the regulatory genes are a combination of immune regulatory genes that work in concert to generate an anti-tumor immune response. Such regulatory genes include various combinations of genes encoding chemoattractants for dendritic cells (e.g., XCL1, XCL2, CCL4, or CCL5), genes encoding growth factors or activators for dendritic cells (e.g., FLT3L, GM-CSF, CD40, or CD40L), and genes encoding lymphocyte signaling proteins (e.g., IL-12, IL-15, CXCL9, or CXCL10). In certain embodiments described herein, the nucleic acid vector encodes a self-replicating RNA molecule in which an RNA replicase is operably linked to one or more regulatory genes to reduce dilution as tumor cells proliferate. Administration of a nucleic acid vector (e.g., a circular DNA vector, e.g., a circular DNA vector encoding a self-replicating RNA molecule) can be accompanied by delivery of an electric field (e.g., a pulsed electric field) into the tumor microenvironment to enhance electrotransfer to target cells.

[0005] In one aspect, the present invention provides a nucleic acid vector (e.g., a DNA vector, e.g., a circular DNA vector) comprising a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding a growth factor or activator for dendritic cells (e.g., a gene encoding FLT3L (e.g., a gene encoding sFLT3L), a gene encoding GM-CSF, or a gene encoding CD40 or a gene encoding CD40L), and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding IL-12, a gene encoding IL-15, a gene encoding CXCL9, or a gene encoding CXCL10). In some embodiments, the nucleic acid vector (e.g., a DNA vector, e.g., a circular DNA vector) comprises a first promoter, a second promoter, and a third promoter that respectively drive expression of a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding a growth factor or activator for dendritic cells (e.g., a gene encoding FLT3L, a gene encoding GM-CSF, or a gene encoding CD40 or a gene encoding CD40L), and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding IL-12, a gene encoding IL-15, a gene encoding CXCL9, or a gene encoding CXCL10). In some embodiments, one or more of the first promoter, the second promoter, and the third promoter are human cytomegalovirus (CMV) promoters. In some embodiments, the first promoter is a CMV promoter, the second promoter is a CMV promoter, and the third promoter is a CMV promoter. In some embodiments, one or more of the first promoter, the second promoter, and the third promoter is a CAG promoter.In some embodiments, the first promoter is a CAG promoter, the second promoter is a CAG promoter, and the third promoter is a CAG promoter. In some embodiments, the nucleic acid vector (e.g., a DNA vector, e.g., a circular DNA vector) comprises a single promoter that drives expression of a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding a growth factor or activator for dendritic cells (e.g., a gene encoding FLT3L, a gene encoding GM-CSF, or a gene encoding CD40 or a gene encoding CD40L), and a gene encoding a signaling protein for lymphocytes (e.g., a gene encoding IL-12, a gene encoding IL-15, a gene encoding CXCL9, or a gene encoding CXCL10). In certain embodiments, the nucleic acid vector is a non-viral nucleic acid vector. In some embodiments, the promoter is a CMV promoter. In other embodiments, the promoter is a CAG promoter.

[0006] In some of any of the above embodiments, the nucleic acid vector is a circular DNA vector that lacks a bacterial origin of replication and / or a drug resistance gene (e.g., lacks both a bacterial origin of replication and / or a drug resistance gene). In some embodiments, the nucleic acid vector (e.g., a DNA vector, e.g., a circular DNA vector) is between 2.5 kb and 20 kb in length. In some embodiments, the nucleic acid vector (e.g., a DNA vector, e.g., a circular DNA vector) is between 3.5 kb and 10 kb in length.

[0007] In some embodiments, the nucleic acid vector (e.g., a circular DNA vector) comprises, in 5' to 3' orientation, a gene encoding a dendritic cell growth factor or activator, a gene encoding a lymphocyte signaling protein, and a gene encoding a dendritic cell chemoattractant. In some embodiments, the circular DNA vector is a synthetic circular DNA vector lacking recombination sites (e.g., a synthetic circular DNA vector lacking recombination sites and a bacterial backbone).

[0008] In another aspect, the invention features a circular DNA vector that lacks a bacterial origin of replication and / or a drug resistance gene (e.g., lacks both a bacterial origin of replication and / or a drug resistance gene). The circular DNA vector has the following elements arranged in a 5' to 3' direction (e.g., operably linked): (a) a promoter (e.g., a CMV promoter or a CAG promoter); (b) a sequence encoding a self-replicating RNA molecule that includes (i) a sequence encoding a replicase that transcribes RNA from the self-replicating RNA molecule and (ii) a sequence (e.g., a gene) encoding one or more heterologous proteins; and (c) a polyadenylation sequence. In some embodiments, the sequence (e.g., a gene) encoding one or more heterologous proteins includes a gene encoding one or more immunomodulatory proteins. In some embodiments, the genes encoding one or more (e.g., one, two, three, or more) immunomodulatory proteins are selected from genes encoding chemoattractants for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), genes encoding growth factors or activators for dendritic cells (e.g., a gene encoding FLT3L (e.g., a gene encoding sFLT3L), a gene encoding GM-CSF, or a gene encoding CD40 or a gene encoding CD40L), and genes encoding lymphocyte signaling proteins (e.g., a gene encoding IL-12, a gene encoding IL-15, a gene encoding CXCL9, or a gene encoding CXCL10).In some embodiments, the genes encoding one or more immunomodulatory proteins include all three of a gene encoding a dendritic cell chemoattractant (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding a dendritic cell growth factor or activator (e.g., a gene encoding FLT3L (e.g., a gene encoding sFLT3L), a gene encoding GM-CSF, or a gene encoding CD40 or a gene encoding CD40L), and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding IL-12, a gene encoding IL-15, a gene encoding CXCL9, or a gene encoding CXCL10). In some embodiments of any of the above aspects, the replicase is an alphavirus replicase (e.g., a VEE replicase or a variant thereof (e.g., a replicase having the amino acid sequence of one or more (one, two, three, or all four) of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and / or SEQ ID NO:8, or a variant thereof having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and / or SEQ ID NO:8)).

[0009] In another aspect, the invention features a pharmaceutical composition (e.g., as a liquid composition or a dry (e.g., lyophilized) composition) that includes any of the nucleic acid vectors or circular DNA vectors of the above aspects and a pharma- ceutically acceptable carrier.

[0010] In another aspect, provided herein is a method of treating cancer in an individual (e.g., a human cancer patient) in need of such treatment. In some embodiments, the method comprises administering to the individual a nucleic acid vector (e.g., a non-viral nucleic acid vector), a circular DNA vector, or a pharmaceutical composition of any one of the above aspects in an amount effective to treat the cancer. In some embodiments, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered intratumorally. In other embodiments, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered systemically. In some embodiments, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered in combination with delivery of an electric field to the tumor microenvironment (e.g., pulsed electric field therapy, e.g., pulsed electric field therapy administered using electrodes placed in the tumor). In some embodiments, the method comprises delivering an electric field into the tumor microenvironment, where the electric field promotes introduction of the nucleic acid vector (e.g., the circular DNA vector) into a cell (e.g., a tumor cell), thereby delivering a heterologous regulatory gene to the cell (e.g., a tumor cell) to treat the cancer. Additionally or alternatively, the nucleic acid vector, circular DNA vector, or pharmaceutical composition can be administered in combination with an additional anti-cancer therapy (e.g., radiation therapy (e.g., cytotoxic radiation therapy), photodynamic therapy, hyperthermia, oncolytic therapy (e.g., administration of an oncolytic virus), or an anti-cancer agent (e.g., a chemotherapeutic agent, a checkpoint inhibitor, a cytotoxic agent, a growth inhibitory agent, an anti-angiogenic agent, a cytokine, a cytokine antagonist, an antibody-drug conjugate, a cancer vaccine, or a combination thereof)).

[0011] In another aspect, the invention provides a method of treating cancer in an individual (e.g., a human cancer patient) to whom a nucleic acid vector (e.g., a non-viral nucleic acid vector), circular DNA vector, or pharmaceutical composition of any embodiment of the invention has been administered or will be administered by delivering an electric field to the tumor microenvironment (e.g., by delivering a pulsed electric field to the tumor microenvironment using, for example, electrodes placed within the tumor).

[0012] In another aspect, the invention features a method of modulating a tumor microenvironment in an individual in need thereof (e.g., a human cancer patient, e.g., a human cancer patient having a solid tumor). In some embodiments, the method includes administering to the individual a nucleic acid vector (e.g., a non-viral nucleic acid vector), a circular DNA vector, or a pharmaceutical composition of any one of the above aspects in an amount effective to modulate the tumor microenvironment. In some embodiments, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered intratumorally. In other embodiments, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered systemically. In some embodiments, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered in combination with delivery of an electric field to the tumor microenvironment (e.g., pulsed electric field therapy, e.g., pulsed electric field therapy administered using electrodes placed in the tumor). In some embodiments, the method includes modulating the tumor microenvironment by delivering an electric field into the tumor microenvironment, where the electric field promotes introduction of the nucleic acid vector (e.g., the circular DNA vector) into a cell (e.g., a tumor cell), thereby delivering a heterologous regulatory gene to the cell (e.g., a tumor cell) to modulate the tumor microenvironment in the individual. Additionally or alternatively, the nucleic acid vector, circular DNA vector, or pharmaceutical composition can be administered in combination with an additional anti-cancer therapy (e.g., radiation therapy (e.g., cytotoxic radiation therapy), photodynamic therapy, hyperthermia, oncolytic therapy (e.g., administration of an oncolytic virus), or an anti-cancer agent (e.g., a chemotherapeutic agent, a checkpoint inhibitor, a cytotoxic agent, a growth inhibitory agent, an anti-angiogenic agent, a cytokine, a cytokine antagonist, an antibody-drug conjugate, a cancer vaccine, or a combination thereof)).

[0013] In another aspect, the invention provides methods of modulating the tumor microenvironment in an individual (e.g., a human cancer patient) to which a nucleic acid vector (e.g., a non-viral nucleic acid vector), circular DNA vector, or pharmaceutical composition of any embodiment of the invention has been or will be administered, by delivering an electric field to the tumor microenvironment (e.g., delivering a pulsed electric field to the tumor microenvironment, e.g., using electrodes placed in the tumor), thereby delivering a heterologous regulatory gene to a cell (e.g., a tumor cell) to modulate the tumor microenvironment in the individual.

[0014] In another aspect, the invention features inducing expression of a regulatory protein in a tumor microenvironment in an individual in need thereof (e.g., a human cancer patient, e.g., a human cancer patient having a solid tumor). In some embodiments, the method comprises administering to the individual a nucleic acid vector (e.g., a non-viral nucleic acid vector), a circular DNA vector, or a pharmaceutical composition of any one of the above aspects in an amount effective to induce expression of a regulatory protein encoded thereby. In some embodiments, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered intratumorally. In other embodiments, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered systemically. In some embodiments, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered in combination with delivery of an electric field to the tumor microenvironment (e.g., pulsed electric field therapy, e.g., pulsed electric field therapy administered using electrodes placed in the tumor). In some embodiments, the method comprises modulating the tumor microenvironment by delivering an electric field into the tumor microenvironment, where the electric field promotes introduction of the nucleic acid vector (e.g., the circular DNA vector) into a cell (e.g., a tumor cell), thereby delivering a heterologous regulatory gene to the cell (e.g., a tumor cell) and inducing its expression. Additionally or alternatively, the nucleic acid vector, circular DNA vector, or pharmaceutical composition can be administered in combination with an additional anti-cancer therapy (e.g., radiation therapy (e.g., cytotoxic radiation therapy), photodynamic therapy, hyperthermia, oncolytic therapy (e.g., administration of an oncolytic virus), or an anti-cancer agent (e.g., a chemotherapeutic agent, a checkpoint inhibitor, a cytotoxic agent, a growth inhibitory agent, an anti-angiogenic agent, a cytokine, a cytokine antagonist, an antibody-drug conjugate, a cancer vaccine, or a combination thereof)).

[0015] In another aspect, the invention provides a method of inducing expression of a regulatory protein in the tumor microenvironment in an individual (e.g., a human cancer patient) to whom a nucleic acid vector (e.g., a non-viral nucleic acid vector), circular DNA vector, or pharmaceutical composition of any embodiment of the invention has been administered or will be administered, by delivering an electric field to the tumor microenvironment (e.g., delivering a pulsed electric field to the tumor microenvironment, e.g., using electrodes placed in the tumor), thereby delivering a heterologous regulatory gene to a cell (e.g., a tumor cell) and inducing expression of the regulatory protein encoded by the vector in the tumor microenvironment.

[0016] In another aspect, provided herein are methods of treating cancer in an individual in need of treatment (e.g., a human cancer patient) by administering a non-viral nucleic acid vector comprising a gene encoding a dendritic cell chemoattractant in an amount effective to treat the cancer. In some embodiments, the non-viral nucleic acid vector is administered intratumorally. In other embodiments, the non-viral nucleic acid vector is administered systemically. In some embodiments, the non-viral nucleic acid vector is administered in combination with delivery of an electric field to the tumor microenvironment (e.g., pulsed electric field therapy, e.g., pulsed electric field therapy administered using electrodes placed in the tumor). In some embodiments, the method includes delivering an electric field into the tumor microenvironment, where the electric field promotes introduction of the non-viral nucleic acid vector, thereby delivering the gene encoding a dendritic cell chemoattractant to a cell (e.g., a tumor cell) to treat the cancer. Additionally or alternatively, the non-viral nucleic acid vector can be administered in combination with an additional anti-cancer therapy (e.g., radiation therapy (e.g., cytotoxic radiation therapy), photodynamic therapy, hyperthermia, oncolytic therapy (e.g., administration of an oncolytic virus), or an anti-cancer agent (e.g., a chemotherapeutic agent, a checkpoint inhibitor, a cytotoxic agent, a growth inhibitory agent, an anti-angiogenic agent, a cytokine, a cytokine antagonist, an antibody-drug conjugate, a cancer vaccine, or a combination thereof)).

[0017] In another aspect, the invention provides a method of treating cancer in an individual (e.g., a human cancer patient) who has been administered or will be administered a non-viral nucleic acid vector having a gene encoding a chemoattractant for dendritic cells by delivering an electric field to the tumor microenvironment (e.g., by delivering a pulsed electric field to the tumor microenvironment using, for example, electrodes placed within the tumor).

[0018] In another aspect, the invention features a method of modulating a tumor microenvironment in an individual in need thereof (e.g., a human cancer patient, e.g., a human cancer patient having a solid tumor) by administering to the individual a non-viral nucleic acid vector having a gene encoding a dendritic cell chemoattractant in an amount effective to modulate the tumor microenvironment. In some embodiments, the non-viral nucleic acid vector is administered intratumorally. In other embodiments, the non-viral nucleic acid vector is administered systemically. In some embodiments, the non-viral nucleic acid vector is administered in combination with delivery of an electric field to the tumor microenvironment (e.g., pulsed electric field therapy, e.g., pulsed electric field therapy administered using electrodes placed in the tumor). In some embodiments, the method includes modulating the tumor microenvironment by delivering an electric field into the tumor microenvironment, where the electric field promotes introduction of the non-viral nucleic acid vector into a cell (e.g., a tumor cell), thereby delivering a gene encoding a dendritic cell chemoattractant to the cell (e.g., a tumor cell) and modulating the tumor microenvironment in the individual. Additionally or alternatively, the non-viral nucleic acid vector can be administered in combination with an additional anti-cancer therapy (e.g., radiation therapy (e.g., cytotoxic radiation therapy), photodynamic therapy, hyperthermia, oncolytic therapy (e.g., administration of an oncolytic virus), or an anti-cancer agent (e.g., a chemotherapeutic agent, a checkpoint inhibitor, a cytotoxic agent, a growth inhibitory agent, an anti-angiogenic agent, a cytokine, a cytokine antagonist, an antibody-drug conjugate, a cancer vaccine, or a combination thereof)).

[0019] In another aspect, the invention provides a method of modulating the tumor microenvironment in an individual (e.g., a human cancer patient) to which a non-viral nucleic acid vector carrying a gene encoding a dendritic cell chemoattractant has been or will be administered, by delivering an electric field to the tumor microenvironment (e.g., delivering a pulsed electric field to the tumor microenvironment, e.g., using electrodes placed in the tumor), thereby delivering a gene encoding a dendritic cell chemoattractant to a cell (e.g., a tumor cell) and modulating the tumor microenvironment in the individual.

[0020] In another aspect, the invention features inducing expression of a regulatory protein in a tumor microenvironment in an individual in need thereof (e.g., a human cancer patient, e.g., a human cancer patient having a solid tumor) by administering to the individual a non-viral nucleic acid vector having a gene encoding a dendritic cell chemoattractant in an amount effective to induce expression of the gene encoding the dendritic cell chemoattractant. In some embodiments, the non-viral nucleic acid vector is administered intratumorally. In other embodiments, the non-viral nucleic acid vector is administered systemically. In some embodiments, the non-viral nucleic acid vector is administered in combination with delivery of an electric field to the tumor microenvironment (e.g., pulsed electric field therapy, e.g., pulsed electric field therapy administered using electrodes placed in the tumor). In some embodiments, the method includes modulating the tumor microenvironment by delivering an electric field into the tumor microenvironment, where the electric field promotes introduction of the non-viral nucleic acid vector into a cell (e.g., a tumor cell), thereby delivering and inducing expression of a gene encoding a dendritic cell chemoattractant to the cell (e.g., a tumor cell). Additionally or alternatively, the non-viral nucleic acid vector can be administered in combination with an additional anti-cancer therapy (e.g., radiation therapy (e.g., cytotoxic radiation therapy), photodynamic therapy, hyperthermia, oncolytic therapy (e.g., administration of an oncolytic virus), or an anti-cancer agent (e.g., a chemotherapeutic agent, a checkpoint inhibitor, a cytotoxic agent, a growth inhibitory agent, an anti-angiogenic agent, a cytokine, a cytokine antagonist, an antibody-drug conjugate, a cancer vaccine, or a combination thereof)).

[0021] In another aspect, the invention provides a method of inducing expression of a dendritic cell chemoattractant within the tumor microenvironment in an individual (e.g., a human cancer patient) to whom a non-viral nucleic acid vector carrying a gene encoding a dendritic cell chemoattractant has been administered or will be administered, by delivering an electric field to the tumor microenvironment (e.g., delivering a pulsed electric field to the tumor microenvironment, e.g., using electrodes placed in the tumor), thereby delivering the gene encoding a dendritic cell chemoattractant to a cell (e.g., a tumor cell) and inducing expression of the dendritic cell chemoattractant within the tumor microenvironment.

[0022] In another aspect, the invention provides a method of expressing a protein in a tumor microenvironment in an individual in need of expression. In some embodiments, the method includes (a) administering a synthetic circular DNA vector encoding the protein; and (b) transmitting an electric field into the tumor microenvironment, such that the electric field promotes introduction of the synthetic circular DNA vector into tumor cells, thereby delivering the protein-encoding sequence to the tumor cells for expression in the tumor microenvironment in the individual. In some embodiments, step (b) includes transmitting a pulsed electric field. In some embodiments, the pulsed electric field is transmitted via electrodes disposed in the tumor. In some embodiments, the synthetic circular DNA vector is administered intratumorally. In some embodiments, the synthetic circular DNA vector is administered systemically.

[0023] In some embodiments, the synthetic circular DNA vector is administered in combination with an additional anti-cancer therapy, hi some embodiments, the protein is selected from the group consisting of a gene encoding a chemoattractant for dendritic cells, a gene encoding a growth factor or activator for dendritic cells, and a gene encoding a lymphocyte signaling protein.

[0024] In some embodiments, the synthetic circular DNA vector encodes a gene encoding a chemoattractant for dendritic cells, a gene encoding a growth factor or activator for dendritic cells, and a gene encoding a lymphocyte signaling protein. In some embodiments, the synthetic circular DNA vector does not include an origin of replication, a drug resistance gene, or a recombination site. In some embodiments, the synthetic circular DNA vector does not include a bacterial backbone. [Brief description of the drawings]

[0025] [Figure 1A] 1 is a schematic diagram showing an exemplary DNA vector of the present invention having a single transcription unit. The DNA vector includes a 5'CAG promoter (PCAG), a gene encoding sFlt3L, a first Furin-P2A sequence, a gene encoding IL-12, a second Furin-P2A sequence, a gene encoding XCL1, and a 3' polyA tail. The circularized DNA vector has a size of about 3.5 kb to 5 kb. [Figure 1B] Figure 1 shows an image of a gel showing a band representing the synthetic circular DNA vector of Figure 1A, with a size of 4537 bp. The asterisk indicates the position of the supercoiled monomeric form, which is 78% of the sample shown here. Lane 1 shows a sample loaded at 200 ng, while lane 2 shows the same sample loaded at 800 ng. The ladder is supercoiled ladder (New England Biosciences). [Figure 2A] 1 is a schematic diagram showing an exemplary multiple transcription unit DNA vector of the present invention. The DNA vector comprises a first transcription unit having a first PCAG, a gene encoding sFlt3L, and a first 3' polyA tail, a second transcription unit having a second PCAG, a gene encoding IL-12, and a second 3' polyA tail, and a third transcription unit having a third PCAG, a gene encoding XCL1, and a third 3' polyA tail. The circularized DNA vector has a size of about 5 kb to 10 kb. [Figure 2B]Figure 2 shows an image of a gel showing a band representing the synthetic circular DNA vector of Figure 2A (SEQ ID NO: 37) with a size of 8035 bp. The asterisk indicates the position of the supercoiled monomeric form, which is 85% of the sample shown here. Lane 1 shows a sample loaded at 200 ng, while lane 2 shows the same sample loaded at 800 ng. The ladder is a supercoiled ladder (New England Biosciences). [Figure 3A-3C] 3A-3C are graphs showing protein expression of immune-modulating proteins from the 8035 bp multiple transcription unit synthetic circular DNA vector (c3-Tx) shown in FIG. 2A, normalized to medium and measured by ELISA. FIG. 3A shows expression of sFlt3L, FIG. 3B shows expression of IL-12p70, and FIG. 3C shows expression of XCL1. [Figure 4] FIG. 13 is a graph showing expression of functional IL-12p70 by c3-Tx by SEAP-QuantiBlue assay. [Figure 5A-5C] 5A-5C are flow cytometry plots showing differentiation of bone marrow-derived dendritic cells (BMDCs) into conventional DCs (cDCs) and plasmacytoid DCs (pDCs) under various conditions: (A) differentiation in 20 ng / mL sFLT3L expressed by c3-Tx-transfected cells (in conditioned medium), (B) differentiation in 20 ng / mL recombinant sFLT3L, and (C) differentiation in recombinant soluble GMCSF (rsGMCSF). [Figures 6A-6C] 6A-6C are bar graphs showing differentiation of BMDCs into cDCs and pDCs under various conditions: (A) differentiation in 20 ng / mL sFLT3L expressed by c3-Tx-transfected cells (in conditioned medium), (B) differentiation in 20 ng / mL recombinant sFLT3L, and (C) differentiation in recombinant soluble GMCSF (rsGMCSF). [Figure 7A-7E]Figures 7A and 7B show in-life images showing fLuc luminescence over time in tumor-bearing mice under various treatment conditions. Figures 7A and 7B show PBS control mice at 3 days and 17 days after treatment, respectively. Figures 7C and 7D show mice treated with a plasmid DNA vector encoding fLuc (p-fLuc) at 3 days and 17 days after treatment, respectively. Figures 7E and 7F show mice treated with a synthetic circular DNA vector encoding fLuc (c3-fLuc) at 3 days and 17 days after treatment, respectively. [Figure 8] FIG. 13 shows a graph showing fLuc radiance relative to PBS control for the experiment of FIGS. 7A-7F, including additional time points of 7, 10, and 14 days. [Figure 9] FIG. 13 is a graph showing tumor growth over time in mice treated with c3-Tx by electrotransfer compared to PBS control mice. [Figure 10] 1 shows a graph depicting immune cell infiltration in tumors administered a single dose of c3-Tx or PBS and subjected to electrical energy, with each data point representing the percentage of cells within the tumor expressing CD45 in c3-Tx-treated or PBS control mice. [Figure 11] FIG. 13 is a graph showing survival over time of mice treated with two doses of c3-Tx compared to mice injected with PBS or c3-fLuc. [Figure 12] FIG. 13 shows a graph depicting the mean tumor volume over time in mice treated with two doses of c3-Tx compared to mice injected with PBS or c3-fLuc. [Figure 13] FIG. 13 is a graph showing GFP expression in tumors injected with mRNA GFP and then electrotransferred compared to tumors injected with a synthetic circular DNA vector (c3-GFP) and then electrotransferred, and tumors injected with PBS and then electrotransferred. [Figure 14A]FIG. 1 is a schematic diagram showing a tricistronic self-replicating RNA molecule encoding sFLT3L, IL-12, and XCL1, having a length of 10429 bp. [Figure 14B] FIG. 1 is a schematic diagram showing a plasmid encoding a tricistronic self-replicating RNA molecule encoding sFLT3L, IL-12, and XCL1 having a length of 13978. [Figures 15A-15C] 15A-C are graphs showing protein expression of immune-modulating proteins from tricistronic self-replicating RNA molecules and plasmids encoding the tricistronic self-replicating RNA molecules, normalized to medium and measured by ELISA: FIG. 15A shows expression of sFlt3L, FIG. 15B shows expression of IL-12p70, and FIG. 15C shows expression of XCL1. [Figure 16] FIG. 1 shows a graph depicting expression of functional IL-12p70 by tricistronic self-replicating RNA molecules and plasmids encoding the tricistronic self-replicating RNA molecules by SEAP-QuantiBlue assay. [Figure 17] FIG. 13 is a bar graph showing differentiation of BMDCs into cDCs and pDCs under various conditions, including treatment with tricistronic self-replicating RNA molecules and conditioned medium from a plasmid encoding a tricistronic self-replicating RNA molecule, compared to treatment with rFLT3L and rGMCSF. [Figure 18A-18B]18A is a schematic diagram showing an exemplary method for synthesizing a circular DNA vector encoding a self-replicating RNA molecule of the present invention. The relative organization of each element is shown. FIG. 18A is a schematic diagram showing individual DNA fragments useful as starting materials. The 5' sequence, which has a CAG promoter (PCAG), a sequence encoding a nonstructural protein (replicase), and a subgenomic promoter (SGP), is divided into three parts. The gene encoding soluble Flt3 ligand (sFlt3L), the gene encoding IL-12, and the gene encoding XCL1 are operably connected via a Furin-P2A sequence. The 3' polyadenylation signal sequence (polyA) is shown. FIG. 18B is a schematic diagram showing a circular DNA plasmid produced by golden gate assembly of the DNA fragments shown in FIG. 18A. The circular plasmid has a size of about 13 kb. The relative organization of each element is shown. The DNA vector contains a PCAG for the 5'UTR, a sequence encoding a nonstructural protein (replicase), an SGP, a gene encoding sFlt3L, a first Furin-P2A sequence, a gene encoding IL-12, a second Furin P2A sequence, a gene encoding XCL1, a ribozyme, and a polyA tail. [Figures 19A-19E]19A is a schematic diagram showing an exemplary method for synthesizing a self-replicating RNA molecule of the present invention. The relative organization of each element is shown. FIG. 19A is a schematic diagram showing individual DNA fragments useful as starting materials. The 5' sequence, which has a T7 promoter (PT7), a sequence encoding a nonstructural protein (replicase), and a subgenomic promoter (SGP), is divided into three parts. The gene encoding XCL1, the gene encoding soluble Flt3 ligand (sFlt3L), and the gene encoding IL-12 are operably connected via a Furin-P2A sequence. The 3' polyadenylation sequence (AAAA) is shown. FIG. 19B is a schematic diagram showing a circular DNA plasmid produced by Golden Gate assembly of the DNA fragments shown in FIG. 19A. The circular plasmid has a size of about 13.5 kb. FIG. 19C is a schematic diagram showing a linearized DNA molecule generated by digestion with I-SceI. The linearized DNA molecule has a size of about 10.5 kb. Figure 19D is a schematic diagram showing a self-replicating RNA molecule generated by in-vitro transcription of the linearized DNA molecule of Figure 19C. Figure 19E is a schematic diagram showing a 5'-capped self-replicating RNA molecule with an extended polyadenylation sequence at the 3' end. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention relates to compositions and methods for treating cancer by administering a nucleic acid vector (e.g., a circular DNA vector) carrying a regulatory gene to modulate the tumor microenvironment. The present invention is based, at least in part, on the discovery that the nucleic acid vector (e.g., a circular DNA vector) of the present invention can be effective in generating an anti-tumor immune response through the delivery of multiple immunomodulatory genes. The compositions and methods of the present invention can effectively modulate the tumor microenvironment through the recruitment and / or activation of dendritic cells, which can direct anti-tumor immunity by presenting tumor antigens to lymphocytes (e.g., T cells). In some instances, the nucleic acid vector (e.g., a circular DNA vector) is administered to an individual suffering from cancer upon delivery of an electric field to the tumor microenvironment (e.g., by delivering a pulsed electric field via an electrode placed in the tumor). In certain embodiments of the present invention, the compositions and methods of the present invention can mitigate dilution of regulatory gene expression as a result of rapid tumor cell proliferation by providing self-replicating RNA molecules to the tumor microenvironment.

[0027] I. Definition Unless otherwise specified, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published documents which provide such persons with general guidance to many of the terms used in this application. In the event of any conflict between the definitions set forth herein and those in the referenced publications, the definitions set forth herein shall control.

[0028] As used herein, the term "circular DNA vector" refers to a DNA molecule in a circular form. Such circular forms can typically be amplified into concatemers by rolling circle amplification. A linear double-stranded nucleic acid with end-stranded linkages (e.g., a backbone covalently linked by a hairpin loop or other structure) is not a circular vector as used herein. The term "circular DNA vector" is used interchangeably herein with the term "covalently closed circular DNA vector." Those skilled in the art will appreciate that such circular vectors include vectors that are covalently closed and have supercoiled and complex DNA topology as described herein. In certain embodiments, the circular DNA vector is supercoiled. In certain instances, the circular DNA vector lacks a bacterial origin of replication (e.g., when the circular DNA vector encodes a self-replicating RNA molecule, the circular DNA vector lacks a bacterial origin of replication and encodes an RNA origin of replication).

[0029] As used herein, a "cell-free method" for producing a circular DNA vector refers to a method that facilitates all steps of the method from preparation of a template DNA vector (e.g., a plasmid DNA vector) to production of a therapeutic circular DNA vector without relying on the inclusion of any DNA in a host cell, such as a bacterial (e.g., E. coli) host cell. For example, a cell-free method is carried out in one or more synthetic vessels (e.g., glass or plastic tubes, bioreactors, vessels, tanks, or other suitable vessels) in suitable solutions (e.g., buffer solutions) to which enzymes and other agents can be added to facilitate DNA amplification, DNA modification, and DNA isolation.

[0030] As used herein, the terms "recombination site" and "site-specific recombination recognition site" each refer to a nucleic acid sequence that is the product of site-specific recombination and includes a first sequence that corresponds to a portion of a first recombinase attachment site and a second sequence that corresponds to a portion of a second recombinase attachment site. An example of a hybrid recombination site is attR, which is the product of site-specific recombination and includes a first sequence that corresponds to a portion of attP and a second sequence that corresponds to a portion of attB. Alternatively, a recombination site can be generated from Cre / Lox recombination. Thus, a vector generated from Cre / Lox recombination (e.g., a vector that includes a LoxP site) includes a recombination site as used herein. Other site-specific recombination events that generate recombination sites include, for example, lambda integrase, FLP recombinase, and Kw recombinase. A nucleic acid sequence resulting from a non-site-specific recombination event (e.g., ITR-mediated intermolecular recombination) is not a recombination site as defined herein.

[0031] As used herein, the term "self-replicating RNA molecule" refers to a self-replicating genetic element that contains RNA that replicates from a single origin of replication. The terms "self-replicating RNA", "replicon RNA" and "self-amplifying replicon RNA" are used interchangeably herein.

[0032] As used herein, the term "protein" refers to multiple amino acids bound together via peptide bonds (i.e., as a primary structure) and includes non-covalently associated multimeric (e.g., dimeric, trimeric, etc.) proteins (e.g., proteins having quaternary structure). Thus, the term "protein" encompasses peptides, native proteins, recombinant proteins, and fragments thereof. In some embodiments, a protein has a primary structure and no secondary, tertiary, or quaternary structure under physiological conditions. In some embodiments, a protein has a primary structure and a secondary structure and no tertiary or quaternary structure under physiological conditions. In certain embodiments, a protein has a primary structure, a secondary structure, and a tertiary structure, but no quaternary structure under physiological conditions (e.g., a monomeric protein having one or more folded alpha-helices and / or beta-sheets). In some embodiments, any of the proteins described herein has a length of at least 25 amino acids (e.g., between 50 amino acids and 1000 amino acids).

[0033] As used herein, the term "regulatory sequence" refers to a nucleic acid sequence that encodes one or more proteins that engage and regulate a cell by binding and signaling through a receptor (e.g., a surface receptor) expressed by the cell. Regulatory sequences can be monocistronic or polycistronic (e.g., bicistronic or tricistronic).

[0034] As used herein, the term "immune modulatory sequence" refers to a nucleic acid sequence that encodes one or more proteins that engage and modulate an immune cell (e.g., an innate or adaptive immune cell), for example, by binding and signaling through a receptor (e.g., a surface receptor) on the immune cell. Immune modulatory sequences can be monocistronic or polycistronic (e.g., bicistronic or tricistronic).

[0035] As used herein, the term "polycistronic" refers to a nucleic acid sequence (e.g., a DNA vector or an RNA sequence) that contains two or more protein-coding genes downstream of a single promoter (i.e., one promoter drives the expression of two or more proteins). Polycistronic sequences include bicistronic and tricistronic sequences. In some instances, a polycistronic sequence contains two, three, four, or more immunomodulatory protein-coding genes. Methods for synthesizing polycistronic nucleic acid sequences are known in the art. In some instances, the multiple protein-coding genes are separated by termination signals and / or cleavage sites (e.g., furin-P2A sites).

[0036] The terms "first", "second" and "third" used herein to identify promoters in a nucleic acid vector do not describe the positioning of the promoters on the vector, i.e., the first promoter, the second promoter and the third promoter may, but need not, be arranged in a 5'→3' orientation. For example, a nucleic acid vector comprising a first promoter, a second promoter and a third promoter driving the expression of XCL1, FLT3L and IL-12, respectively, covers each of the following 5'→3' orientations: promoter (P)-XCL1-P-FLT3L-P-IL-12; P-FLT3L-P-IL-12-P-XCL1; P-IL-12-P-XCL1-P-FLT3L; P-XCL1-P-IL-12-P-FLT3L; P-FLT3L-P-XCL1-P-IL-12; and P-IL-12-P-FLT3L-P-XCL1. The first promoter, the second promoter, and the third promoter may be the same promoter (eg, CAG).

[0037] As used herein, the term "dendritic cell chemoattractant" refers to a protein that attracts (e.g., promotes infiltration of) dendritic cells (e.g., conventional dendritic cells (e.g., cDC1 or cDC2) or plasmacytoid DCs (e.g., pDCs)) by binding to a receptor expressed thereon (e.g., by binding to a dendritic cell surface receptor). Dendritic cell chemoattractants can promote dendritic cell infiltration into an area of ​​tissue by forming a concentration gradient along which dendritic cells migrate toward a higher concentration of the chemoattractant. In some embodiments, the dendritic cell chemoattractant is an inflammatory dendritic cell chemoattractant. Exemplary dendritic cell chemoattractants include XCL1, XCL2, CCL5, and CCL4.

[0038] As used herein, "XCL1" refers to any native XCL1 (also known as lymphotactin, Ltn, ATAC, SCYC-α, or SCYC-1, a member of the C chemokine subfamily) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., natural or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent and improved variants can be determined based on XCR1 signaling (e.g., by XCL1 binding to DC-expressed XCR1). In some embodiments, functionally equivalent or improved variants exhibit improved stability (e.g., muteins that confer structural (e.g., folding) stability of XCL1). XCL1 encompasses full-length unprocessed XCL1 and all forms of XCL1 resulting from native processing in cells. An exemplary human XCL1 sequence is provided as National Center for Biotechnology Information (NCBI) Gene ID: 6375. In some instances, XCL1 is encoded by a heterologous gene having at least 95% sequence identity to SEQ ID NO:9 or SEQ ID NO:9A (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9 or SEQ ID NO:9A). In some instances, the XCL1 encoded by the heterologous gene has at least 95% sequence identity to SEQ ID NO:10 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:10).

[0039] As used herein, "XCL2" refers to any native XCL2 (also known as SCM1-β, or SCYC-2, a member of the C chemokine subfamily) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., natural variants or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent and improved variants can be determined based on XCR2 signaling (e.g., by XCL2 binding to DC-expressed XCR2). In some embodiments, functionally equivalent or improved variants exhibit improved stability (e.g., muteins that confer structural (e.g., folding) stability of XCL2). XCL2 encompasses full-length unprocessed XCL2 and all forms of XCL2 resulting from native processing in cells. An exemplary human XCL2 sequence is provided as National Center for Biotechnology Information (NCBI) Gene ID: 6846. In some instances, XCL2 is encoded by a heterologous gene having at least 95% sequence identity to SEQ ID NO:11 or SEQ ID NO:11A (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:11 or SEQ ID NO:11A). In some instances, XCL1 encoded by a heterologous gene has at least 95% sequence identity to SEQ ID NO:12 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:12).

[0040] As used herein, "CCL5" refers to any native CCL5 (also known as RANTES (Regulated on Activation, Normal T sell Expressed and Secreted)) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., naturally occurring variants or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent and improved variants may be determined based on CCR5 signaling and / or its known downstream effects (e.g., CCR5-associated migration and / or tumor infiltration by dendritic cells, eosinophils, basophils, monocytes, effector memory T cells, B cells, or NK cells). In some embodiments, functionally equivalent or improved variants exhibit improved stability. CCL5 encompasses full-length unprocessed CCL5 and all forms of CCL5 resulting from native processing in cells. An exemplary human CCL5 protein sequence is provided as UniProt Accession No. P13501. In some examples, CCL5 is encoded by a heterologous gene having at least 95% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 13A (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 13A). In some examples, CCL5 encoded by a heterologous gene has at least 95% sequence identity to SEQ ID NO: 14 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14).

[0041] As used herein, "CCL4" refers to any native CCL4 (also known as macrophage inflammatory protein-1β (MIP-1β)) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., natural variants or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent and improved variants may be determined based on CCR5 receptor signaling and / or its known downstream effects (e.g., CCR5-associated migration and / or tumor infiltration by dendritic cells, eosinophils, basophils, monocytes, effector memory T cells, B cells, or NK cells). In some embodiments, functionally equivalent or improved variants exhibit improved stability. CCL4 encompasses full-length unprocessed CCL4 and all forms of CCL4 resulting from native processing in cells. An exemplary human CCL4 protein sequence is provided as UniProt Accession No. P13236. In some examples, CCL4 is encoded by a heterologous gene having at least 95% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 15A (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 15A). In some examples, CCL4 encoded by a heterologous gene has at least 95% sequence identity to SEQ ID NO: 16 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16).

[0042] As used herein, the term "dendritic cell growth factor or activator" refers to a protein that promotes the differentiation, activation, and / or mobilization of dendritic cells (e.g., conventional dendritic cells (e.g., cDC1 or cDC2) or plasmacytoid DCs (e.g., pDCs)) by binding to a receptor expressed thereon (e.g., by binding to a dendritic cell surface receptor). Growth factors or activators of dendritic cells include FLT3L, GM-CSF, CD40, and CD40L. As used herein, the gene encoding FLT3L (e.g., the gene encoding sFLT3L), the gene encoding GM-CSF, and the gene encoding CD40 or the gene encoding CD40L are genes encoding growth factors or activators of dendritic cells.

[0043] As used herein, "Fms-related receptor tyrosine kinase 3 ligand (FLT3L)" refers to any native FLT3L from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., natural variants or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. FLT3L includes soluble FLT3L (sFLT3L). Functionally equivalent and improved variants can be determined based on tyrosine kinase III receptor signaling and / or its known downstream effects (e.g., FLT3L-associated hematopoietic regulation). In some embodiments, functionally equivalent or improved variants show improved stability. FLT3L includes full-length unprocessed FLT3L and any form of FLT3L resulting from native processing in cells. An exemplary human FLT3L protein sequence is provided as UniProt Accession No. P49771. In some instances, sFLT3L is encoded by a heterologous gene having at least 95% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 17A (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 17A). In some instances, the FLT3L encoded by the heterologous gene has at least 95% sequence identity to SEQ ID NO: 18 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18).

[0044] As used herein, "granulocyte-macrophage colony-stimulating factor (GM-CSF)" refers to any native GM-CSF (also known as CSF2) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., natural variants or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent and improved variants may be determined based on CSF2R receptor signaling and / or its known downstream effects (e.g., JAK-2 recruitment and activation, STAT5 phosphorylation, pim-1 and / or CIS transcription, PI3K signaling, and / or JAK / STAT-Bcl-2 signaling). In some embodiments, functionally equivalent or improved variants exhibit improved stability. GM-CSF encompasses full-length unprocessed GM-CSF and all forms of GM-CSF resulting from native processing in cells. An exemplary human GM-CSF protein sequence is provided as UniProt Accession No. P04141. In some instances, GM-CSF is encoded by a heterologous gene having at least 95% sequence identity to SEQ ID NO:19 or SEQ ID NO:19A (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:19 or SEQ ID NO:19A). In some instances, GM-CSF encoded by a heterologous gene has at least 95% sequence identity to SEQ ID NO:20 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:20).

[0045] As used herein, "cluster of differentiation 40 ligand (CD40L)" refers to any native CD40L from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., natural variants or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent and improved variants may be determined based on CD40 / TNFRSF5 signaling and / or its known downstream effects (e.g., T cell costimulation in combination with T cell receptor (TCR) binding, IL-4 and / or IL-10 production, NFκB activation, MAPK8 activation in T cells, and / or PAK2 activation in T cells). In some embodiments, functionally equivalent or improved variants exhibit improved stability. CD40L encompasses full-length unprocessed CD40L and all forms of CD40L resulting from native processing in cells. An exemplary human CD40L protein sequence is provided as UniProt Accession No. P29965. In some instances, CD40L is encoded by a heterologous gene having at least 95% sequence identity to SEQ ID NO:21 or SEQ ID NO:21A (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21 or SEQ ID NO:21A). In some instances, CD40L encoded by a heterologous gene has at least 95% sequence identity to SEQ ID NO:22 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:22).

[0046] As used herein, the term "lymphocyte signaling protein" refers to a protein that sends a signal to a lymphocyte by binding to a receptor expressed on the lymphocyte (e.g., by binding to a lymphocyte surface receptor). In some instances, the lymphocyte signaling protein causes activation and / or infiltration (e.g., tumor infiltration) of lymphocytes, such as T cells and / or NK cells. In some instances, the lymphocyte signaling protein is a soluble lymphocyte signaling protein. Lymphocyte signaling proteins include cytokines and chemokines.

[0047] The term "cytokine" refers to a soluble protein that acts as an intercellular mediator (e.g., as a paracrine signal) on immune cells (e.g., innate or adaptive immune cells). Exemplary cytokines include interleukins (ILs), such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, and IL-36-γ; tumor necrosis factors, such as TNF-α or TNF-β; interferons (IFNs), such as IFN-γ and IFN-α; and other protein factors, including leukemia inhibitory factor (LIF) and Kit ligand (KL). In some instances, cytokines cause activation of lymphocytes, such as T cells and / or NK cells.

[0048] As used herein, the term "chemokine" refers to a soluble protein released by cells that acts on immune cells to selectively induce chemotaxis and activation (e.g., tumor invasion and activation). Chemokines can also trigger angiogenesis, inflammation, wound healing, and tumor formation. In some instances, chemokines are inflammatory. Exemplary chemokines include CXCL9 and CXCL10. Other non-limiting examples of chemokines useful as part of the present invention include CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL10.

[0049] As used herein, "interleukin-12 (IL-12)" refers to any native IL-12 (e.g., IL-12p35 / p40 heterodimer; IL-12p70) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., natural variants or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent and improved variants may be determined based on certain proinflammatory and / or Th1-skewing effects (e.g., induction of IFN-γ from T cells and / or NK cells) known in the art. In some embodiments, functionally equivalent or improved variants exhibit improved stability. IL-12 includes full-length unprocessed IL-12 and all forms of IL-12 resulting from native processing in cells. IL-12 includes synthetic fusions (e.g., via a linker) of IL-12 subunits p35 and p40. In some examples, IL-12 is encoded by a heterologous gene having at least 95% sequence identity to SEQ ID NO:23 or SEQ ID NO:23A (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:23 or SEQ ID NO:23A). In some examples, IL-12 encoded by a heterologous gene has at least 95% sequence identity to SEQ ID NO:24 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24).

[0050] As used herein, "interleukin-15 (IL-15)" refers to any native IL-15 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., natural variants or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent and improved variants may be determined based on activation of the IL-15R signaling pathway (e.g., JAK kinase activation, STAT3 activation, STAT5 activation, and / or STAT6 activation; and / or activation of T cells and NK cells). In some embodiments, functionally equivalent or improved variants exhibit improved stability. IL-15 encompasses full-length unprocessed IL-15 and all forms of IL-15 resulting from native processing in cells. IL-15 includes synthetic fusions of IL-15 and its receptor. In some instances, IL-15 is encoded by a heterologous gene having at least 95% sequence identity to SEQ ID NO:25 or SEQ ID NO:25A (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25 or SEQ ID NO:25A). In some instances, IL-15 encoded by a heterologous gene has at least 95% sequence identity to SEQ ID NO:26 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26).

[0051] As used herein, "chemokine (CXC motif) ligand 9 (CXCL9)" refers to any native CXCL9 (also known as monokine induced by gamma interferon (MIG)) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., natural variants or synthetic variants), such as variants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent and improved variants may be determined based on CXCR3 receptor interactions. In some embodiments, functionally equivalent or improved variants exhibit improved stability. CXCL9 encompasses full-length unprocessed CXCL9 and all forms of CXCL9 resulting from native processing in cells. An exemplary human CXCL9 protein sequence is provided as UniProt accession number Q07325. In some examples, CXCL9 is encoded by a heterologous gene having at least 95% sequence identity to SEQ ID NO:27 or SEQ ID NO:27A (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27 or SEQ ID NO:27A). In some examples, CXCL9 encoded by a heterologous gene has at least 95% sequence identity to SEQ ID NO:28 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28).

[0052] As used herein, "chemokine (CXC motif) ligand 10 (CXCL10)" refers to any native CXCL10 (also known as monokine induced by gamma interferon (MIG)) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., natural variants or synthetic variants), such as variants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent and improved variants may be determined based on CXCR3 receptor interaction. In some embodiments, functionally equivalent or improved variants exhibit improved stability. CXCL10 encompasses full-length unprocessed CXCL10 and any form of CXCL10 resulting from native processing in cells. An exemplary human CXCL10 protein sequence is provided as UniProt Accession No. P02778. In some examples, CXCL10 is encoded by a heterologous gene having at least 95% sequence identity to SEQ ID NO:29 or SEQ ID NO:29A (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29 or SEQ ID NO:29A). In some examples, CXCL10 encoded by a heterologous gene has at least 95% sequence identity to SEQ ID NO:30 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:30).

[0053] As used herein, the term "therapeutic sequence" refers to a portion of a DNA molecule (e.g., a plasmid DNA vector or a concatemer thereof) that contains any genetic material necessary for transcription of one or more therapeutic moieties in a target cell, and may include one or more coding sequences, promoters, terminators, introns, and / or other regulatory elements. The therapeutic moiety may be a therapeutic protein (e.g., a replacement protein (e.g., a protein that replaces a defective protein in a target cell) or an endogenous protein (e.g., an immunomodulatory protein such as a cytokine)) and / or a therapeutic nucleic acid (e.g., one or more microRNAs). In a DNA vector with two or more transcription units, the therapeutic sequence includes multiple transcription units. The therapeutic sequence may include one or more genes (e.g., heterologous genes or transgenes) that are administered for therapeutic purposes.

[0054] The term "heterologous gene" refers to a transgene that is administered (e.g., as part of a DNA vector or a self-replicating RNA molecule). A heterologous gene may be a mammalian gene (e.g., a gene encoding a heterologous regulatory (e.g., immunomodulatory) protein) that is endogenously expressed by the individual receiving the heterologous gene. As used herein, a "variant" of a heterologous gene, replicase, or a fragment thereof differs in at least one amino acid residue from a reference amino acid sequence, such as a naturally occurring amino acid sequence or an amino acid sequence. In this context, a difference in at least one amino acid residue may consist, for example, of a mutation, deletion, or insertion of an amino acid residue to another amino acid. A variant may be a homolog, isoform, or transcript variant of a therapeutic protein or a fragment thereof as defined herein, the homolog, isoform, or transcript variant being characterized by a degree of identity or homology, respectively, as defined herein.

[0055] In some instances, the variant of the heterologous gene, replicase, or fragment thereof comprises at least one amino acid substitution (e.g., 1 to 100 amino acid substitutions, 1 to 50 amino acid substitutions, 1 to 20 amino acid substitutions, 1 to 10 amino acid substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions). Substitutions in which amino acids of the same class are exchanged are called conservative substitutions. In particular, these are amino acids with aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chain or amino acids whose side chains can form hydrogen bridges, e.g., side chains with hydroxyl functions. A conservative constitution may, for example, replace an amino acid having a polar side chain by another amino acid having a corresponding polar side chain, or, for example, replace an amino acid characterized by a hydrophobic side chain by another amino acid having a corresponding hydrophobic side chain (e.g., serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). In certain embodiments, variants of a protein or fragment thereof may be encoded by an RNA according to the invention, wherein at least one amino acid residue of the amino acid sequence comprises at least one conservative substitution compared to a reference sequence, such as a respective naturally occurring sequence.

[0056] In some instances, for example, insertions, deletions, and / or non-conservative substitutions at positions that do not cause substantial alteration of the three-dimensional structure of the protein are also encompassed by the term variant. Alterations in the three-dimensional structure due to insertion(s) or deletion(s) can be easily determined by those skilled in the art, for example, using CD spectroscopy (circular dichroism spectroscopy).

[0057] To determine the percentage of identity of two sequences (e.g., nucleic acid sequences, e.g., DNA, RNA, or amino acid sequences), the sequences can be aligned for subsequent comparison with each other. For this purpose, gaps can be inserted into the sequence of the first sequence and the elements at the corresponding positions of the second sequence can be compared. If a position in the first sequence is occupied by the same element as the corresponding position in the second sequence, the two sequences are identical at this position. The percentage of identity of two sequences is a function of the number of identical positions divided by the total number of positions. The percentage of identity of two sequences can be determined using a mathematical algorithm. A preferred but non-limiting example of a mathematical algorithm that can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm can be integrated, for example, into the BLAST program. Sequences that are identical to the sequences of the present invention to a certain extent can be identified by this program. In any embodiment described herein, the sequence may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the particular sequence number described (e.g., any one of SEQ ID NO: 1 to SEQ ID NO: 36). Those skilled in the art will understand that, thanks to the genetic code, the DNA sequences described herein allow for the deduction of the corresponding RNA sequence, and similarly, the RNA sequences allow for the deduction of the corresponding DNA sequence. All sequences described herein are listed in Table 1.

[0058] As used herein, the term "operably linked" or "operatively linked" refers to an arrangement of elements where the components so described are configured to perform their normal functions. A nucleic acid is "operably linked" or "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to one or more heterologous genes if it affects the transcription of the one or more heterologous genes. In addition, a control element operably linked to a coding sequence can affect the expression of the coding sequence. Control elements need not be contiguous with the coding sequence, so long as they function to direct its expression. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0059] As used herein, the term "isolated" means artificially produced and not integrated into a native host genome. For example, isolated nucleic acid vectors include nucleic acid vectors encapsulated in lipid envelopes (e.g., liposomes) or polymer matrices. In some embodiments, the term "isolated" refers to a DNA vector that is (i) amplified in vitro (e.g., in a cell-free environment), e.g., by rolling circle amplification or polymerase chain reaction (PCR); (ii) recombinantly produced by molecular cloning; (iii) purified by restriction endonuclease cleavage and gel electrophoretic fractionation, or column chromatography; or (iv) synthesized, e.g., by chemical synthesis. An isolated nucleic acid vector is one that can be readily manipulated by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector for which the 5' and 3' restriction sites are known or for which the polymerase chain reaction (PCR) primer sequences are disclosed is considered to be isolated, while a nucleic acid sequence naturally present in a natural host is not isolated. An isolated nucleic acid vector may, but need not, be substantially purified. An isolated self-replicating molecule is one that can be easily manipulated by recombinant techniques well known in the art. An isolated self-replicating RNA molecule may be substantially purified, but need not be. For example, an isolated self-replicating RNA molecule includes a self-replicating RNA molecule that is encapsulated in a lipid envelope (e.g., a liposome) or a polymer matrix.

[0060] As used herein, the term "naked" refers to a nucleic acid molecule (e.g., a circular DNA vector) that is not encapsulated in a lipid envelope (e.g., a liposome) or a polymer matrix and is not physically associated (e.g., covalently or non-covalently bound) with a solid structure (e.g., a particulate structure) upon administration to an individual. In some examples of the present invention, the pharmaceutical composition comprises a naked circular DNA vector (e.g., a naked synthetic circular DNA vector).

[0061] The term "pharmaceutical composition" refers to an agent in a form such that the biological activity of one or more active ingredients contained therein is effective and does not contain additional ingredients that are unacceptably toxic to a patient to whom the formulation is administered. A pharmaceutical composition may include a pharma- ceutical acceptable carrier, e.g., in liquid or dried (e.g., lyophilized) form.

[0062] The phrase "pharmacologically acceptable" indicates that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation, and / or with the mammal being treated therewith.

[0063] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the vector or composition of the present invention is administered. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences," Academic Press, 23 rd It will be published in the 2020 edition.

[0064] As used herein, a "vector" refers to a nucleic acid molecule capable of carrying a sequence of interest (e.g., a heterologous or therapeutic sequence) and is linked to a target cell, which then allows the heterologous gene to be replicated, processed, and / or expressed in the target cell. Once the target or host cell processes the sequence of interest (e.g., genome) of the vector, the sequence of interest (e.g., genome) is not considered a vector. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop that includes a bacterial backbone into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors" or "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0065] As used herein, "target cell" refers to a cell that expresses a regulatory protein encoded by a heterologous gene. Target cells include tumor cells (i.e., cancer cells) and tumor-resident cells (e.g., healthy cells present in the tumor microenvironment (e.g., tumor-resident antigen-presenting cells such as dendritic cells, and tumor-infiltrating lymphocytes such as T cells)).

[0066] As used herein, the term "individual" includes any mammal in need of the methods of treatment or prevention described herein (e.g., a mammal having cancer). In some embodiments, the individual is a human. In other embodiments, the individual is a non-human mammal (e.g., a non-human primate (e.g., monkey), mouse, pig, rabbit, cat, or dog). The subject may be male or female. In one embodiment, the subject has cancer, e.g., a cancer characterized by the presence of one or more solid tumors.

[0067] "Cancer" refers to the abnormal proliferation of malignant cancer cells, including cancers characterized by the presence of solid tumors, such as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, gallbladder and biliary tract cancer, thyroid cancer, thymus cancer, bone cancer, and brain cancer. Cancer cells (e.g., tumor cells) in a cancer patient can be immunologically distinguishable from normal somatic cells in the subject (e.g., cancer tumors can be immunogenic). For example, cancer cells can elicit a systemic immune response in a cancer patient against one or more antigens expressed by the cancer cells. The antigens that elicit the immune response may be tumor antigens or may be shared by normal cells. A patient with cancer may exhibit at least one identifiable indication, symptom, or test result sufficient to diagnose cancer according to clinical criteria known in the art. Examples of such clinical criteria are described in medical textbooks, such as Harrison's Principles of Internal Medicine (Longo DL, Fauci AS, Kasper DL, Hauser SL, Jameson J, Loscalzo J. eds. 18e. New York, NY McGraw-Hill; 2012). In some cases, diagnosing cancer in an individual may include identifying a particular cell type (e.g., cancer cells) in a sample of bodily fluid or tissue obtained from the individual.

[0068] As used herein, a "solid tumor" is a cancer of any body tissue other than blood, bone marrow, or lymphatic system. Solid tumors can be further divided into those of epithelial cell origin and those of non-epithelial cell origin. Examples of epithelial cell or solid tumors include tumors of the head and neck, gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gallbladder, labia, nasopharynx, skin, uterus, male reproductive organs, urinary organs, bladder, and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors.

[0069] As used herein, "tumor microenvironment" refers to the collection of cells and extracellular materials (e.g., matrix proteins, vesicles, and soluble factors (e.g., cytokines)) within a solid tumor.

[0070] As used herein, an "effective amount" or "effective dose" of a nucleic acid vector or composition thereof refers to an amount sufficient to achieve a desired biological and / or pharmacological effect when administered to an individual, for example, according to a selected form, route, and / or schedule of administration. As will be appreciated by those of skill in the art, the absolute amount of a particular composition that is effective may vary depending on factors such as the desired biological or pharmacological endpoint, the agent being delivered, the target tissue, etc. Those of skill in the art will further appreciate that an "effective amount" may be contacted with a cell or administered to a subject in a single dose or through the use of multiple doses. An effective amount of a composition for treating cancer may slow or stop disease progression (e.g., tumor growth and / or metastasis), increase partial or complete responses, and increase overall survival (e.g., compared to a reference population, e.g., an untreated or placebo population).

[0071] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention that seeks to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or alleviation of the disease state, and remission or improved prognosis. In some embodiments, the nucleic acid vectors of the invention (e.g., circular DNA vectors) are used to delay the onset of disease or to slow the progression of disease.

[0072] In particular, the treatment may include reducing or inhibiting the growth of cancer, including complete remission of cancer and / or inhibition of cancer metastasis. Cancer growth generally refers to any of several indicators that indicate the transformation of cancer into a more developed form. Thus, indicators for measuring the inhibition of cancer growth may be reduction in cancer cell viability, tumor volume or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging methods). Slowing tumor growth, destruction of tumor vasculature, improved performance in delayed hypersensitivity skin tests, increased cytolytic T lymphocyte activity, and reduced tumor-specific antigen levels. Reducing immune suppression in a cancerous tumor in a subject may improve the subject's ability to resist cancer growth, particularly cancer growth already present in the subject, and / or reduce the tendency of cancer growth in the subject.

[0073] By "reducing or inhibiting" is meant the ability to cause an overall decrease, preferably by 20% or more, more preferably by 50% or more, and most preferably by 75% or more, 85%, 90%, 95% or more. Reducing or inhibiting can refer to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.

[0074] As used herein, "modulating" or "modulating" the tumor microenvironment means causing a measurable change in any tumor biomarker known to correlate with or affect (i.e., directly or indirectly) the balance between tumor progression and tumor clearance. For example, the tumor microenvironment is modulated by a treatment if the treatment increases the relative expression of a biomarker associated with tumor clearance, such as an immunogenic protein in the tumor microenvironment (e.g., a proinflammatory cytokine or a dendritic cell activation protein, or a protein expressed on the cell surface, such as activated tumor-infiltrating lymphocytes), and / or an immunogenic cell profile in the tumor microenvironment (e.g., an increased presence of tumor-infiltrating lymphocytes). In some embodiments, the tumor microenvironment is said to be modulated by a treatment if the treatment decreases the relative expression of a biomarker associated with tumor progression (e.g., a biomarker of T cell exhaustion, such as PD-1 or PD-L1 expression in the tumor microenvironment). Biomarkers associated with tumor clearance or tumor progression can be determined by any suitable means, such as Western blot, immunohistochemistry (IHC), polymerase chain reaction (PCR), single cell sequencing, flow cytometry, mesoscale discovery (MSD), or other conventional assays performed, for example, on a biopsy of the tumor microenvironment or blood (e.g., circulating cells, plasma, or serum). In some embodiments, the measurable change is any statistically significant change, for example, in the expression level of an immunogenic protein or other biomarker associated with tumor progression. For example, a measurable change in expression level can be at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90% change in the expression level of an immunogenic protein or other biomarker associated with tumor progression relative to a baseline expression level (e.g., expression in a reference cell, a reference tissue, or a subject without or prior to treatment). In some instances, the tumor microenvironment is determined to be modulated by a treatment if the treatment extends progression-free survival, results in an objective response, including a partial or complete response, extends overall survival, and / or ameliorates one or more symptoms of cancer.

[0075] As used herein, the term "biomarker" refers to a protein, DNA, RNA, carbohydrate, or glycolipid-based molecular marker, the expression or presence of which can be detected by standard methods (or the methods disclosed herein) in an individual, subject, or patient sample, and is useful for monitoring the responsiveness or sensitivity of the tumor environment to a circular DNA vector, or a composition thereof. The expression of such a biomarker can be determined to be higher or lower in a sample obtained from an individual treated according to the method of the present invention relative to a reference level (including, for example, the median expression level of the biomarker in samples from a group / population of patients, e.g., patients with solid tumors; the median expression level of the biomarker in samples from a group / population of patients, e.g., patients with solid tumors; or the level in a sample previously obtained from the individual). An individual having an expression level higher or lower than the reference expression level of the biomarker can be characterized as an individual having a tumor microenvironment that has been modulated by the treatment. For example, such individuals exhibiting gene expression levels that are the most extreme 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% (i.e., higher or lower) relative to a reference level (such as the median level described above) may be identified as individuals having a tumor microenvironment that is modulated by the treatment.

[0076] The terms "level of expression" or "expression level" are used interchangeably and generally refer to the amount of a polynucleotide, or an amino acid product or protein, in a biological sample (e.g., a tumor sample). "Expression" generally refers to the process by which genetically coded information is converted into structures present and operational in a cell. Thus, according to the present invention, "expression" of a gene may refer to transcription into a polynucleotide, translation into a protein, or post-translational modification of a protein. Transcribed polynucleotides, translated proteins, or post-translationally modified protein fragments shall also be considered expressed, regardless of whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of a protein, e.g., by proteolysis. "Expressed genes" include those that are transcribed into polynucleotides as mRNA and then translated into proteins, and also include those that are transcribed into RNA but not translated into proteins (e.g., transfer RNA and ribosomal RNA).

[0077] As used herein, the term "expression persistence" refers to the period during which a sequence of interest (e.g., a heterologous gene or therapeutic sequence) or a functional portion thereof (e.g., one or more coding sequences of a therapeutic DNA vector) is expressible in a transfected cell ("intracellular persistence") or in any descendants of the transfected cell ("intergenerational persistence"). A sequence of interest (e.g., a heterologous gene or therapeutic sequence) or a functional portion thereof may be expressible when it is not silenced, for example, by DNA methylation and / or histone methylation and compaction. Expression persistence can be assessed by detecting or quantifying (i) the mRNA transcribed from the sequence of interest (e.g., a heterologous gene or therapeutic sequence) in the target cell or its descendants (e.g., via qPCR, RNA-seq, or any other suitable method), and (ii) the protein translated from the sequence of interest (e.g., a heterologous gene or therapeutic sequence) in the target cell or its descendants (e.g., via Western blot, ELISA, or any other suitable method). In some instances, expression persistence is assessed by detecting or quantifying the therapeutic DNA in the target cell or its progeny (e.g., the presence of the therapeutic circular DNA vector in the target cell, e.g., by episomal DNA copy number analysis) in conjunction with either or both of (i) the mRNA transcribed from the sequence of interest (e.g., heterologous gene or therapeutic sequence) in the target cell or its progeny, and (ii) the protein translated from the sequence of interest (e.g., heterologous gene or therapeutic sequence) in the target cell or its progeny. Expression persistence of the sequence of interest (e.g., heterologous gene or therapeutic sequence), or a functional portion thereof, can be quantified against a reference vector, such as a control vector produced in bacteria (e.g., a circular vector (e.g., a plasmid) produced in bacteria or having one or more bacterial signatures not present in the vector of the invention), using any method known in the art to characterize gene expression. Expression persistence can be quantified at any given time point after administration of the vector.For example, in some embodiments, expression of a therapeutic circular DNA vector of the invention persists for at least two weeks after administration if it is detectable in the target cell or its progeny two weeks after administration of the therapeutic circular DNA vector. In some embodiments, expression of a gene is "sustained" in a target cell if it is detectable in the target cell for one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer after administration. In some embodiments, expression of a sequence of interest (e.g., a heterologous gene or therapeutic sequence) is said to persist for a given period of time after administration if any detectable percentage of the original expression level (e.g., at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, or at least 100%) remains after the given period of time (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer after administration).

[0078] The terms "sample" and "biological sample" are used interchangeably to refer to any biological sample obtained from an individual, including bodily tissue (e.g., tumor tissue), bodily fluid, cells, or other sources. Bodily fluids are, for example, lymph, serum, whole fresh blood, peripheral blood mononuclear cells, frozen whole blood, plasma (including fresh or frozen), urine, saliva, semen, synovial fluid, and cerebrospinal fluid. Samples also include breast tissue, kidney tissue, colon tissue, brain tissue, muscle tissue, synovial tissue, skin, hair follicles, bone marrow, and tumor tissue. Methods for obtaining tissue biopsies and bodily fluids from mammals are well known in the art.

[0079] As used herein, "administering" refers to a method of giving a dosage of a nucleic acid vector of the invention or a composition thereof (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising a nucleic acid vector) to an individual. The compositions utilized in the methods described herein can be administered, for example, intratumorally, peritumorally, intravenously, subcutaneously, intradermally, percutaneously, intramuscularly, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, topically, transdermally, periocularly, intraconjunctivally, subtenonly, intracamerally, subretinally, retrobulbarly, intralacrimally, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localized perfusion directly bathing the target cells, by catheter, by lavage, in a cream, or in a lipid composition. The compositions utilized in the methods described herein can be administered systemically or locally (e.g., intratumorally or peritumorally). Methods of administration can vary depending on a variety of factors, such as the compound or composition being administered and the severity of the condition, disease, or disorder being treated.

[0080] "Administered in combination with" refers to administration of multiple therapeutic components as part of the same treatment regimen. The nucleic acid vectors of the present invention (e.g., circular DNA vectors) can be administered in combination with pulsed electric field therapy, for example, as part of the same outpatient treatment or over several days. Additionally or alternatively, the nucleic acid vectors of the present invention (e.g., circular DNA vectors) can be administered in combination with another therapeutic agent (e.g., as part of the same pharmaceutical composition or as separate pharmaceutical compositions, at the same time or at different times).

[0081] As used herein, "electrotransfer" refers to the movement of a molecule (e.g., a nucleic acid vector, e.g., a circular DNA vector) across the membrane of a target cell (e.g., from the outside to the inside of a target cell) caused by the delivery of an electric field (e.g., a pulsed electric field) to the microenvironment in which the cell resides. Electrotransfer can occur as a result of electrophoresis, i.e., the movement of a molecule (e.g., a nucleic acid vector, e.g., a circular DNA vector) along an electric field (e.g., in the direction of the electric current) based on the charge of the molecule. Electrophoresis can induce electrotransfer, for example, by moving the molecule (e.g., a nucleic acid vector, e.g., a circular DNA vector) into the vicinity of the cell membrane to allow biological transport processes (e.g., endocytosis, including pinocytosis or phagocytosis) or passive transport (e.g., diffusion or lipid partitioning) to carry the molecule into the cell. Additionally or alternatively, electrotransfer may occur as a result of electroporation, i.e., the generation of pores in the target cells caused by the transmission of an electric field (e.g., a pulsed electric field), the size, shape, and duration of the pores being suitable for accommodating the transfer of molecules (e.g., nucleic acid vectors, e.g., circular DNA vectors) from the outside of the target cells to the inside of the target cells. Thus, in some instances, electrotransfer occurs as a result of a combination of electrophoresis and electroporation.

[0082] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metoledopa, and uredopa; ethylenimines and methylaminamines, including itretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine. hylamelamines; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL™); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including the synthetic analogs topotecan (HYCAMTIN™), CPT-11 (irinotecan, CAMPTOSAR™), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; C C-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, meclostatin, Nitrogen mustards such as retinol, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin); CDP323, an oral alpha-4 integrin inhibitor; dynemycins, including dynemycin A; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, austramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN™, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL™), liposomal doxorubicin TLC D-99 (including MYOCET™, pegylated liposomal doxorubicin (CAELYX™, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate, gemcitabine (GEMZAR™), tegafur (UFTORAL™), capecitabine (XELODA™), epothilones, and 5-fluoxetine; antimetabolites such as lauracil (5-FU); combretastatins; folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; fluorinic acid folic acid supplements such as aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine;Diazicon; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; phenamet; pirarubicin; rosoxantrone; 2-ethylhydrazide; procarbazine; PSK™ polysaccharide complex (JHS Natural, Eugene, Oregon) Products); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A and anguidin); urethanes; vindesine (ELDISINE™, FILDESIN™); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, NJ), albumin-modified nanoparticle formulation of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE™, Rhome-Poulene, Antony, France). Rorer; chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN™), and carboplatin; vincas that prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN™), vincristine (ONCOVIN™), vindesine (ELDISINE™, FILDESIN™), and vinorelbine (NAVELBINE™); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN™);bisphosphonates such as clodronate (e.g., BONEFOS™ or OSTAC™), etidronate (DIDROCAL™), NE-58095, zoledronic acid / zoledronate (ZOMETA™), alendronate (FOSAMAX™), pamidronate (AREDIA™), tiludronate (SKELID™), or risedronate (ACTONEL™); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides; nucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R) (e.g., erlotinib (Tarceva™)); and VEGF-A, which reduces cell proliferation; vaccines, such as the THERATOOPE™ vaccine and gene therapy vaccines, such as the ALLOVECTIN™ vaccine, the LEUVECTIN™ vaccine, and the VAXID™ vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN™); rmRH (e.g., ABARELIX™); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT™, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE™); CCI-779; tipifarnib (R11577); orafenib, ABT510; oblimersen sodium tyrosine kinase inhibitors; serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE™); farnesyltransferase inhibitors such as lonafamib (SCH6636, SARASAR™); and pharmacologic acceptable salts, acids, or derivatives of any of the above; as well as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone;Combinations of two or more of the above, such as FOLFOX, an abbreviation for a treatment regimen that combines oxaliplatin (ELOXATIN™) with 5-FU and leucovorin, as well as pharmacologic acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0083] Chemotherapeutic agents, as defined herein, include "anti-hormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit the effects of hormones that may promote the growth of cancer. These include, but are not limited to, antiestrogens with a mixed agonist / antagonist profile, including selective estrogen receptor modulators (SERMs) such as tamoxifen (NOLVADEX™), 4-hydroxytamoxifen, toremifene (FARESTON™), idoxifene, droloxifene, raloxifene (EVISTA™), trioxyfene, keoxifene, and SERM3; pure antiestrogens without agonist properties, such as fulvestrant (FASLODEX™), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN™), and anastrazole (ARIMIDEX™), letrozole (FEMARA™), and aminoglutethamine (AMIDEX™). The aromatase inhibitors may be hormones themselves, including nonsteroidal aromatase inhibitors such as thymide; other aromatase inhibitors include vorozole (RIVISOR™), megestrol acetate (MEGASE™), fadrozole, and 4(5)-imidazole; luteinizing hormone releasing hormone agonists including leuprolide (LUPRON™ and ELIGARD™), goserelin, buserelin, and tripterelin; megestrol acetate and medroxyprogesterone acetate, etc. sex steroids, including progestins such as diethylstilbestrol and premarin, and androgens / retinoids such as fluoxymesterone, all trans retinoic acid and fenretinide; onapristone; antiprogesterones; estrogen receptor downregulators (ERDs); antiandrogens such as flutamide, nilutamide and bicalutamide; and pharma- ceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above.

[0084] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed herein.

[0085] "Growth inhibitors" as used herein refer to compounds or compositions that inhibit cell growth either in vitro or in vivo. Thus, growth inhibitors may significantly reduce the percentage of cells in S phase. Examples of growth inhibitors include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that arrest G1 also extend to S phase arrest, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and araC. Further information can be found, for example, on page 13, in Mendelsohn et al. eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antitineoplastic drugs" by Murakami et al. (WB Saunders, Philadelphia, 1995). The taxanes (paclitaxel and docetaxel) are anticancer drugs that are both derived from the yew tree. Docetaxel (TAXOTERE™, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL™, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers, stabilize microtubules by preventing depolymerization, and inhibit mitosis in cells.

[0086] The singular forms "a" and "an" mean "one or more." For example, "a gene" is understood to refer to one or more such genes. Thus, the singular forms "one or more of," and "at least one of" are used interchangeably herein.

[0087] As used herein, the term "about" refers to a value within ±10% variation from a reference value, unless otherwise indicated.

[0088] II. Therapeutic composition The present invention includes nucleic acid vectors (e.g., circular DNA vectors, e.g., naked circular DNA vectors; or self-replicating RNA molecules), and pharmaceutical compositions thereof, which are useful, for example, in methods of treating cancer through modulation of the tumor microenvironment (e.g., immunomodulation of the tumor microenvironment). Such nucleic acid vectors include circular DNA vectors (e.g., circular DNA vectors having multiple transcription units, each encoding an immunomodulatory gene; circular DNA vectors having a single transcription unit encoding multiple immunomodulatory genes; and circular DNA vectors encoding a replicase and a self-replicating RNA molecule encoding one or more immunomodulatory genes). In some examples, the circular DNA vector (e.g., naked circular DNA vector) is a synthetic circular DNA vector, which refers to a circular DNA vector whose production from a template is produced using a cell-free process that does not involve bacterial cells.

[0089] Nucleic Acid Vectors Provided herein are nucleic acid vectors (e.g., DNA vectors, e.g., circular DNA vectors, or self-replicating RNA molecules) having regulatory sequences, each of which encodes multiple regulatory genes. In embodiments involving self-replicating RNA molecules, the regulatory sequences can be operably linked to a replicase. The regulatory sequences can be immunomodulatory, i.e., modulate immune cells, e.g., by encoding an immunomodulatory protein that binds to the surface of immune cells to mobilize and / or activate the immune cells. The regulatory sequences (e.g., immunomodulatory sequences) can be monocistronic or polycistronic (i.e., bicistronic, tricistronic, etc.). The immunomodulatory sequences include genes encoding one or more immunomodulatory proteins that encode peptides and proteins capable of binding to various immune cells, e.g., genes encoding chemoattractants for dendritic cells, genes encoding growth factors for dendritic cells, and genes encoding lymphocyte signaling proteins.

[0090] In some instances, the immunomodulatory protein binds to and signals through an antigen-presenting cell (e.g., a cross-presenting antigen-presenting cell, e.g., a professional antigen-presenting cell such as a dendritic cell (e.g., a conventional dendritic cell (e.g., cDC1 or cDC2) or a plasmacytoid DC (e.g., pDC)). Such antigen-presenting cell-associated immunomodulatory proteins include dendritic cell chemoattractants, e.g., XCL1, XCL2, CCL5, or CCL4.Thus, in some embodiments of the invention, the immune modulatory sequence includes a gene encoding XCL1 (e.g., a gene encoding an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 10; and / or a gene encoding an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 9 or SEQ ID NO: 9A). or 100% identical to SEQ ID NO:12); a gene encoding XCL2 (e.g., a gene encoding an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:12; and / or a gene encoding an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:11 or SEQ ID NO:11A). a gene encoding CCL5 (e.g., a gene encoding an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:14; and / or a gene having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:13 or SEQ ID NO:13A) ), or a gene encoding CCL4 (e.g., a gene encoding an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:16; and / or a gene having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:15 or SEQ ID NO:15A).Antigen-presenting cell-associated immunomodulatory proteins also include dendritic cell growth factors and activators, such as FLT3L (e.g., soluble FLT3L (sFLT3L)), GM-CSF, CD40, or CD40L). Thus, in some examples of the invention, the immunomodulatory sequence includes a gene encoding FLT3L (e.g., a gene encoding sFLT3L (e.g., a gene encoding an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 18; and / or a gene having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 17 or SEQ ID NO: 17A)). In other examples, immune modulatory sequences include genes encoding GM-CSF (e.g., genes encoding an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:20; and / or genes having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:19 or SEQ ID NO:19A). In other examples, immune modulatory sequences include genes encoding CD40L (e.g., genes encoding an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:22; and / or genes having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:21 or SEQ ID NO:21A).In some instances, the nucleic acid vector features a single promoter driving expression of multiple genes in the immune regulatory sequence (e.g., a single promoter driving expression of a gene encoding a dendritic cell chemoattractant, a gene encoding a dendritic cell growth factor or activator, and / or a gene encoding a lymphocyte signaling protein). In other embodiments, the nucleic acid vector includes a promoter driving each gene in the immune regulatory sequence (e.g., a first promoter, a second promoter, and a third promoter driving expression of a gene encoding a dendritic cell chemoattractant, a gene encoding a dendritic cell growth factor or activator, and a gene encoding a lymphocyte signaling protein, respectively).

[0091] In some instances, the immune regulating sequence comprises a gene encoding a dendritic cell chemoattractant (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4) and a gene encoding a dendritic cell growth factor or activator (e.g., a gene encoding FLT3L (e.g., a gene encoding sFLT3L), a gene encoding GMCSF, or a gene encoding CD40L). In certain embodiments, such immune regulating sequences comprise no more than two genes. In some embodiments, the immune regulating sequence has multiple transcription units and comprises a first promoter 5' to a gene encoding a dendritic cell chemoattractant and a second promoter 5' to a gene encoding a dendritic cell growth factor or activator. In other embodiments, the immune regulating sequence is bicistronic and comprises a single promoter 5' to a gene encoding a dendritic cell chemoattractant and a gene encoding a dendritic cell growth factor or activator.

[0092] Immunomodulatory proteins can also bind to and signal through lymphocytes (e.g., T cells, NK cells, or B cells). In some instances, the self-replicating RNA molecules of the invention comprise an immunomodulatory sequence having a gene encoding a lymphocyte signaling protein. Lymphocyte signaling proteins include cytokines and chemokines, which can activate or stimulate lymphocytes when bound to their receptors. Thus, in some embodiments, the immunomodulatory sequence comprises a gene encoding a lymphocyte signaling protein, such as a cytokine or chemokine. Cytokines include interleukins (IL), such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, and IL-36-γ; tumor necrosis factors, such as TNF-α or TNF-β; interferons (IFN), such as IFN-β, IFN-γ, and IFN-α; and other protein factors including leukemia inhibitory factor (LIF) and Kit ligand (KL).In certain embodiments, the cytokine encoded by the immune modulatory sequence is IL-12 (e.g., a cytokine having an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:24; and / or a cytokine having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:23 or SEQ ID NO:23A). and / or an immune regulating sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:26), IL-15 (e.g., a cytokine having an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:26; and / or an immune regulating sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:25 or SEQ ID NO:25A. and / or an immunomodulatory sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:32; and / or an immunomodulatory sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:31 or SEQ ID NO:31A. sequence), or IL-36-γ (e.g., a cytokine having an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:36; and / or an immune modulating sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:35 or SEQ ID NO:35A).Chemokines include CXCL9, CXCL10, CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, and CCL11. In some embodiments, the chemokine encoded by the immune modulatory sequence is CXCL9 (e.g., a cytokine having an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:28; and / or a cytokine having an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:27 or SEQ ID NO:27A). or CXCL10 (e.g., a cytokine having an amino acid sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:30; and / or an immune regulatory sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:29 or SEQ ID NO:29A).

[0093] In some instances, the immune modulatory sequence includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4) and a gene encoding a signaling protein for lymphocytes (e.g., a gene encoding a lymphocyte activating cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In other examples, the immune regulating sequence includes a gene encoding a growth factor or activator of dendritic cells (e.g., a gene encoding FLT3L (e.g., a gene encoding sFLT3L), a gene encoding GM-CSF, or a gene encoding CD40L) and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activating cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In certain embodiments, such immune regulating sequences include no more than two genes. In some embodiments, the immune regulating sequence has multiple transcription units and includes a first promoter 5' to a gene encoding a dendritic cell chemoattractant and a second promoter 5' to a gene encoding a lymphocyte signaling protein. In other embodiments, the immune regulating sequence is bicistronic and includes a single promoter 5' to a gene encoding a dendritic cell chemoattractant and a gene encoding a lymphocyte signaling protein.

[0094] In some instances, the immune regulatory sequence is a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding a growth factor or activator for dendritic cells (e.g., a gene encoding FLT3L (e.g., a gene encoding sFLT3L), a gene encoding GMCSF, or a gene encoding CD40L), and a gene encoding a signaling protein for lymphocytes (e.g., a gene encoding a lymphocyte activating cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CCL4). In some embodiments, the immune regulatory sequence comprises a first promoter 5' to a gene encoding a dendritic cell chemoattractant, a second promoter 5' to a gene encoding a dendritic cell growth factor or activator, and a third promoter 5' to a gene encoding a lymphocyte signaling protein. In other embodiments, the immune regulatory sequence is tricistronic and comprises a single promoter 5' to a gene encoding a dendritic cell chemoattractant, a gene encoding a dendritic cell growth factor or activator, and a gene encoding a lymphocyte signaling protein.

[0095] For example, an immune regulatory sequence (e.g., an immune regulatory sequence having three transcription units as illustrated in FIG. 2, or a tricistronic immune regulatory sequence as illustrated in FIG. 1) can include a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding a growth factor or activator of dendritic cells (e.g., a gene encoding FLT3L (e.g., a gene encoding sFLT3L), a gene encoding GM-CSF, or a gene encoding CD40L), and a gene encoding IL-12. Alternatively, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcription units, or a tricistronic immune regulatory sequence) may include a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding a growth factor or activator of dendritic cells (e.g., a gene encoding FLT3L (e.g., a gene encoding sFLT3L), a gene encoding GM-CSF, or a gene encoding CD40L), and a gene encoding IL-15. Alternatively, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcription units, or a tricistronic immune regulatory sequence) may include a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding a growth factor or activator of dendritic cells (e.g., a gene encoding FLT3L (e.g., a gene encoding sFLT3L), a gene encoding GM-CSF, a gene encoding CD40L), and a gene encoding CCL4.In other specific embodiments, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcription units, or a tricistronic immune regulatory sequence) may include a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding a growth factor or activator of dendritic cells (e.g., a gene encoding FLT3L (e.g., a gene encoding sFLT3L), a gene encoding GM-CSF, or a gene encoding CD40L), and a gene encoding CCL5.

[0096] In some examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcription units, or a tricistronic immune regulatory sequence) includes a gene encoding a dendritic cell chemoattractant (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)).

[0097] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding an activating cytokine for lymphocytes (e.g., a gene encoding IL-12, a gene encoding IL-15). Alternatively, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In some examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding IL-12. In other examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding IL-15.In yet another example, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding CXCL9. In an alternative example, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding CXCL10.

[0098] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL1, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL1, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding IL-12. In other examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL1, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding IL-15. In yet another example, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL1, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding CXCL9. In an alternative example, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL1, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding CXCL10.

[0099] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL2, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL2, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding IL-12. In other examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL2, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding IL-15. In yet another example, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL2, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding CXCL9. In an alternative example, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL2, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding CXCL10.

[0100] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding CCL4, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding IL-12. In other examples, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding IL-15. In yet other examples, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding CXCL9. In an alternative embodiment, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding FLT3L (eg, a gene encoding sFLT3L), and a gene encoding CXCL10.

[0101] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding CCL5, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding IL-12. In other examples, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding IL-15. In yet other examples, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding FLT3L (e.g., a gene encoding sFLT3L), and a gene encoding CXCL9. In an alternative embodiment, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding FLT3L (eg, a gene encoding sFLT3L), and a gene encoding CXCL10.

[0102] In some instances, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) comprises a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding GM-CSF, and a gene encoding a lymphocyte activating cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15). Alternatively, the immune regulatory sequence comprises a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding GM-CSF, and a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In some examples, the immune regulating sequence includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding GM-CSF, and a gene encoding IL-12. In other examples, the immune regulating sequence includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding GM-CSF, and a gene encoding IL-15. In yet other examples, the immune regulating sequence includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding GM-CSF, and a gene encoding CXCL9. In alternative examples, the immune modulatory sequence includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding GM-CSF, and a gene encoding CXCL10.

[0103] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL1, a gene encoding GM-CSF, and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence includes a gene encoding XCL1, a gene encoding GM-CSF, and a gene encoding IL-12. In other examples, the immune regulatory sequence includes a gene encoding XCL1, a gene encoding GM-CSF, and a gene encoding IL-15. In yet other examples, the immune regulatory sequence includes a gene encoding XCL1, a gene encoding GM-CSF, and a gene encoding CXCL9. In alternative examples, the immune regulatory sequence includes a gene encoding XCL1, a gene encoding GM-CSF, and a gene encoding CXCL10.

[0104] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL2, a gene encoding GM-CSF, and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence includes a gene encoding XCL2, a gene encoding GM-CSF, and a gene encoding IL-12. In other examples, the immune regulatory sequence includes a gene encoding XCL2, a gene encoding GM-CSF, and a gene encoding IL-15. In yet other examples, the immune regulatory sequence includes a gene encoding XCL2, a gene encoding GM-CSF, and a gene encoding CXCL9. In alternative examples, the immune regulatory sequence includes a gene encoding XCL2, a gene encoding GM-CSF, and a gene encoding CXCL10.

[0105] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding CCL4, a gene encoding GM-CSF, and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding GM-CSF, and a gene encoding IL-12. In other examples, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding GM-CSF, and a gene encoding IL-15. In yet other examples, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding GM-CSF, and a gene encoding CXCL9. In alternative examples, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding GM-CSF, and a gene encoding CXCL10.

[0106] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding CCL5, a gene encoding GM-CSF, and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding GM-CSF, and a gene encoding IL-12. In other examples, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding GM-CSF, and a gene encoding IL-15. In yet other examples, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding GM-CSF, and a gene encoding CXCL9. In alternative examples, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding GM-CSF, and a gene encoding CXCL10.

[0107] In some instances, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) comprises a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding CD40L, and a gene encoding a lymphocyte activating cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15). Alternatively, the immune regulatory sequence comprises a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding CD40L, and a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In some examples, the immune regulating sequence includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding CD40L, and a gene encoding IL-12. In other examples, the immune regulating sequence includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding CD40L, and a gene encoding IL-15. In yet other examples, the immune regulating sequence includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding CD40L, and a gene encoding CXCL9. In alternative examples, the immune modulatory sequence includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding CD40L, and a gene encoding CXCL10.

[0108] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL1, a gene encoding CD40L, and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence includes a gene encoding XCL1, a gene encoding CD40L, and a gene encoding IL-12. In other examples, the immune regulatory sequence includes a gene encoding XCL1, a gene encoding CD40L, and a gene encoding IL-15. In yet other examples, the immune regulatory sequence includes a gene encoding XCL1, a gene encoding CD40L, and a gene encoding CXCL9. In alternative examples, the immune regulatory sequence includes a gene encoding XCL1, a gene encoding CD40L, and a gene encoding CXCL10.

[0109] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding XCL2, a gene encoding CD40L, and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence includes a gene encoding XCL2, a gene encoding CD40L, and a gene encoding IL-12. In other examples, the immune regulatory sequence includes a gene encoding XCL2, a gene encoding CD40L, and a gene encoding IL-15. In yet other examples, the immune regulatory sequence includes a gene encoding XCL2, a gene encoding CD40L, and a gene encoding CXCL9. In alternative examples, the immune regulatory sequence includes a gene encoding XCL2, a gene encoding CD40L, and a gene encoding CXCL10.

[0110] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding CCL4, a gene encoding CD40L, and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding CD40L, and a gene encoding IL-12. In other examples, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding CD40L, and a gene encoding IL-15. In yet other examples, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding CD40L, and a gene encoding CXCL9. In alternative examples, the immune regulatory sequence includes a gene encoding CCL4, a gene encoding CD40L, and a gene encoding CXCL10.

[0111] In certain examples, the immune regulatory sequence (e.g., an immune regulatory sequence having three transcriptional units, or a tricistronic immune regulatory sequence) includes a gene encoding CCL5, a gene encoding CD40L, and a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a lymphocyte activation cytokine (e.g., a gene encoding IL-12, a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding CD40L, and a gene encoding IL-12. In other examples, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding CD40L, and a gene encoding IL-15. In yet other examples, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding CD40L, and a gene encoding CXCL9. In alternative examples, the immune regulatory sequence includes a gene encoding CCL5, a gene encoding CD40L, and a gene encoding CXCL10.

[0112] In certain embodiments, the immune regulating sequence has three transcription units, where a first promoter is 5' to the gene encoding XCL1, a second promoter is 5' to the gene encoding sFLT3L, and a third promoter is 5' to the gene encoding IL-12. In alternative embodiments, the immune regulating sequence is tricistronic, where a single promoter is 5' to the gene encoding XCL1, the gene encoding sFLT3L, and the gene encoding IL-12.

[0113] In certain embodiments, the immune regulating sequence has three transcription units, where a first promoter is 5' to the gene encoding XCL1, a second promoter is 5' to the gene encoding sFLT3L, and a third promoter is 5' to the gene encoding IL-15. In alternative embodiments, the immune regulating sequence is tricistronic, where a single promoter is 5' to the gene encoding XCL1, the gene encoding sFLT3L, and the gene encoding IL-15.

[0114] Alternatively, in some instances, the immune regulating sequence has three transcription units, where a first promoter is 5' to the gene encoding XCL1, a second promoter is 5' to the gene encoding GM-CSF, and a third promoter is 5' to the gene encoding IL-12. In alternative embodiments, the immune regulating sequence is tricistronic, where a single promoter is 5' to the gene encoding XCL1, the gene encoding GM-CSF, and the gene encoding IL-12.

[0115] In certain embodiments, the immune regulating sequence has three transcription units, where a first promoter is 5' to the gene encoding XCL1, a second promoter is 5' to the gene encoding GM-CSF, and a third promoter is 5' to the gene encoding IL-15, In alternative embodiments, the immune regulating sequence is tricistronic, where a single promoter is 5' to the gene encoding XCL1, the gene encoding GM-CSF, and the gene encoding IL-15.

[0116] In some embodiments of any of the above nucleic acid vectors (e.g., circular DNA vectors), the immune modulatory sequence comprises, operably linked in a 5' to 3' orientation, a gene encoding a dendritic cell chemoattractant, a gene encoding a dendritic cell growth factor or activator, and a gene encoding a lymphocyte signaling protein (e.g., a sequence encoding a replicase, a gene encoding a dendritic cell chemoattractant, a gene encoding a dendritic cell growth factor or activator, and a gene encoding a lymphocyte signaling protein). In other embodiments, the immune modulatory sequence comprises, operably linked in a 5' to 3' orientation, a gene encoding a dendritic cell growth factor or activator, a gene encoding a dendritic cell chemoattractant, and a gene encoding a lymphocyte signaling protein (e.g., a sequence encoding a replicase, a gene encoding a dendritic cell growth factor or activator, a gene encoding a dendritic cell chemoattractant, and a gene encoding a lymphocyte signaling protein). In other embodiments, the immune modulatory sequence comprises, operably linked in a 5' to 3' orientation, a gene encoding a dendritic cell chemoattractant, a gene encoding a lymphocyte signaling protein, and a gene encoding a dendritic cell growth factor or activator (e.g., a sequence encoding a replicase, a gene encoding a dendritic cell chemoattractant, a gene encoding a lymphocyte signaling protein, and a gene encoding a dendritic cell growth factor or activator). In other embodiments, the immune modulatory sequence comprises, operably linked in a 5' to 3' orientation, a gene encoding a dendritic cell growth factor or activator, a gene encoding a lymphocyte signaling protein, and a gene encoding a dendritic cell chemoattractant (e.g., a sequence encoding a replicase, a gene encoding a dendritic cell growth factor or activator, a gene encoding a lymphocyte signaling protein, and a gene encoding a dendritic cell chemoattractant).In other embodiments, the immune modulatory sequence comprises, operably linked in a 5' to 3' orientation, a gene encoding a lymphocyte signaling protein, a gene encoding a dendritic cell chemoattractant, and a gene encoding a dendritic cell growth factor or activator. In other embodiments, the immune modulatory sequence comprises, operably linked in a 5' to 3' orientation, a gene encoding a lymphocyte signaling protein, a gene encoding a dendritic cell chemoattractant, and a gene encoding a dendritic cell growth factor or activator. In other embodiments, the immune modulatory sequence comprises, operably linked in a 5' to 3' orientation, a gene encoding a lymphocyte signaling protein, a gene encoding a dendritic cell growth factor or activator, and a gene encoding a dendritic cell chemoattractant.

[0117] In certain embodiments, the immune modulatory sequence encodes an amino acid sequence having at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% identity, or 100% identity to SEQ ID NO: 34. Additionally or alternatively, in some embodiments, the immune modulatory sequence comprises a nucleic acid sequence having at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% identity, or 100% identity to SEQ ID NO: 33 or SEQ ID NO: 33A.

[0118] In certain examples of any of the above-described embodiments of the invention, the tricistronic immune regulatory sequence comprises no more than three genes.

[0119] The heterologous gene of any of the self-replicating RNA molecules described herein may encode a functionally equivalent fragment of any of the proteins described herein, or variants thereof (e.g., dendritic cell chemoattractants, dendritic cell growth factors or activators, and / or lymphocyte signaling proteins). The fragment of a protein or variant thereof encoded by a self-replicating RNA molecule according to the invention may comprise an amino acid sequence having at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity) with a reference amino acid sequence (e.g., the amino acid sequence of the respective naturally occurring full-length protein or variant thereof).

[0120] Genes encoding other immune regulating proteins useful within the immune regulating sequence include genes encoding other cytokines, chemokines, and growth factors. Other cytokines useful as part of the present invention include TNFα, IFN-γ, IFN-α, TGF-β, IL-1, IL-2, IL-4, IL-10, IL-13, IL-17, and IL-18. Non-limiting examples of chemokines useful as part of the present invention include CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL10.Non-limiting examples of growth factors include adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), macrophage colony-stimulating factor (m-CSF), granulocyte colony-stimulating factor (G-CSF), epidermal growth factor (EGF), ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3, erythropoietin ... EPO, fibroblast growth factor 1 (FGF1), FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, fetal bovine somatotropin (FBS), glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, artemin, Long-term differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatocellular carcinoma-derived growth factor (HDGF), insulin, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, keratinocyte growth factor (KGF), migration stimulating factor (MSF), macrophage stimulating protein (MSP), myostatin (GDF-8), neuregulin 1 (NR G1), NRG2, NRG3, NRG4, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), NT-4, placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

[0121] In any of the polycistronic nucleic acid vectors (e.g., DNA vectors, e.g., circular DNA vectors) described herein, cleavage sites can be designed between protein-coding regions. For example, a Furin-P2A site can separate any of the protein-coding genes described herein. Ribozymes can also be incorporated into the self-replicating RNA molecule to cleave a site downstream of the protein-coding gene. In some embodiments, a T2A, E2A, F2A, or any other suitable self-cleavage site (e.g., a viral-derived cleavage site) can separate any of the protein-coding genes described herein.

[0122] Circular DNA vector Some embodiments of the invention include circular DNA vectors (e.g., circular DNA vectors having multiple transcription units each encoding an immune-modulating gene, circular DNA vectors having a single transcription unit encoding multiple immune-modulating genes, and circular DNA vectors encoding self-replicating RNA molecules encoding a replicase and one or more immune-modulating genes). In some examples, a circular DNA vector useful for encoding an immune-modulating sequence described herein may be a plasmid DNA vector. In certain examples of the invention, a circular DNA vector differs from a conventional plasmid DNA vector in that it lacks a plasmid backbone element (e.g., bacterial elements such as (i) a bacterial origin of replication and / or (ii) a drug resistance gene). In some embodiments, a circular DNA vector described herein (e.g., a DNA vector encoding any of the self-replicating RNA molecules described herein) lacks a recombination site (e.g., a synthetic circular DNA vector made by a cell-free process). In alternative embodiments, a circular DNA vector described herein (e.g., encoding any of the self-replicating RNA molecules described herein) includes a recombination site (e.g., a minicircle DNA vector).

[0123] The circular DNA vector of the present invention may include a promoter operably linked to the 5' of the sequence encoding the self-replicating RNA molecule. A promoter is operably linked to a sequence encoding the self-replicating RNA molecule if the promoter can effect transcription of the sequence encoding the self-replicating RNA molecule. Promoters that can be used as part of the circular DNA vector include constitutive promoters, inducible promoters, native promoters, and tissue-specific promoters. Examples of constitutive promoters include the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with a RSV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter. In certain embodiments of the present invention, the circular DNA vector includes a CMV promoter. In some embodiments, the circular DNA vector includes a CAG promoter.

[0124] Alternatively, the circular DNA vector of the present invention comprises an inducible promoter. Inducible promoters allow the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, or specific physiological conditions, such as acute phase, specific differentiation state of cells, or only in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources. Many other systems have been described and can be easily selected by those skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone-inducible mouse mammary tumor virus promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline repression system, the tetracycline induction system, the RU486 induction system, and the rapamycin induction system. Still other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, specific differentiation state of cells, or only in replicating cells.

[0125] The circular DNA vector of the present invention may contain a polyadenylation sequence 3' of the sequence encoding the self-replicating RNA molecule. Useful polyadenylation sequences include extended polyadenylation sequences of more than 20 nt (e.g., more than 25 nt, more than 30 nt, more than 35 nt, more than 40 nt, more than 50 nt, more than 60 nt, more than 70 nt, or more than 80 nt, e.g., 20 nt to 100 nt, 30 nt to 100 nt, 40 nt to 100 nt, 50 nt to 100 nt, 60 nt to 100 nt, 70 nt to 100 nt, 80 nt to 100 nt, 100 nt to 200 nt, 200 nt to 300 nt, or 300 nt to 400 nt, or more).

[0126] Circular DNA vectors, which lack bacterial elements such as DNA replication origins and / or drug resistance genes, can persist in an individual longer than traditional DNA vectors (e.g., plasmids) and longer than naked RNA. In certain embodiments involving self-replicating RNA, circular DNA vectors provide enhanced stability to the self-replicating RNA molecules encoded therein.

[0127] Circular DNA vectors may have a variety of sizes and shapes. The circular DNA vectors encoding the self-replicating RNA molecules of the present invention may be 5 kb to 20 kb in length (e.g., 6 kb to 18 kb, 7 kb to 16 kb, 8 kb to 14 kb, or 9 kb to 12 kb in length, e.g., 5 kb to 6 kb, 6 kb to 7 kb, 7 kb to 8 kb, 8 kb to 9 kb, 9 kb to 10 kb, 10 kb to 11 kb, 11 kb to 12 kb, 12 kb to 13 kb, 13 kb to 14 kb, 1 kb to 1 ... The length may be 4 kb to 15 kb, 15 kb to 16 kb, 16 kb to 18 kb, or 18 kb to 20 kb, for example, about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 10.5 kb, about 11 kb, about 11.5 kb, about 12 kb, about 12.5 kb, about 13 kb, about 14 kb, about 15 kb, about 16 kb, about 17 kb, about 18 kb, about 19 kb, or about 20 kb.

[0128] Circular DNA vectors useful as part of the present invention can be readily synthesized by various means known in the art and described herein. For example, circular DNA vectors lacking plasmid backbone elements (e.g., bacterial elements such as (i) bacterial origin of replication and / or (ii) drug resistance genes) can be made using in vitro (cell-free) methods that can result in purer compositions compared to bacterial-based methods. Such in vitro synthesis methods can involve, for example, the use of phage polymerases such as Phi29 polymerase as a replication tool using rolling circle amplification. Certain methods of in vitro synthesis of circular DNA vectors are further described in WO 2019 / 178500, which is incorporated herein by reference.

[0129] Self-replicating RNA molecules and DNA vectors encoding the same Provided herein are self-replicating RNA molecules useful as regulatory therapies, e.g., immunomodulatory therapies. When delivered to a vertebrate cell, the self-replicating RNA molecule can result in the production of multiple daughter RNAs by transcription from itself (through antisense copies generated from itself). In some instances, the self-replicating RNA molecule is a positive-stranded molecule that can be directly translated after delivery to a cell, which translation produces a replicase that then produces antisense and sense transcripts from the delivered RNA. This results in the delivery of multiple daughter RNAs. These daughter RNAs, like subgenomic transcripts, can be translated to result in the in situ expression of the encoded protein (e.g., a regulatory protein, e.g., an immunomodulatory protein), or can be transcribed to give additional transcripts with the same sense as the delivered RNA that are translated to result in the in situ expression of a regulatory protein. The overall result of this series of transcriptions is an amplification of the number of self-replicating RNA molecules, and the encoded regulatory protein can become the major polypeptide product of the target cell (e.g., tumor cells and / or tumor-resident cells).

[0130] In some instances, any of the above-mentioned DNA vectors (e.g., circular DNA vectors) encode a self-replicating RNA molecule that includes a regulatory sequence. Such a self-replicating RNA molecule is characterized by having a positive-strand replicon that includes a replicase sequence derived from an alphavirus, which is translated into a replicase (or replicase-transcriptase) after delivery to a target cell. The replicase is translated as a polyprotein that self-cleaves to give a replication complex that creates a genomic negative-strand copy of the delivered positive-strand RNA. These negative-strand transcripts can themselves be transcribed to give further copies of the positive-strand parent RNA and to give subgenomic transcripts (e.g., regulatory sequences). Thus, translation of the subgenomic transcripts results in in situ expression of the regulatory protein by the infected cell.

[0131] Non-limiting examples of alphaviruses from which the replicase-encoding sequences of the invention may be derived include Venezuelan equine encephalitis virus (VEE), Semliki Forest virus (SF), Sindbis virus (SIN), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Everglades virus (EVE), Mucambo virus (MUC), Pixuna virus (PIX), Semliki Forest virus (SF), Middelburg virus (MID), Chikungunya virus (CHIK), O'Nyong-nyong virus (ONN), Ross River virus (RR), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAG), Bebaru virus (BEB), Mayaro virus (MAY), Una virus (UNA), Aura virus (AURA), Babanki virus (BAB), Highlands J virus (HJ), and Fort Morgan virus (FM). In certain examples of the invention, the self-replicating RNA molecule comprises a VEE replicase or a variant thereof. In some embodiments, the self-replicating RNA molecule comprises a sequence encoding the replicase of SEQ ID NO:1 or SEQ ID NO:1A. In some embodiments, the self-replicating RNA molecule comprises a sequence encoding the replicase comprising a nucleic acid sequence at least 95% identical to SEQ ID NO:1 or SEQ ID NO:1A (e.g., at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to SEQ ID NO:1 or SEQ ID NO:1A). In some embodiments, the self-replicating RNA molecule comprises a sequence encoding a replicase that comprises a nucleic acid sequence at least 95% identical to SEQ ID NO:3 or SEQ ID NO:3A (e.g., at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to SEQ ID NO:3 or SEQ ID NO:3A).In some embodiments, the self-replicating RNA molecule comprises a sequence encoding a replicase that comprises a nucleic acid sequence at least 95% identical to SEQ ID NO:5 or SEQ ID NO:5A (e.g., at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to SEQ ID NO:5 or SEQ ID NO:5A). In some embodiments, the self-replicating RNA molecule comprises a sequence encoding a replicase that comprises a nucleic acid sequence at least 95% identical to SEQ ID NO:7 or SEQ ID NO:7A (e.g., at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to SEQ ID NO:7 or SEQ ID NO:7A). In some embodiments, the self-replicating RNA molecule comprises a sequence encoding a replicase that includes a first nucleic acid sequence at least 95% identical to SEQ ID NO:1 or SEQ ID NO:1A (at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:1 or SEQ ID NO:1A), a second nucleic acid sequence at least 95% identical to SEQ ID NO:3 or SEQ ID NO:3A (at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3 or SEQ ID NO:3A), a third nucleic acid sequence at least 95% identical to SEQ ID NO:5 or SEQ ID NO:5A (at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:5 or SEQ ID NO:5A), and a fourth nucleic acid sequence at least 95% identical to SEQ ID NO:7 or SEQ ID NO:7A (at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:7 or SEQ ID NO:7A). In some embodiments, the self-replicating RNA molecule comprises a nucleic acid sequence comprising SEQ ID NO:1, SEQ ID NO:1A, SEQ ID NO:3, SEQ ID NO:3A, SEQ ID NO:5, SEQ ID NO:5A, SEQ ID NO:7, and SEQ ID NO:7A.In some embodiments, the replicase-encoding sequence encodes an amino acid sequence that is at least 95% identical to SEQ ID NO:2 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to SEQ ID NO:2). In some embodiments, the replicase-encoding sequence encodes an amino acid sequence that is at least 95% identical to SEQ ID NO:4 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to SEQ ID NO:4). In some embodiments, the replicase-encoding sequence encodes an amino acid sequence that is at least 95% identical to SEQ ID NO:6 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to SEQ ID NO:6). In some embodiments, the replicase-encoding sequence encodes an amino acid sequence that is at least 95% identical to SEQ ID NO:8 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to SEQ ID NO:8).In some embodiments, the replicase-encoding sequence encodes a first amino acid sequence at least 95% identical to SEQ ID NO:2 (at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2), a second amino acid sequence at least 95% identical to SEQ ID NO:4 (at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4), a third amino acid sequence at least 95% identical to SEQ ID NO:6 (at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:6), and a fourth amino acid sequence at least 95% identical to SEQ ID NO:8 (at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:8). In some embodiments, the replicase-encoding sequence encodes an amino acid sequence comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

[0132] Mutant or wild-type viral sequences can be used. For example, in some embodiments, the self-replicating RNA comprises the attenuated TC83 mutant of VEE replicase. Other mutations in replicase are contemplated herein, including replicase mutant replicase (e.g. mutant VEE replicase) obtained by in vitro generation method, for example, as taught in Yingzhong et al., Sci Rep. 2019, 9: 6932 (the methodology of which is incorporated herein by reference).

[0133] In some instances, the self-replicating RNA molecule comprises (i) a sequence encoding a replicase (e.g., an RNA sequence encoding an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule) and (ii) a heterologous regulatory gene. The polymerase can be an alphavirus replicase, e.g., an alphavirus replicase that includes one, two, three, or all four alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4. In some instances, the polymerase is a VEE replicase, e.g., a VEE replicase that includes one, two, three, or all four alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4.

[0134] In some examples of the invention, the self-replicating RNA molecule does not encode an alphavirus structural protein (e.g., capsid protein). Such a self-replicating RNA may result in the production of its own genomic RNA copy in a cell, but cannot result in the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecule is unable to persist itself in an infectious form. The alphavirus structural proteins can be replaced with a gene(s) encoding the heterologous regulatory protein(s) of interest such that the subgenomic transcript encodes the heterologous regulatory protein(s) rather than the structural alphavirus virion proteins.

[0135] Thus, in some instances, a self-replicating RNA molecule of the invention may have two open reading frames: a first (5') open reading frame that encodes a replicase and a second (3') open reading frame that encodes one or more (e.g., two or three) heterologous regulatory proteins (e.g., any of the immunomodulatory proteins described herein, or a combination thereof). In some embodiments, the RNA may have additional (e.g., downstream) open reading frames, e.g., to encode additional heterologous genes or to encode auxiliary polypeptides.

[0136] Suitable self-replicating RNA molecules may have a variety of lengths. In some embodiments of the invention, the length of the self-replicating RNA molecule is between 5,000 nucleotides and 50,000 nucleotides (i.e., between 5 kb and 50 kb). In some examples, the self-replicating RNA molecule is between 5 kb and 20 kb in length (e.g., between 6 kb and 18 kb, between 7 kb and 16 kb, between 8 kb and 14 kb, or between 9 kb and 12 kb in length, e.g., between 5 kb and 6 kb, between 6 kb and 7 kb, between 7 kb and 8 kb, between 8 kb and 9 kb, between 9 kb and 10 kb, between 10 kb and 11 kb, between 11 kb and 12 kb, between 12 kb and 13 kb, between 13 kb and 14 kb, between 14 kb and 15 kb). The length of the nucleic acid sequence is about 15 kb, 15 kb to 16 kb, 16 kb to 18 kb, or 18 kb to 20 kb, for example, about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 10.5 kb, about 11 kb, about 11.5 kb, about 12 kb, about 12.5 kb, about 13 kb, about 14 kb, about 15 kb, about 16 kb, about 17 kb, about 18 kb, about 19 kb, or about 20 kb.

[0137] The self-replicating RNA molecule may have a 3' poly A tail. Additionally, the self-replicating RNA molecule may contain a poly A polymerase recognition sequence (e.g., AAUAAA).

[0138] Self-replicating RNA molecules can be produced by any method known in the art or described herein, for example, in vitro transcription (IVT). IVT can use (cDNA) templates produced and propagated in bacteria in plasmid form or produced synthetically (e.g., by gene synthesis and / or polymerase chain reaction (PCR) engineering methods). For example, a DNA-dependent RNA polymerase (such as bacteriophage T7, T3 or SP6 RNA polymerase) can be used to transcribe the RNA from a DNA template. Appropriate capping and polyA addition reactions can be used as needed (although the polyA of the replicon is usually encoded within the DNA template). These RNA polymerases can have strict requirements for the 5' nucleotide(s) transcribed, and in some embodiments, these requirements must be matched with those of the encoded replicase to ensure that the IVT-transcribed RNA can function efficiently as a substrate for the self-encoded replicase.

[0139] In some instances, the self-replicating RNA may include (in addition to any 5' cap structure) one or more nucleotides having a modified nucleobase.For example, self-replicating RNA molecules can be expressed as m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N-6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine), m2A (2-methyladenosine), Am (2'-β-methyladenosine), ms2m6A (2-methylthio-N-6-methyladenosine), i6A (N-6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io6A (N-6-isopenten ... (cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N-6-(cis-hydroxyisopentenyl)adenosine), g6A (N-6-glycinylcarbamoyl adenosine), t6A (N-6-threonylcarbamoyl adenosine), ms2t6A (2-methylthio-N-6-threonylcarbamoyl adenosine), m6t6A (N-6-methyl-N-6-threonylcarbamoyl adenosine), hn6A (N6-hydroxynorvalylcarbamoyl adenosine), ms2hn6A (2- methylthio-N-6-hydroxynorvalylcarbamoyl adenosine), Ar(p)(2'-O-ribosyladenosine (phosphate)), I(inosine), m11(1-methylinosine), m'Im(1,2'-O-dimethylinosine), m3C(3-methylcytidine), Cm(2T-O-methylcytidine), s2C(2-thiocytidine), ac4C(N-4-acetylcytidine), f5C(5-formylcytidine), m5Cm(5,2-O-dimethylcytidine), ac4Cm(N4-acetyl-2T-O-methylcytidine) ), k2C (lysidine), m1G (1-methylguanosine), m2G (N2-methylguanosine), m7G (7-methylguanosine), Gm (2'-O-methylguanosine), m22G (N2,N2-dimethylguanosine), m2Gm (N2,2'-O-dimethylguanosine), m22Gm (N2,N2,2'-β-trimethylguanosine), Gr(p) (2'-O-ribosylguanosine (phosphate)), yW (wibutosine), o2yW (peroxywibutosine), OHyW (hydroxywibutosine), OHyW. *(undermodified hydroxywibutosine), imG (wibutosine), mimG (methylguanosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G (archaeosine), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-1,2'-dimethyluridine), m5s3U (5-methyl-1,2'-dimethyluridine), m5s4U (5-methyl-1,2'-dimethyluridine), m5s5U (5-methyl-1,2'-dimethyluridine), m5s6U (5-methyl-1,2'-dimethyluridine), m5s7U (5-methyl-1,2'-dimethyluridine), m5s8U (5-methyl-1,2'-dimethyluridine), m5s9U (5-methyl-1,2'-dimethyluridine), m5s1U (5-methyl-1,2'-dimethyluridine), m5s2 ... (2-thio-2'-O-methyluridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl)uridine)), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), uridine), mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5s2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (5-carbamoylmethyl-2'-O-methyluridine), c mnm5U (5-carboxymethylaminomethyluridine), cnmm5Um (5-carboxymethylaminomethyl-2-LO-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m62A (N6,N6-dimethyladenosine), Tm (2'-O-methylinosine), m4C (N-4-methylcytidine), m4Cm (N4,2-O-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6 Am (N6,TO-dimethyladenosine), rn62 Am (N6,N6,O-2-trimethyladenosine), m2'7G (N2,7-dimethylguanosine), m2'2'7G (N2,N2,7-trimethylguanosine), m3Um (3,2T-O-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), m1Gm (1,2'-O-dimethylguanosine), m'Am ((1,2-O-dimethyladenosine) irinomethyluridine )), tm5s2U (S-taurinomethyl-2-thiouridine), imG-14 (4-demethylguanosine), imG2 (isoguanosine), or ac6A (N-6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkane-2-yl ... Nyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine , 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, or abasic nucleotides. For example, the self-replicating RNA molecule may include one or more modified pyrimidine nucleobases, such as pseudouridine residues and / or 5-methylcytosine residues.

[0140] In other embodiments of the invention, the self-replicating RNA molecule is substantially free of nucleotides having modified nucleases, for example, in some instances, the self-replicating RNA molecule is free of modified nucleobases and may be free of modified nucleotides, i.e., all of the nucleotides in the RNA are standard A, C, G and U ribonucleotides (except for any 5' cap structure, which may include 7'-methylguanosine).

[0141] In some instances, the self-replicating RNA contains only phosphodiester bonds between nucleosides. In some instances, the self-replicating RNA contains phosphoramidate, phosphorothioate, and / or methylphosphonate linkages. In any of the polycistronic self-replicating RNA molecules described herein, a cleavage site can be engineered between the protein coding regions.

[0142] In certain embodiments, the RNA according to the invention does not encode a reporter molecule, such as luciferase, or a fluorescent protein, such as green fluorescent protein (GFP).

[0143] In some embodiments, the heterologous protein encoded by the self-replicating RNA is a variant of any of the heterologous proteins described herein. Additionally or alternatively, the replicase encoded by the self-replicating RNA can be a variant of any of the replicases described herein. In some embodiments, the variant is a functional fragment (e.g., a fragment of a protein that is functionally similar or functionally equivalent to the protein).

[0144] Pharmaceutical Compositions Provided herein is a pharmaceutical composition comprising any of the nucleic acid vectors (e.g., circular DNA vectors or self-replicating RNA molecules) described herein in a pharma- ceutically acceptable carrier. Thus, in some examples, the pharmaceutical composition of the present invention comprises a nucleic acid vector (e.g., circular DNA vectors or self-replicating RNA molecules) comprising an immune regulatory sequence (e.g., an immune regulatory sequence having multiple transcription units, or a polycistronic immune regulatory sequence) including a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4) and a gene encoding one or more immune regulatory proteins (e.g., a gene encoding a growth factor or activator for dendritic cells (e.g., a gene encoding FLT3L, a gene encoding GMCSF, or a gene encoding CD40L) or a gene encoding a signaling protein for lymphocytes (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)) in a pharma- ceutically acceptable carrier.

[0145] In other examples, the pharmaceutical compositions of the invention comprise one or more nucleic acid vectors (e.g., one or more circular DNA vectors, each encoding a different immune-modulating gene, e.g., a heterogeneous nucleic acid vector (e.g., a circular DNA vector)). For example, in such examples, the pharmaceutical compositions of the invention comprise two or more (e.g., two or three) nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, wherein each vector comprises an immune-modulating sequence (e.g., a monocistronic immune-modulating sequence) comprising a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), a gene encoding a dendritic cell growth factor or activator (e.g., a gene encoding FLT3L, a gene encoding GMCSF, or a gene encoding CD40L), or a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)).

[0146] In some embodiments, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) a first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., a monocistronic immune regulating sequence) including a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4) or a gene encoding a growth factor or activator for dendritic cells (e.g., a gene encoding FLT3L, a gene encoding GMCSF, or a gene encoding CD40L), and (ii) a second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)).

[0147] In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) a first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., a monocistronic immune regulating sequence) including a gene encoding a dendritic cell chemoattractant (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), and (ii) a second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-12.In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-15. In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL9. In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) a first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., a monocistronic immune regulating sequence) that comprises a gene encoding XCL1, and (ii) a second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL10.

[0148] In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL2, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL2, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-12. In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL2, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-15. In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL2, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL9.In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) a first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., a monocistronic immune regulating sequence) that comprises a gene encoding XCL2, and (ii) a second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL10.

[0149] In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding CCL4, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma-ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding CCL4, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-12. In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding CCL4, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-15. In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding CCL4, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL9.In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) a first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., a monocistronic immune regulating sequence) that comprises a gene encoding CCL4, and (ii) a second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL10.

[0150] In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding CCL5, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In some examples, the pharmaceutical composition comprises a nucleic acid vector (e.g., circular DNA vector) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding CCL5, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-12. In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding CCL5, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-15. In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding CCL5, and (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL9.In some examples, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) a first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., a monocistronic immune regulating sequence) that comprises a gene encoding CCL5, and (ii) a second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL10.

[0151] In some embodiments, the pharmaceutical composition comprises two nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) a first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., a monocistronic immune regulating sequence) including a gene encoding a growth factor or activator of dendritic cells (e.g., a gene encoding FLT3L, a gene encoding GMCSF, or a gene encoding CD40L), and (ii) a second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)).

[0152] In some embodiments, the pharmaceutical composition comprises three nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, wherein (i) a first nucleic acid vector (e.g., circular DNA vector) comprises (a) an immune regulating sequence (e.g., a monocistronic immune regulating sequence) including a gene encoding a dendritic cell chemoattractant (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4); (ii) a second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding a dendritic cell growth factor or activator (e.g., a gene encoding FLT3L, a gene encoding GMCSF, or a gene encoding CD40L); and (iii) a third nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)).

[0153] In some embodiments, the pharmaceutical composition comprises three nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) a first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., a monocistronic immune regulating sequence) including a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding FLT3L, and (iii) a third nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some embodiments, the pharmaceutical composition comprises three nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) a first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., a monocistronic immune regulating sequence) including a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding GM-CSF, and (iii) a third nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)).

[0154] In some embodiments, the pharmaceutical composition comprises three nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding FLT3, and (iii) the third nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-12. In some embodiments, the pharmaceutical composition comprises three nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding FLT3, and (iii) the third nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-15. In some embodiments, the pharmaceutical composition comprises three nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding FLT3, and (iii) the third nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL9. In some embodiments, the pharmaceutical composition comprises three nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding FLT3, and (iii) the third nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL10.

[0155] In some embodiments, the pharmaceutical composition comprises three nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding GM-CSF, and (iii) the third nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-12. In some embodiments, the pharmaceutical composition comprises three nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding GM-CSF, and (iii) the third nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding IL-15. In some embodiments, the pharmaceutical composition comprises three nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding GM-CSF, and (iii) the third nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL9. In some embodiments, the pharmaceutical composition comprises three nucleic acid vectors (e.g., circular DNA vectors) in a pharma- ceutically acceptable carrier, where (i) the first nucleic acid vector (e.g., circular DNA vector) comprises an immune regulating sequence (e.g., monocistronic immune regulating sequence) comprising a gene encoding XCL1, (ii) the second nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding GM-CSF, and (iii) the third nucleic acid vector (e.g., circular DNA vector) comprises a gene encoding CXCL10.

[0156] In any of the above described pharmaceutical compositions, the immune modulatory sequence can be within a self-replicating RNA coding sequence that includes a replicase coding sequence that transcribes RNA from the self-replicating RNA molecule.

[0157] In some examples, the pharmaceutical composition comprises two self-replicating RNA molecules in a pharma- ceutically acceptable carrier, where (i) the first self-replicating RNA molecule comprises (a) a sequence encoding a replicase that transcribes RNA from the self-replicating RNA molecule, and (b) an immune regulating sequence (e.g., a monocistronic immune regulating sequence) comprising a gene encoding a dendritic cell chemoattractant (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), and (ii) the second self-replicating RNA molecule comprises (a) a sequence encoding a replicase that transcribes RNA from the self-replicating RNA molecule, and (b) a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)).

[0158] In some embodiments, the pharmaceutical composition comprises three self-replicating RNA molecules in a pharma- ceutically acceptable carrier, where (i) a first self-replicating RNA molecule comprises (a) a sequence encoding a replicase that transcribes RNA from the self-replicating RNA molecule, and (b) an immune-regulating sequence (e.g., a monocistronic immune-regulating sequence) that comprises a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), and (ii) a second self-replicating RNA molecule comprises (a) a sequence encoding a replicase that transcribes RNA from the self-replicating RNA molecule, and and (b) a gene encoding a growth factor or activator of dendritic cells (e.g., a gene encoding FLT3L, a gene encoding GMCSF, or a gene encoding CD40L), and (iii) the third self-replicating RNA molecule comprises (a) a sequence encoding a replicase that transcribes RNA from the self-replicating RNA molecule, and (b) a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). A pharmaceutically acceptable carrier may include excipients and / or stabilizers that are non-toxic to an individual at the dosages and concentrations used. In some embodiments, the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharma- ceutically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as tweens, polyethylene glycols (PEGs), and pluronics.

[0159] The pharmaceutical composition having the self-replicating RNA molecule of the present invention may contain a pharma- ceutically acceptable carrier. When the composition is provided in liquid form, the carrier may be water (e.g., pyrogen-free water), isotonic saline, or an aqueous buffer solution, such as a phosphate buffer solution or a citrate buffer solution. The injection of the pharmaceutical composition may be performed in water or a buffer solution, such as an aqueous buffer solution containing, for example, a sodium salt (e.g., at least 50 mM of a sodium salt), a calcium salt (e.g., at least 0.01 mM of a calcium salt), or a potassium salt (e.g., at least 3 mM of a potassium salt). According to certain embodiments, the sodium, calcium, or potassium salts may occur in the form of their halides, such as chlorides, iodides, or bromides, their hydroxides, carbonates, bicarbonates, or sulfates, etc. Examples of sodium salts include, but are not limited to, NaCl, NaI, NaBr, Na2CO2, NaHCO2, and Na2SO4. Examples of potassium salts include, for example, KCl, KI, KBr, K2CO2, KHCO2, and K2SO4. Examples of calcium salts include, for example, CaCl2, CaI2, CaBr2, CaCO2, CaSO4, and Ca(OH)2. Furthermore, organic anions of the above-mentioned cations may be contained in the buffer. According to a particular embodiment, the buffer suitable for injection purposes as defined above may contain a salt selected from sodium chloride (NaCl), calcium chloride (CaCl2) or potassium chloride (KCl), and further anions may be present. CaCl2 may also be replaced by another salt, such as KCl. In some embodiments, the salts in the injection buffer are present in a concentration of at least 50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl), and at least 0.01 mM calcium chloride (CaCl2). The injection buffer may be hypertonic, isotonic or hypotonic relative to a particular reference medium, i.e. the buffer may have a higher, the same or a lower salt content relative to a particular reference medium, preferably using the abovementioned salts in concentrations that do not cause cell damage due to osmotic or other concentration effects. The reference medium may be a liquid such as blood, lymph, intercellular fluid, other body fluids or a common buffer solution.Such common buffers or fluids are known to those of skill in the art, with lactated Ringer's solution being particularly preferred as the liquid base.

[0160] One or more compatible solid or liquid fillers, diluents, or encapsulating compounds may be suitable for administration to a person. The components of the pharmaceutical composition according to the invention may be mixed with the nucleic acid vector according to the invention as defined herein in such a way that there is no interaction that would significantly reduce the pharmaceutical efficacy of the (pharmaceutical) composition according to the invention under typical conditions of use. Pharmaceutically acceptable carriers, fillers and diluents may have a sufficiently high purity and a sufficiently low toxicity to make them suitable for administration to the individual being treated. Some examples of compounds that can be used as pharma- ceutically acceptable carriers, fillers, or components thereof are sugars such as lactose, glucose, trehalose, and sucrose; starches such as corn starch or potato starch; dextrose; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and oil derived from cocoa; polyols such as polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; or alginic acid.

[0161] The choice of pharma- ceutically acceptable carrier can depend on the way the pharmaceutical composition is to be administered.

[0162] Suitable unit dosage forms for injection include sterile solutions of water, saline, and mixtures thereof. The pH of such solutions can be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid, and collagen matrices. Suitable pharma- ceutically acceptable carriers for topical application include those suitable for use in lotions, creams, gels, etc. For oral administration of pharmaceutical compositions, tablets, capsules, etc. are preferred unit dosage forms.

[0163] Further additives which may be included in the pharmaceutical composition are emulsifiers such as tweens, wetting agents such as sodium lauryl sulfate, colorants, pharmaceutical carriers, stabilizers, antioxidants, and preservatives.

[0164] The pharmaceutical composition according to the present invention can be provided in liquid or dry (e.g., lyophilized) form. In a particular embodiment, the nucleic acid vector of the pharmaceutical composition is provided in lyophilized form. The lyophilized composition comprising the nucleic acid vector of the present invention can be reconstituted before administration in a suitable buffer, preferably based on an aqueous carrier, such as lactated Ringer's solution, Ringer's solution, or phosphate buffer solution.

[0165] In certain embodiments of the invention, any of the nucleic acid vectors of the invention can be complexed with one or more cationic or polycationic compounds, such as cationic or polycationic polymers, cationic or polycationic peptides or proteins, such as protamine, cationic or polycationic polysaccharides, and / or cationic or polycationic lipids.

[0166] According to certain embodiments, the nucleic acid vectors of the invention can be complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles. Thus, in one embodiment, the compositions of the invention comprise liposomes, lipoplexes, and / or lipid nanoparticles comprising the nucleic acid vector.

[0167] Lipid-based formulations can be effective delivery systems for nucleic acid vectors due to their biocompatibility and ease of their large-scale production. Cationic lipids have been widely studied as synthetic materials for the delivery of nucleic acids. After mixing, the nucleic acid is condensed by the cationic lipid to form lipid / nucleic acid complexes known as lipoplexes. These lipid complexes can protect the genetic material from the action of nucleases and deliver the genetic material into cells by interacting with the negatively charged cell membrane. Lipoplexes can be created by directly mixing positively charged lipids with negatively charged nucleic acids at physiological pH.

[0168] Conventional liposomes contain a lipid bilayer that may be composed of cationic, anionic, or neutral phospholipids and cholesterol, which surrounds an aqueous core. Both the lipid bilayer and the aqueous space can incorporate hydrophobic or hydrophilic compounds, respectively. The properties and in vivo behavior of liposomes can be modified by adding hydrophilic polymer coatings, such as polyethylene glycol (PEG), to the liposome surface to provide steric stabilization. Furthermore, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the ends of the attached PEG chains.

[0169] Liposomes are colloidal lipid- and surfactant-based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment. Liposomes can exist as spherical vesicles and can range in size from 20 nm to several microns. Cationic lipid-based liposomes can be complexed with negatively charged nucleic acids through electrostatic interactions, resulting in complexes that provide the biocompatibility, low toxicity, and potential for large-scale production required for in vivo clinical applications. Liposomes can fuse with the plasma membrane for uptake. Once inside the cell, the liposomes are processed by the endocytic pathway and the genetic material is subsequently released from the endosome / carrier into the cytoplasm.

[0170] Cationic liposomes can function as a delivery system for RNA. Cationic lipids such as MAP, (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate) can form complexes or lipoplexes with negatively charged nucleic acids through electrostatic interactions to form nanoparticles, resulting in high in vitro transfection efficiency. In addition, neutral lipid-based nanoliposomes for nucleic acid vector delivery are available, such as neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based nanoliposomes.

[0171] Thus, in one embodiment of the present invention, the nucleic acid vectors of the present invention are complexed with cationic lipids and / or neutral lipids, thereby forming liposomes, lipid nanoparticles, lipoplexes or neutral lipid-based nanoliposomes.

[0172] In a particular embodiment, the pharmaceutical composition according to the invention comprises a nucleic acid vector of the invention formulated with a cationic or polycationic compound and / or a polymeric carrier.Accordingly, in a further embodiment of the invention, the nucleic acid vector as defined herein is optionally formulated with a ratio of the nucleic acid vector and the cationic or polycationic compound selected from the range of about 5:1 (w / w) to about 0.25:1 (w / w), such as about 5:1 (w / w) to about 0.5:1 (w / w), such as about 4:1 (w / w) to about 1:1 (w / w) or about 3:1 (w / w) to about 1:1 (w / w), such as about 3:1 (w / w) to about 2:1 (w / w). and / or polymeric carrier, or optionally with a nitrogen / phosphate (N / P) ratio between the nucleic acid vector and the cationic or polycationic compound and / or polymeric carrier in the range of about 0.1 to 10, e.g., about 0.3 to 4 or 0.3 to 1, e.g., about 0.5 to 1 or 0.7 to 1, e.g., about 0.3 to 0.9 or 0.5 to 0.9. For example, the N / P ratio between the nucleic acid vector and one or more polycations is in the range of about 0.1 to 10, including the ranges of about 0.3 to 4, about 0.5 to 2, about 0.7 to 2, and about 0.7 to 1.5.

[0173] The nucleic acid vectors described herein may also be associated with vehicles, transfection agents or complexing agents to increase transfection efficiency and / or expression of the regulated genes according to the present invention.

[0174] In some instances, the nucleic acid vectors of the present invention are complexed with one or more polycations, preferably protamine or oligofectamine. Further cationic or polycationic compounds which may be used as transfection or complexing agents include cationic polysaccharides such as chitosan, polybrene, cationic polymers such as polyethyleneimine (PEI), cationic lipids such as DOTMA: [1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPE, LEAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristoxypropyldimethylhydroxyethylammonium bromide, MAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diene, ethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers such as modified polyamino acids, such as β-amino acid polymers or inverse polyamides, modified polyethylenes, such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), modified acrylates, such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), modified amidoamines, such as pAMAM (poly(amidoamine)), modified polyβ-aminoesters (PBAE), such as diamine end-modified 1,Examples of the polymer include 4-butanediol diacrylate-co-5-amino-1-pentanol polymers, dendrimers such as polypropylamine dendrimers or pAMAM-based dendrimers, polyimines such as PEI (poly(ethyleneimine), poly(propyleneimine), polyallylamine, sugar-based polymers such as cyclodextrin-based polymers, dextran-based polymers, chitosan, silane-based polymers such as PMOXA-PDMS copolymers, and block polymers consisting of a combination of one or more cationic blocks (e.g., selected from the cationic polymers described above) and one or more hydrophilic or hydrophobic blocks (e.g., polyethylene glycol).

[0175] According to a particular embodiment, the pharmaceutical composition of the invention comprises a nucleic acid vector (e.g., a circular DNA vector) encapsulated in or attached to a polymeric carrier. The polymeric carrier used according to the invention may be a polymeric carrier formed by disulfide-bridged cationic moieties. The disulfide-bridged cationic moieties may be the same as each other or different. The polymeric carrier may also contain further components. It is also particularly preferred that the polymeric carrier used according to the invention comprises a mixture of cationic peptides, proteins or polymers, crosslinked by disulfide bonds as described herein, and optionally further components as defined herein. In this context, the disclosure of WO 2012 / 013326 is incorporated herein by reference. In this context, the cationic moiety forming the basis of the polymeric carrier by disulfide bridges is usually selected from any suitable cationic or polycationic peptide, protein or polymer suitable for this purpose, in particular any cationic or polycationic peptide, protein or polymer capable of complexing with the further nucleic acid contained in the nucleic acid vector or composition defined herein, thereby preferably condensing the nucleic acid vector. The cationic or polycationic peptides, proteins or polymers may be linear molecules, although branched cationic or polycationic peptides, proteins or polymers can also be used.

[0176] All disulfide-bridged cationic or polycationic proteins, peptides or polymers of the polymeric carrier that may be used to complex the nucleic acid vectors according to the invention, included as part of the pharmaceutical composition of the present invention, may comprise at least one SH moiety (e.g. at least one cysteine ​​residue, or any further chemical group that exhibits an SH moiety) capable of forming a disulfide bond upon condensation with at least one further cationic or polycationic protein, peptide or polymer as the cationic component of the polymeric carrier as mentioned herein.

[0177] Such polymeric carriers used to complex the nucleic acid vectors of the invention may be formed by disulfide-bridged cationic (or polycationic) components. In particular, such cationic or polycationic peptides or proteins or polymers of the polymeric carriers containing at least one SH moiety or additionally modified to contain at least one SH moiety may be selected from proteins, peptides and polymers as complexing agents.

[0178] In other embodiments, the pharmaceutical compositions according to the present invention may be administered naked, without association with any additional vehicles, transfection agents, or complexing agents.

[0179] The pharmaceutical compositions provided herein may contain two or more active ingredients as necessary for the particular cancer being treated. For example, in some instances, any of the pharmaceutical compositions described herein may contain an additional therapeutic agent, such as an anti-cancer agent (e.g., a chemotherapeutic agent, a checkpoint inhibitor, a cytotoxic agent, a growth inhibitor, an anti-angiogenic agent, a cytokine, a cytokine antagonist, an antibody drug conjugate, a cancer vaccine, or a combination thereof). In certain embodiments, the additional therapeutic agent is a checkpoint inhibitor, such as a PD-1 axis binding antagonist (e.g., an anti-PD-1 antagonist or an anti-PD-L1 antibody), such as pembrolizumab (MK-3475), nivolumab (ONO-4538 / BMS-936558, MDX1106), pidilizumab (CT-011), atezolizumab (MPDL3280A), or AMP-224).In some embodiments, the anti-cancer agent is a BCL-2 inhibitor (such as GDC-0199 / ABT-199), lenalidomide (REVLIMID™), a PI3K-δ inhibitor (such as idelalisib (ZYDELIG™)), an agonist (e.g., an activating costimulatory molecule, such as CD40, CD226, CD28, OX40 (e.g., AgonOX), GITR, CD137 (TNFRSF9, 4-1BB, or ILA), CD27 (e.g., CDX-1127), HVEM, or agonist antibodies against CD127), antagonists, e.g., inhibitory costimulatory molecules, e.g., CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1-methyl-D-tryptophan (also known as 1-D-MT), antagonists against TGFβ, such as methimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells (e.g., cytotoxic T cells, or CTLs) expressing a chimeric antigen receptor (CAR), for example, adoptive transfer of T cells comprising a dominant negative TGFβ receptor, e.g., a dominant negative TGFβ type II receptor.

[0180] III. Treatment method Provided herein are methods of treating cancer in an individual in need of treatment by administering to the individual a therapeutically effective amount of either a nucleic acid vector (e.g., a circular DNA vector or a self-replicating RNA molecule), or a composition thereof. In certain instances, the therapeutic effect of the nucleic acid vector (e.g., immune modulation of the tumor microenvironment) occurs following delivery of an electric field to the tumor microenvironment.

[0181] Administration of Nucleic Acid Vectors and Compositions Thereof The invention includes administering the nucleic acid vectors described herein (e.g., circular DNA vectors and self-replicating RNA molecules), or pharmaceutical compositions thereof, to an individual with cancer (e.g., a human cancer patient). In some embodiments, the nucleic acid vector (e.g., circular DNA vector) administered (e.g., as part of a pharmaceutical composition or any of the therapeutic methods described herein) is any of the polycistronic nucleic acid vectors described above. For example, the methods of the invention include administering a polycistronic (e.g., bicistronic or tricistronic) nucleic acid vector comprising a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4) and a gene encoding one or more immunomodulatory proteins (e.g., a gene encoding a growth factor or activator of dendritic cells (e.g., a gene encoding FLT3L, a gene encoding GMCSF, or a gene encoding CD40L) or a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). The method of the present invention includes administering to an individual (e.g., a human cancer patient) an immune modulatory nucleic acid vector comprising a tricistronic immune modulatory sequence comprising a gene encoding XCL1, a gene encoding FLT3L, and a gene encoding IL-12; an immune modulatory nucleic acid vector comprising a tricistronic immune modulatory sequence comprising a gene encoding XCL1, a gene encoding FLT3L, and a gene encoding IL-15; or an immune modulatory nucleic acid vector comprising a tricistronic immune modulatory sequence comprising a gene encoding XCL1, a gene encoding GM-CSF, and a gene encoding IL-15 (e.g., a circular DNA vector) to an individual (e.g., a human cancer patient).

[0182] An alternative method of the invention involves administering to an individual (e.g., a human cancer patient) an immune modulatory nucleic acid vector that includes an immune modulatory sequence that includes multiple transcription units, where one transcription unit includes a gene encoding a chemoattractant for dendritic cells (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4) and another transcription unit includes a gene encoding one or more immune modulatory proteins (e.g., a gene encoding a growth factor or activator of dendritic cells (e.g., a gene encoding FLT3L, a gene encoding GMCSF, or a gene encoding CD40L) or a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). Thus, in certain examples, the methods of the invention include administering to an individual (e.g., a human cancer patient) an immune regulating nucleic acid vector (e.g., a circular DNA vector) comprising an immune regulating sequence comprising three transcription units, where a first transcription unit comprises a gene encoding XCL1, a second transcription unit comprises a gene encoding FLT3L, and a third transcription unit comprises a gene encoding IL-12; an immune regulating nucleic acid vector (e.g., a circular DNA vector) comprising an immune regulating sequence comprising three transcription units, where a first transcription unit comprises a gene encoding XCL1, a second transcription unit comprises a gene encoding FLT3L, and a third transcription unit comprises a gene encoding IL-15; or an immune regulating nucleic acid vector (e.g., a circular DNA vector) comprising an immune regulating sequence comprising three transcription units, where a first transcription unit comprises a gene encoding XCL1, a second transcription unit comprises a gene encoding GM-CSF, and a third transcription unit comprises a gene encoding IL-15.

[0183] In other examples, the therapeutic methods of the invention include administering one or more monocistronic nucleic acid vectors (e.g., circular DNA vectors). For example, in such examples, the methods of the invention include administering to an individual (e.g., a human cancer patient) one or more immune modulatory nucleic acid vectors (e.g., circular DNA vectors) having immune modulatory sequences (e.g., monocistronic immune modulatory sequences) including genes encoding chemoattractants for dendritic cells (e.g., genes encoding XCL1, XCL2, CCL5, or CCL4), genes encoding growth factors or activators for dendritic cells (e.g., genes encoding FLT3L, GMCSF, or CD40L), or genes encoding signaling proteins for lymphocytes (e.g., genes encoding cytokines (e.g., genes encoding IL-12 or IL-15) or genes encoding chemokines (e.g., genes encoding CXCL9 or CXCL10)).

[0184] In some embodiments, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) including a gene encoding a dendritic cell chemoattractant (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4) or a gene encoding a dendritic cell growth factor or activator (e.g., a gene encoding FLT3L, a gene encoding GMCSF, or a gene encoding CD40L), and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)).

[0185] In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) including a gene encoding a dendritic cell chemoattractant (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding XCL1, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding XCL1, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-12. In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding XCL1, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-15.In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) the first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., monocistronic immune regulatory sequence) that includes a gene encoding XCL1, and (ii) the second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL9. In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) the first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., monocistronic immune regulatory sequence) that includes a gene encoding XCL1, and (ii) the second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL10.

[0186] In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding XCL2, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding XCL2, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-12. In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) the first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., monocistronic immune regulatory sequence) that includes a gene encoding XCL2, and (ii) the second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-15. In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) the first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., monocistronic immune regulatory sequence) that includes a gene encoding XCL2, and (ii) the second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL9. In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding XCL2, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL10.

[0187] In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding CCL4, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding CCL4, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-12. In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) the first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding CCL4, and (ii) the second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-15. In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) the first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding CCL4, and (ii) the second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL9. In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding CCL4, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL10.

[0188] In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding CCL5, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10). In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding CCL5, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-12. In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) the first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding CCL5, and (ii) the second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-15. In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) the first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding CCL5, and (ii) the second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL9. In some examples, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) that includes a gene encoding CCL5, and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL10.

[0189] In some embodiments, the method includes administering two nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) including a gene encoding a growth factor or activator of dendritic cells (e.g., a gene encoding FLT3L, a gene encoding GMCSF, or a gene encoding CD40L), and (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)).

[0190] In some embodiments, the method includes administering three nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes (a) an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) including a gene encoding a dendritic cell chemoattractant (e.g., a gene encoding XCL1, a gene encoding XCL2, a gene encoding CCL5, or a gene encoding CCL4), (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding a dendritic cell growth factor or activator (e.g., a gene encoding FLT3L, a gene encoding GMCSF, or a gene encoding CD40L), and (iii) a third nucleic acid vector (e.g., circular DNA vector) includes a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)).

[0191] In some embodiments, the method includes administering three nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) including a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding FLT3L, and (iii) a third nucleic acid vector (e.g., circular DNA vector) includes a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)). In some embodiments, the method includes administering three nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulatory sequence (e.g., a monocistronic immune regulatory sequence) including a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding GM-CSF, and (iii) a third nucleic acid vector (e.g., circular DNA vector) includes a gene encoding a lymphocyte signaling protein (e.g., a gene encoding a cytokine (e.g., a gene encoding IL-12 or a gene encoding IL-15) or a gene encoding a chemokine (e.g., a gene encoding CXCL9 or a gene encoding CXCL10)).

[0192] In some embodiments, the method includes administering three nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding FLT3, and (iii) a third nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-12. In some embodiments, the method includes administering three nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding FLT3, and (iii) a third nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-15. In some embodiments, the method includes administering three nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding FLT3, and (iii) a third nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL9. In some embodiments, the method includes administering three nucleic acid vectors (e.g., circular DNA vectors) in a pharmaceutically acceptable carrier, where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding FLT3, and (iii) a third nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL10.

[0193] In some embodiments, the method includes administering three nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding GM-CSF, and (iii) a third nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-12. In some embodiments, the method includes administering three nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding GM-CSF, and (iii) a third nucleic acid vector (e.g., circular DNA vector) includes a gene encoding IL-15. In some embodiments, the method includes administering three nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding GM-CSF, and (iii) a third nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL9. In some embodiments, the method includes administering three nucleic acid vectors (e.g., circular DNA vectors), where (i) a first nucleic acid vector (e.g., circular DNA vector) includes an immune regulating sequence (e.g., monocistronic immune regulating sequence) that includes a gene encoding XCL1, (ii) a second nucleic acid vector (e.g., circular DNA vector) includes a gene encoding GM-CSF, and (iii) a third nucleic acid vector (e.g., circular DNA vector) includes a gene encoding CXCL10.

[0194] In any of the above methods, the immune modulatory sequence can be within a self-replicating RNA coding sequence that includes a replicase coding sequence that transcribes RNA from the self-replicating RNA molecule.

[0195] A variety of administration routes are provided by the present invention. In certain embodiments, the nucleic acid vector (e.g., a circular DNA vector) or pharmaceutical composition thereof is administered intratumorally. In other embodiments, the nucleic acid vector (e.g., a circular DNA vector) or pharmaceutical composition thereof is administered peritumorally. In other embodiments, the nucleic acid vector (e.g., a circular DNA vector) or pharmaceutical composition thereof is administered subdermally (e.g., subdermally adjacent to a tumor). In other embodiments, the nucleic acid vector (e.g., a circular DNA vector) or pharmaceutical composition thereof is administered systemically (e.g., intravenously).

[0196] In some instances, a single injection of the nucleic acid vector (e.g., a circular DNA vector) or pharmaceutical composition thereof is administered to the individual over the course of treatment. In some embodiments, a single injection of the nucleic acid vector (e.g., a circular DNA vector) or pharmaceutical composition thereof is administered intratumorally to the individual.

[0197] The nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof can be administered as a single dose. Alternatively, the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof can be administered in multiple doses (e.g., 2 or more doses, 3 or more doses, 4 or more doses, 5 or more doses, 6 or more doses, e.g., 2 doses, 3 doses, 4 doses, 5 doses, or 6 doses) over the course of treatment.

[0198] In some embodiments, the frequency of administration is once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks, or once a month, once every two months, or once every three months, or less frequently. The progress of therapy (e.g., modulation of tumor microenvironment) can be easily monitored (e.g., detected or quantified) according to methods known in the art and described herein. The administration schedule of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof can be changed over time.

[0199] The dosage of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof described herein can be empirically determined in an individual who has received one or more administrations of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof. The individual is given increasing doses of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof. To assess the effectiveness of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof, indicators of disease / disorder can be monitored. For repeated administration over several days or more, depending on the pathology, treatment is continued until a desired level of tumor microenvironment regulation is achieved.

[0200] In some instances, the appropriate dosage of a nucleic acid vector (e.g., circular DNA vector) described herein will depend on the particular nucleic acid vector (e.g., circular DNA vector), the type and severity of the cancer, previous therapy, the individual's medical history and response to the nucleic acid vector (e.g., circular DNA vector), and the discretion of the attending physician. The clinician can administer the nucleic acid vector (e.g., circular DNA vector), or pharmaceutical composition thereof, until a dosage is reached that achieves a desired result (e.g., modulation of the tumor microenvironment, as defined herein). In some embodiments, the desired result is a decrease in the expression level of a biomarker associated with tumor progression, or an increase in the expression level of a biomarker associated with tumor clearance (e.g., an immune-modulating protein). In other embodiments, the desired result is a decrease in tumor burden, a decrease in tumor cell number or metabolic activity, or an increase in immune cell activity. Further methods for determining whether a dosage has produced a desired result will be apparent to one of skill in the art. Administration of one or more nucleic acid vectors (e.g., circular DNA vectors) can be continuous or intermittent, for example, depending on the individual's physiological state and other factors known to one of skill in the art. Administration of the nucleic acid vector (e.g., a circular DNA vector) may be essentially continuous over a preselected period of time, or may be in a series of spaced administrations, for example, either before, during, or after the onset of the targeted disease or disorder.

[0201] As a general proposition, a therapeutically effective amount of a nucleic acid vector (e.g., a circular DNA vector) administered to a human may be between 1.0 pg and 1 mg of nucleic acid (e.g., 0.01 ng to 100 μg, 0.1 ng to 50 μg, 1 ng to 10 μg, or 10 ng to 1 μg, e.g., 0.01 ng to 0.05 ng, 0.05 ng to 0.1 ng, 0.1 ng to 0.5 ng, 0.5 ng to 1 ng, 1 ng to 5 ng, 5 ng to 10 ng, 10 ng to 50 ng, 50 ng to 100 ng, 100 ng to 500 ng, 500 ng to 1 μg, 1 μg). The amount of the antibody may be in the range of about 1 μg to 5 μg, or 5 μg to 10 μg, for example, about 1 pg, about 5 pg, about 10 pg, about 20 pg, about 25 pg, about 50 pg, about 75 pg, about 100 pg, about 1 ng, about 5 ng, about 10 ng, about 20 ng, about 25 ng, about 50 ng, about 75 ng, about 100 ng, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 50 μg, about 75 μg, about 100 μg, or about 500 μg.

[0202] Treatments of the present invention may also include administration of one or more additional therapeutic agents (e.g., anti-cancer agents) appropriate to the particular cancer being treated. For example, it may be desirable to further administer an additional therapeutic agent (e.g., anti-cancer agent) selected from a chemotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, and / or an anti-hormonal agent.

[0203] Electric field transmission In some aspects of the invention, an electric field is delivered into the tumor microenvironment. The electric field delivered into the tumor microenvironment can promote the introduction of a nucleic acid vector (e.g., a circular DNA vector) into a target cell (e.g., a tumor cell or a tumor-infiltrating immune cell (e.g., a tumor-infiltrating lymphocyte or a tumor-resident antigen-presenting cell (e.g., a tumor-resident dendritic cell)) in the tumor microenvironment. Such electric field-mediated nucleic acid delivery (electrotransfer) can be performed through any one (and combinations thereof) of several mechanisms, including electrophoresis, electrokinetically driven drug uptake, and / or electroporation. Without being bound by theory, in certain instances, the electric field delivered into the tumor microenvironment can promote anti-tumor adaptive immunity conveyed by the nucleic acid vector (e.g., a circular DNA vector). Suitable means for generating an electric field for electrotransfer of nucleic acids in mammalian tissues are known in the art, and any suitable means known in the art or described herein can be adapted for use as part of the present invention.

[0204] An electric field suitable for electrotransfer can be delivered to the tumor microenvironment at or close to the time of administration of the nucleic acid vector (e.g., a circular DNA vector) or a pharmaceutical composition thereof (e.g., as part of the same procedure). For example, the present invention includes methods in which the electric field is delivered within 1 hour of administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof (e.g., within 55 minutes, within 50 minutes, within 45 minutes, within 40 minutes, within 35 minutes, within 30 minutes, within 25 minutes, within 20 minutes, within 15 minutes, within 10 minutes, within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, within 90 seconds, within 60 seconds, within 45 seconds, within 30 seconds, within 20 seconds, within 15 seconds, within 10 seconds, within 9 seconds, within 8 seconds, within 7 seconds, within 6 seconds, within 5 seconds, within 4 seconds, within 3 seconds, within 2 seconds, or within 1 second of administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof) (e.g., simultaneously with or after administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof, but within any of the time periods discussed above). In some embodiments, the electric field is delivered within 24 hours of administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof (e.g., within 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 8 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, or 2 minutes of administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof). In some embodiments, the electric field is delivered within 7 days of administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof (e.g., within 6 days, 5 days, 4 days, 3 days, or 2 days of administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof).

[0205] Various means of generating and transmitting an electric field to the tumor microenvironment are contemplated herein as part of the method of the present invention. Devices and systems with electrodes suitable for transmitting an electric field (e.g., under conditions suitable for electrotransfer) in mammalian tissue are commercially available and may be useful in the methods disclosed herein. In some examples, the electric field is transmitted through an electrode with one or more needles (e.g., one or more needles placed inside (intratumoral) or near (peritumoral) the tumor, e.g., a single-needle electrode, a multi-needle electrode, or a needle array electrode). Suitable needle electrodes include the CLINIPORATOR™ electrode sold by IGEA™ and the needle electrodes sold by AMBU™. Such needle electrodes may be configured to deliver an electric field within a superficial tumor or a tumor accessible beyond the epithelial surface (e.g., outside the body) (e.g., a melanoma, carcinoma, or sarcoma (e.g., a head and neck tumor)), for example, a tumor located (wholly or partially) within 4 cm of the epithelial surface (e.g., within 3 cm of the epithelial surface, within 2 cm of the epithelial surface, or within 1 cm of the epithelial surface, e.g., 1 cm to 4 cm from the epithelial surface, 1 cm to 3 cm from the epithelial surface, 1 cm to 2 cm from the epithelial surface, 2 cm to 4 cm from the epithelial surface, 2 cm to 3 cm from the epithelial surface, or 3 cm to 4 cm from the epithelial surface).

[0206] In other examples, the electric field is delivered via a non-needle electrode (e.g., a tweezers-type electrode (e.g., BTX™ tweezertrodes), a plate-type electrode, or a vernier caliper-type electrode). Such a non-needle electrode may be configured to deliver the electric field minimally or non-invasively into a tumor (e.g., a superficial tumor or a tumor accessible beyond the epithelial surface (e.g., outside the body) (e.g., a melanoma, carcinoma, or sarcoma (e.g., a head and neck tumor)), for example, a tumor located (in whole or in part) within 4 cm of the epithelial surface (e.g., within 3 cm of the epithelial surface, within 2 cm of the epithelial surface, or within 1 cm of the epithelial surface, e.g., between 1 cm and 4 cm of the epithelial surface, between 1 cm and 3 cm of the epithelial surface, between 1 cm and 2 cm of the epithelial surface, between 2 cm and 4 cm of the epithelial surface, between 2 cm and 3 cm of the epithelial surface, or between 3 cm and 4 cm of the epithelial surface). Suitable electrodes include monopolar electrodes (eg, a monopolar needle electrode) and bipolar electrodes (eg, a bipolar tweezers-type electrode, a bipolar plate-type electrode, a bipolar caliper-type electrode, or a bipolar multi-needle electrode).

[0207] The electric field suitable for electrotransfer can have any suitable voltage, waveform, frequency, etc. In some embodiments of any of the methods of transmitting an electric field described herein, a pulsed electric field is transmitted. The pulsed electric field suitable for electrotransfer can be transmitted at a variety of voltages, waveforms, and frequencies. In some examples, the pulsed electric field is transmitted for a period of time ranging from 0.001 milliseconds to 100 milliseconds (e.g., 0.005 milliseconds to 50 milliseconds, 0.05 milliseconds to 25 milliseconds, or 0.1 milliseconds to 5 milliseconds, such as 0.001 milliseconds to 0.01 milliseconds, 0.01 milliseconds to 0.1 milliseconds, 0.1 milliseconds to 1.0 milliseconds, 1 millisecond to 2 milliseconds, 2 milliseconds to 4 milliseconds, 4 milliseconds to 6 milliseconds, 6 milliseconds to 10 milliseconds, 10 milliseconds to 20 milliseconds, 20 milliseconds to 50 milliseconds, or 50 milliseconds to 100 milliseconds, such as about 0.005 milliseconds, about 0.01 milliseconds, about 0.05 milliseconds, about 0. In some embodiments, the pulse duration is about 1 millisecond, about 0.5 milliseconds, about 0.6 milliseconds, about 0.7 milliseconds, about 0.8 milliseconds, about 0.9 milliseconds, about 1 millisecond, about 1.5 milliseconds, about 2 milliseconds, about 2.5 milliseconds, about 3 milliseconds, about 4 milliseconds, about 5 milliseconds, about 6 milliseconds, about 7 milliseconds, about 8 milliseconds, about 9 milliseconds, about 10 milliseconds, about 12 milliseconds, about 15 milliseconds, about 20 milliseconds, about 25 milliseconds, about 30 milliseconds, about 35 milliseconds, about 40 milliseconds, about 50 milliseconds, about 60 milliseconds, about 70 milliseconds, about 80 milliseconds, about 90 milliseconds, or about 100 milliseconds.

[0208] In some embodiments, the pulsed electric field includes 2-100 pulses (e.g., 4-50 pulses, 5-40 pulses, 6-30 pulses, 7-25 pulses, or 8-20 pulses, e.g., 1-5 pulses, 5-10 pulses, 8-12 pulses, 10-15 pulses, 15-20 pulses, 20-50 pulses, or 50-100 pulses). The pulses may have any suitable waveform, such as a square wave, a sine wave, or a sawtooth wave. In some embodiments, the pulsed electric field is delivered at a frequency.

[0209] In any of the above-described embodiments of the method comprising delivery of a pulsed electric field, the energy (e.g., the voltage at the target tissue) of one or more pulses may be in the range of 50V to 10,000V (e.g., 100V to 5,000V, 200V to 4,000V, 300V to 3,000V, 400V to 2,000V, 500V to 1,500V, or 600V to 100V, e.g., 100V to 200V, 200V to 300V, 300V to 400V, 400V to 500V, 500V to 600V, 600V to 700V, 700V to 800V, 800V 100V, about 150V, about 200V, about 3000V, about 400V, 500V, 600V, 700V, 800V, 900V, 1000V, 1500V, about 2000V, about 3000V, about 4000V, about 5000V, about 6000V, about 7000V, about 8000V, about 9000V, or about 10000V. In some examples, the amplitude of one or more (e.g., all) of the pulses is between 50V and 10,000V (e.g., between 100V and 5,000V, 200V and 4,000V, 300V and 3,000V, 400V and 2,000V, 500V and 1,500V, or 600V and 100V, e.g., between 100V and 200V, 200V and 300V, 300V and 400V, 400V and 500V, 500V and 600V, 600V and 700V, 700V and 800V, 800V and 900V, 900V and 1,000V, 1000V to 1500V, 1500V to 2000V, 2000V to 3000V, 3000V to 5000V, or 5000V to 10000V, for example, about 100V, about 150V, about 200V, about 250V, about 300V, 400V, 500V, 600V, 700V, 800V, 900V, 1000V, 1500V, about 2000V, about 3000V, about 4000V, about 5000V, about 6000V, about 7000V, about 8000V, about 9000V, or about 10000V). Any of the above voltages may be the top of a square waveform, the peak of a sine waveform, the peak of a sawtooth waveform, the root mean square (RMS) voltage of a sine waveform, or the RMS voltage of a sawtooth waveform.

[0210] An electric field suitable for electrotransfer may be delivered at or near the site of administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof. For example, in some embodiments, the electric field is delivered to the same tumor microenvironment as the tumor microenvironment to which the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof is administered. In some examples, the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof is delivered to a location that is exposed to an electric field (or will be exposed to an electric field if electric field delivery is subsequent). In some embodiments ...e.g., at least 5% of the maximum tissue voltage, at least 10% of the maximum tissue voltage, at least 20% of the maximum tissue voltage, at least 30% of the maximum tissue voltage, at least 40% of the maximum tissue voltage, at least 50% of the maximum tissue voltage, at least 60% of the maximum tissue voltage, at least 70% of the maximum tissue voltage, at least 80% of the maximum tissue voltage, or at least 10% of the maximum tissue voltage). The electric field is delivered to a location that is exposed (or will be exposed to) a voltage that is at least 90% of the maximum tissue voltage, for example, 1%-10% of the maximum tissue voltage, 10%-20% of the maximum tissue voltage, 20%-30% of the maximum tissue voltage, 30%-40% of the maximum tissue voltage, 40%-50% of the maximum tissue voltage, 50%-60% of the maximum tissue voltage, 60%-70% of the maximum tissue voltage, 70%-80% of the maximum tissue voltage, 80%-90% of the maximum tissue voltage, 90%-95% of the maximum tissue voltage, or 95%-100% of the maximum tissue voltage. In some examples, the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof is administered intratumorally (e.g., administered intratumorally in the same tumor (e.g., in the same tumor microenvironment) as the tumor to which the electric field is delivered). In some examples, the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof is administered peritumorally.

[0211] Alternatively, the administration site can be a tissue area away from the electric field. For example, the administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof can be systemically administered (e.g., intravenously), while the electric field is delivered to the tumor. In some embodiments, the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof is administered to a non-tumor tissue (e.g., intramuscularly, subcutaneously, or subdermally).

[0212] Additional Therapies In certain embodiments of the therapeutic methods provided herein, one or more additional therapies are administered in conjunction with the administration of the nucleic acid vector (e.g., circular DNA vector) or composition thereof, for example, instead of or in combination with the delivery of an electric field.Exemplary additional therapies include anti-cancer therapies such as radiation therapy (e.g., cytotoxic radiation therapy), photodynamic therapy, hyperthermia, oncolytic therapy (e.g., administration of oncolytic viruses), or anti-cancer agents (e.g., chemotherapeutic agents, checkpoint inhibitors, cytotoxic agents, growth inhibitors, anti-angiogenic agents, cytokines, cytokine antagonists, antibody-drug conjugates, cancer vaccines, or combinations thereof). Thus, in some instances, the invention includes any method of administering a nucleic acid vector (e.g., any of the circular DNA vectors described herein (e.g., monocistronic or polycistronic (e.g., bicistronic or tricistronic) nucleic acid vectors (e.g., circular DNA vectors), or nucleic acid vectors having multiple transcription units (e.g., circular DNA vectors)) in combination with radiation therapy (e.g., cytotoxic radiation therapy), photodynamic therapy, hyperthermia, or oncolytic therapy (e.g., oncolytic viruses), where radiation therapy, photodynamic therapy, hyperthermia, or oncolytic therapy is administered according to any known or readily available protocol (e.g., in place of delivery of an electric field).

[0213] In some embodiments, the additional therapy is an additional therapeutic agent such as an anti-cancer agent (e.g., a chemotherapeutic agent, a checkpoint inhibitor, a cytotoxic agent, a growth inhibitory agent, an anti-angiogenic agent, a cytokine, a cytokine antagonist, an antibody-drug conjugate, a cancer vaccine, or a combination thereof). In certain embodiments, the additional therapy is a chemotherapeutic agent. In other certain embodiments, the additional therapy is a checkpoint inhibitor, e.g., a PD-1 axis binding antagonist (e.g., an anti-PD-1 antagonist or an anti-PD-L1 antibody), such as pembrolizumab (MK-3475), nivolumab (ONO-4538 / BMS-936558, MDX1106), pidilizumab (CT-011), atezolizumab (MPDL3280A), or AMP-224).In some embodiments, the additional therapy is a BCL-2 inhibitor (such as GDC-0199 / ABT-199), lenalidomide (REVLIMID™), a PI3K-δ inhibitor (such as idelalisib (ZYDELIG™)), an agonist (e.g., an activating costimulatory molecule, such as CD40, CD226, CD28, OX40 (e.g., AgonOX), GITR, CD137 (TNFRSF9, 4-1BB, or antagonists, such as inhibitory co-stimulatory molecules, e.g., CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1-methyl-D-tryptophan (also known as 1-D-MT)), and an additional therapeutic agent selected from antagonist antibodies against TIGIT, MICA / B, GITR (e.g., TRX518) or arginase, ipilimumab (also known as MDX-010, MDX-101, or YERVOY™), tremelimumab (also known as ticilimumab or CP-675,206), urelumab (also known as BMS-663513), MGA271, antagonists against TGFβ, e.g., meterimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells (e.g., cytotoxic T cells, or CTLs) expressing a chimeric antigen receptor (CAR), e.g., adoptive transfer of T cells comprising a dominant negative TGFβ receptor, e.g., a dominant negative TGFβ type II receptor.

[0214] In further examples, the additional therapy includes an anti-CD20 antibody, such as rituximab or a humanized B-Ly1 antibody (e.g., obinituzumab). In some embodiments, the anti-CD20 antibody is ofatumumab, ublituximab, and / or ibritumomab tiuxetan.

[0215] In some instances, the additional therapy includes an alkylating agent such as 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid or a salt thereof. In some embodiments, the alkylating agent is bendamustine.

[0216] In a further aspect of the invention, the additional therapy comprises a BCL-2 inhibitor. In some embodiments, the BCL-2 inhibitor is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide and salts thereof. In one embodiment, the BCL-2 inhibitor is venetoclax (CAS number: 1257044-40-8).

[0217] In a further aspect of the invention, the additional therapy comprises a phosphoinositide 3 kinase (PI3K) inhibitor. In one embodiment, the PI3K inhibitor inhibits the delta isoform PI3K (i.e., P110δ). In some embodiments, the PI3K inhibitor is 5-fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone and salts thereof. In some embodiments, the PI3K inhibitor is idelalisib (CAS number: 870281-82-6). In one embodiment, the PI3K inhibitor inhibits the alpha and delta isoforms of PI3K. In some embodiments, the PI3K inhibitor is 2-{3-[2-(1-isopropyl-3-methyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl]-1H-pyrazol-1-yl}-2-methylpropanamide and salts thereof.

[0218] In a further aspect of the invention, the additional therapy comprises a Bruton's tyrosine kinase (BTK) inhibitor. In one embodiment, the BTK inhibitor is 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one and salts thereof. In one embodiment, the BTK inhibitor is ibrutinib (CAS number: 936563-96-1).

[0219] In a further aspect of the invention, the additional therapy comprises thalidomide or a derivative thereof. In one embodiment, the thalidomide or a derivative thereof is (RS)-3-(4-amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione and its salts. In one embodiment, the thalidomide or a derivative thereof is lendalidomide (CAS number: 191732-72-6).

[0220] In further aspects of the invention, the additional therapy comprises one or more of cyclophosphamide, doxorubicin, vincristine, or prednisolone (CHOP). In one embodiment, the additional therapy further comprises an anti-CD20 antibody as described above (e.g., GA-101 and / or RITUXAN™). Any of the above methods and therapies can be used without limitation for any cancer, including, for example, solid tumors.

[0221] In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an agent used in radiation therapy, an anti-angiogenic agent, an apoptotic agent, an anti-tubulin agent, or other agent, such as an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), a HER1 / EGFR inhibitor (e.g., erlotinib (Tarceva), a platelet derived growth factor inhibitor (e.g., Gleevec (imatinib mesylate)), a COX-2 inhibitor (e.g., celecoxib), an interferon, a cytokine, an antibody other than an anti-CD3 antibody of the invention, such as an antibody that binds to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA VEGF, or the receptor(s) of VEGF, TRAIL / Apo2, PD-1, PD-L1, PD-L2, or another bioactive or organic chemical agent.

[0222] In some embodiments, the present invention provides methods wherein the additional therapeutic agent is a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone.

[0223] Tumors and individuals The compositions and methods described herein are suitable for treating various types of cancer. In certain instances, the cancer to be treated is a solid tumor (e.g., sarcoma, carcinoma, or lymphoma). Solid tumors treatable by the methods of the present invention include solid tumors of epithelial cell origin and solid tumors of non-epithelial cell origin. Examples of solid tumors of epithelial cell origin include tumors of the head and neck, gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gallbladder, labia, nasopharynx, skin, uterus, male genital tract, urinary tract, bladder, and skin tumors. Solid tumors of non-epithelial cell origin include sarcomas and brain tumors.

[0224] In some instances, the compositions and methods of the present invention are suitable for the treatment of melanoma. Melanomas that can be treated by the compositions and methods of the present invention include superficial spreading melanoma, nodular melanoma, lentigo melanoma, acral lentigo melanoma, amelanotic melanoma, nevus melanoma, Spitzian nevus melanoma, and desmoplastic melanoma.

[0225] In some instances, the compositions and methods are suitable for treating head and neck cancer. Head and neck cancers that can be treated by the methods and compositions of the present invention include squamous cell carcinomas (e.g., oral squamous cell carcinomas). In some instances, head and neck cancers treatable by the present invention are cancers (e.g., carcinomas, e.g., squamous cell carcinomas) of the nasopharynx, oropharynx, hypopharynx, larynx, or trachea. In some embodiments, the head and neck cancer is characterized by a tumor in the lip, oral cavity, oropharynx, hypopharynx, nasopharynx, glottic larynx, or supraglottic larynx. In some embodiments, the head and neck cancer is characterized by an ethmoid sinus tumor, a maxillary sinus tumor, a salivary gland tumor, an occult primary tumor, or a mucosal melanoma.

[0226] In other embodiments, the cancer is selected from the group consisting of desmoid tumor, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, esophageal cancer, mesothelioma, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, or lymphoma. In some embodiments, the tumor is a Clarkin tumor, hilar tumor, germ cell tumor, Ewing tumor, Askin tumor, primitive neuroectodermal tumor, Leydig cell tumor, Wilms tumor, or Sertoli cell tumor. In some embodiments, the tumor is a carcinoma selected from the group consisting of squamous cell carcinoma, cloacal carcinoma, adenocarcinoma, adenosquamous carcinoma, cholangiocarcinoma, hepatocellular carcinoma, invasive papillary urothelial carcinoma, and squamous urothelial carcinoma.

[0227] In some embodiments, the tumor is a resectable tumor. In other embodiments, the tumor is an unresectable tumor. In some embodiments, the tumor is an advanced stage tumor. In other embodiments, the tumor is an early stage tumor. In some embodiments, the cancer is a recurrent and / or refractory cancer (e.g., the method is a second or third line therapy).

[0228] The nucleic acid vectors (e.g., circular DNA vectors) and pharmaceutical compositions prepared according to the invention can be used to treat children or adults. Thus, an individual (e.g., a human patient) can be less than 1 year old, less than 5 years old, between 1 and 5 years old, between 5 and 15 years old, between 15 and 55 years old, or at least 55 years old.

[0229] Detection of tumor microenvironment modulation The modulation of tumor microenvironment by the therapeutic methods provided herein can be detected using any method known in the art (e.g., by detecting gene or protein biomarkers for the modulation of tumor microenvironment). Genetic and protein biomarkers useful for determining whether a treatment modulates a particular tumor microenvironment are readily available to those skilled in the art, for example, through information available from the National Comprehensive Cancer Network (NCCN). Additionally or alternatively, the modulation of tumor microenvironment can be detected or quantified in terms of clinical response criteria, such as the readings defined in the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines.

[0230] In some instances, the tumor microenvironment is modulated by a treatment (e.g., a treatment of the invention, e.g., a treatment comprising administration of a nucleic acid vector (e.g., a circular DNA vector) and / or delivery of an electric field) if the treatment increases the relative expression of a biomarker associated with tumor clearance (e.g., an immunogenic protein (e.g., a proinflammatory cytokine or a dendritic cell activating protein) in the tumor microenvironment). Additionally or alternatively, the tumor microenvironment is considered to be modulated by a treatment if the treatment decreases the relative expression of a biomarker associated with tumor progression (e.g., a biomarker of T cell exhaustion, e.g., PD-1 expression).

[0231] In some embodiments, a measurable change in expression level of a biomarker indicative of modulation of the tumor microenvironment is at least a 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90% change in expression level of the biomarker. For example, a measurable decrease in expression level of a biomarker associated with tumor progression indicates a desirable modulation of the tumor microenvironment if the decrease in expression of the biomarker is at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90% relative to a baseline level. Additionally or alternatively, a measurable increase in expression levels of a biomarker (e.g., an immunogenic protein) associated with tumor clearance can be achieved by increasing expression of the biomarker by at least at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15 ... An increase of at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold (e.g., 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-100%, 2-fold-3-fold, 3-fold-5-fold, 5-fold-10-fold, 10-fold-20-fold, 20-fold-50-fold, 50-fold-100-fold or more relative to a reference level, e.g., 1%-100%, 2-fold-10-fold, 10-fold-50-fold, 50-fold-100-fold or more relative to a reference level) indicates a desirable modulation of the tumor microenvironment.

[0232] In some embodiments, the biomarker associated with tumor clearance is a circulating biomarker such as circulating tumor DNA (ctDNA), e.g., cell-free ctDNA, cell-free RNA, mRNA, or non-coding RNA (e.g., microRNA, small interfering RNA, piwi-interacting RNA, small nuclear RNA, small nucleolar RNA, long non-coding RNA, microRNA).

[0233] In certain instances, modulation of the tumor microenvironment is characterized by detection of an anti-tumor adaptive immune response (e.g., the development, propagation, or enhancement of an anti-tumor adaptive immune response relative to a baseline level), such as an adaptive immune response mounted against one or more tumor antigens expressed by a tumor in the individual undergoing treatment (e.g., where the tumor antigens are generated and recognized in situ and not provided by the treatment). Thus, in some instances, any of the methods of the invention further comprise a step of detecting an anti-tumor adaptive immune response in the individual (e.g., after treatment according to the invention, before treatment according to the invention, or both).

[0234] Tissue or cell samples can be assayed for, for example, mRNA or DNA from genetic biomarkers using Northern, dot blot, polymerase chain reaction (PCR) analysis, array hybridization, RNase protection assay, or commercially available DNA SNP chip microarrays, including snapshots of DNA microarrays. Real-time PCR (RT-PCR) assays, such as quantitative PCR assays, are well known in the art. In some examples of the present invention, a method for detecting mRNA from a genetic biomarker of interest in a biological sample, such as a tumor sample (e.g., a solid tumor sample), includes generating cDNA from the sample by reverse transcription using at least one primer, amplifying the cDNA thus generated, and detecting the presence of the amplified cDNA. Furthermore, such methods can include one or more steps that allow for determining the level of mRNA in the biological sample (e.g., by simultaneously examining the level of a comparative control mRNA sequence of a "housekeeping" gene, such as an actin family member). Optionally, the sequence of the amplified cDNA can be determined.

[0235] In a particular embodiment, expression of the genetic biomarkers described herein can be performed by RT-PCR techniques. The probes used for PCR can be labeled with detectable markers, such as, for example, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes. Such probes and primers can be used to detect the presence of expressed genes (e.g., immune-modulating genes) in a sample. As will be appreciated by those skilled in the art, numerous different primers and probes can be prepared and effectively used to amplify, clone, and / or determine the presence and / or expression level of one or more regulatory (e.g., immune-modulating) genes. Additionally or alternatively, genetic biomarkers from tumor samples can be detected and / or quantified using known methods such as Sanger sequencing, pyrosequencing, allele-specific arrayed primer extension, next generation sequencing (NGS), mutant-enriched liquid chip, amplification refractory mutation system, co-amplification-PCR at lower denaturation temperature, and bead / emulsion / amplification / magnetic PCR.

[0236] Other methods include protocols that test or detect mRNA from at least one gene biomarker in tissue (e.g., tumor tissue, e.g., solid tumor tissue) or cell samples by microarray technology. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the order and position of each element of the array is known. For example, selected genes that may be expressed in a certain disease state can be arrayed on a solid support. Hybridization of a labeled probe with a particular array element indicates that the sample from which the probe is derived expresses that gene. Differential gene expression analysis of disease tissues can provide useful information. Microarray technology utilizes nucleic acid hybridization and computing techniques to evaluate the mRNA expression profile of thousands of genes within a single experiment (see, for example, WO 2001 / 75166). For a discussion of array production, see, e.g., U.S. Pat. Nos. 5,700,637, 5,445,934, and 5,807,522, Lockart, Nat. Biotechnol. 14:1675-1680 (1996), and Cheung et al., Nat. Genet. 21(Suppl):15-19 (1999).

[0237] Additionally, microarray-based DNA profiling and detection methods can be used, for example as described in EP 1753878. Such methods utilize short tandem repeat (STR) analysis and DNA microarrays to rapidly identify and distinguish between different DNA sequences. In one embodiment, labeled STR target sequences are hybridized to DNA microarrays carrying complementary probes. These probes vary in length to cover the range of possible STRs. The labeled single-stranded regions of the DNA hybrids are selectively removed from the microarray surface using post-hybridization enzymatic digestion. The number of repeats in the unknown target is estimated based on the pattern of target DNA that remains hybridized to the microarray. One example of a microarray processor is the commercially available GENECHIP™ system from Affymetrix, which includes arrays made by direct synthesis of oligonucleotides on a glass surface. Other systems may be used, as known to those skilled in the art.

[0238] Other methods for determining the level of biomarkers other than RT-PCR or other PCR-based methods include proteomic techniques as well as personalized genetic profiles that may be useful for treating cancer based on the patient's response at the molecular level.Specialized microarrays herein, such as oligonucleotide microarrays or cDNA microarrays, may include one or more biomarkers with expression profiles that correlate with tumor progression or tumor clearance (e.g., by anti-tumor immunity).Other methods that can be used to detect nucleic acids used in the present invention include high-throughput RNA sequence expression analysis, including RNA-based genomic analysis such as RNASeq.

[0239] In certain instances, modulation of the tumor microenvironment can be detected, quantified, and / or monitored using available NGS technologies, for example tumor-specific NGS kits such as AVENIO™ tumor tissue kits and ctDNA analysis kits.

[0240] A variety of assays are available for detection and quantification of protein biomarkers, including, for example, but not limited to, antibody-based methods including immunohistochemistry (IHC), Western blot analysis, immunoprecipitation, molecular binding assays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofiltration assays (ELIFA), fluorescence activated cell sorting (FACS), MassARRAY, proteomics, blood-based quantitative assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization, fluorescence in situ hybridization (FISH), Southern analysis, or Northern analysis, as well as mass spectrometry and other similar means known in the art. In the case of antibody-based methods, for example, the complex can be detected after contacting the sample with an antibody specific for the biomarker under conditions sufficient for the formation of an antibody-biomarker complex. Detection of the presence of protein biomarkers can be accomplished in a variety of formats, such as immunohistochemistry (IHC), Western blotting (with or without immunoprecipitation), two-dimensional SDS-PAGE, immunoprecipitation, fluorescence-activated cell sorting (FACS), flow cytometry, and ELISA procedures that assay a wide variety of tissues and samples, including plasma or serum. A wide range of immunoassay techniques using such assay formats are available. See, for example, U.S. Pat. Nos. 4,016,043, 4,424,279, and 4,018,653. These assay techniques include both non-competitive single-site and two-site assays, or "sandwich" assays, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to target biomarkers.

[0241] Sandwich assay is one of the most commonly used assays. There are many variations of sandwich assay technology, all of which are intended to be encompassed by the present invention. Briefly, in a typical forward assay, unlabeled antibody is immobilized on a solid substrate, and the sample to be tested is contacted with the binding molecule. After a suitable incubation period for a sufficient time to allow the formation of antibody-antigen complex, a second antibody specific to the antigen, labeled with a reporter molecule capable of generating a detectable signal, is then added and incubated for a time sufficient to form another complex of antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observing the signal generated by the reporter molecule. The results can be qualitative, by simple observation of the visible signal, or quantified, by comparison with a control sample containing a known amount of biomarker.

[0242] Variations of the forward assay include simultaneous assays, in which both the sample and the labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations that will be readily appreciated. In a typical forward sandwich assay, a primary antibody with specificity for a biomarker is covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support may be in the form of a tube, a bead, a disk of a microplate, or any other surface suitable for performing an immunoassay. The binding process is well known in the art and generally consists of cross-linking, covalent binding, or physical adsorption, and the polymer-antibody complex is washed in preparing the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated under appropriate conditions (e.g., room temperature to 40° C., e.g., between 25° C. and 32° C., inclusive) for a sufficient time (e.g., 2 minutes to 40 minutes, or more conveniently, overnight) to allow binding of any subunits present in the antibody. After an incubation period, the antibody subunit solid phase is washed, dried and incubated with a second antibody specific for a portion of the biomarker that is linked to a reporter molecule that is used to indicate binding of the second antibody to the molecular marker.

[0243] An alternative method involves immobilizing the target biomarker in the sample, followed by exposing the immobilized target to a specific antibody that may or may not be labeled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, the bound target may be detectable by direct labeling with the antibody. Alternatively, a second labeled antibody specific to the first antibody is exposed to the target-primary antibody complex to form a ternary complex of target-primary antibody-secondary antibody. This complex is detected by the signal emitted by the reporter molecule. A reporter molecule is a molecule whose chemical nature produces an analytically distinguishable signal that allows the detection of the antibody bound to the antigen. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores, or radionuclide-containing molecules (i.e., radioisotopes) and chemiluminescent molecules.

[0244] In the case of enzyme immunoassays, the enzyme is generally conjugated to the secondary antibody using glutaraldehyde or periodate. However, as will be readily recognized, there is a wide variety of different conjugation techniques readily available to those skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase, and alkaline phosphatase, among others. Substrates used with a particular enzyme are generally selected to produce a detectable color change upon hydrolysis by the corresponding enzyme. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to use fluorogenic substrates that produce fluorescent products rather than the chromogenic substrates mentioned above. In all instances, the enzyme-labeled antibody is added to the primary antibody-molecular marker complex and allowed to bind, after which excess reagent is washed away. A solution containing the appropriate substrate is then added to the antibody-antigen-antibody complex. The substrate reacts with the enzyme linked to the secondary antibody, thereby producing a qualitative visual signal that can be further quantified, usually by spectrophotometry, to provide an indication of the amount of biomarker present in the sample. Alternatively, fluorescent compounds such as fluorescein and rhodamine can be chemically coupled to antibodies without altering their binding capacity. When activated by irradiation with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, thereby inducing an excited state in the molecule, followed by emission of light of a characteristic color that is visually detectable by light microscopy. As in EIA, the fluorescently labeled antibody is bound to the primary antibody-molecular marker complex. After washing away unbound reagents, the remaining ternary complex is exposed to light of the appropriate wavelength, and the observation of fluorescence indicates the presence of the molecular marker of interest. Both immunofluorescence and EIA techniques are very well established in the art. Other reporter molecules such as radioisotopes, chemiluminescent, or bioluminescent molecules can also be used.

[0245] Clinical response criteria can also be used to determine whether tumor microenvironment is modulated.Any established clinical response criteria can be utilized, including RECIST guidelines.For example, in some embodiments, tumor microenvironment is determined to be modulated by a treatment that prolongs progression-free survival, produces objective response, including partial or complete response, prolongs overall survival, and / or improves one or more symptoms of cancer.

[0246] IV. KITS AND MANUFACTURES In another aspect of the invention, an article of manufacture or kit is provided that includes materials useful for the above treatment. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous fluid bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container can house the composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing a condition, and can have a sterile access port (e.g., the container can be an intravenous fluid bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a nucleic acid vector of the invention (e.g., a circular DNA vector or a self-replicating RNA molecule), or a pharmaceutical composition comprising a nucleic acid vector of the invention. The label or package insert indicates that the composition is used to treat a selected cancer. Additionally, the article of manufacture includes (a) a first container in which a composition is housed that includes a nucleic acid vector (e.g., a circular DNA vector) and / or composition of the invention, and (b) a second container in which a composition is housed that includes an additional therapeutic agent (e.g., an additional anti-cancer agent). The article of manufacture may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include a second (or third) container containing a pharma- ceutically acceptable carrier, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, dextrose solution, or any of the pharma- ceutically acceptable carriers disclosed above. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0247] In certain examples of the invention, kits are provided that include (i) any one or more of the above-mentioned materials (e.g., a nucleic acid vector of the invention (e.g., a circular DNA vector or a self-replicating RNA molecule) or a composition comprising a nucleic acid vector (e.g., a circular DNA vector), an additional therapeutic agent (e.g., an additional anti-cancer agent), and / or one or more pharma- ceutically acceptable carriers) and (ii) one or more elements of an energy delivery device (e.g., a device comprising electrodes that transmit an electric field to a tumor microenvironment, such as any suitable device or system described above). In some embodiments, provided herein is a kit that includes a nucleic acid vector of the invention (e.g., a circular DNA vector) and an electrode. In some embodiments, provided herein is a kit that includes a pharmaceutical composition comprising a nucleic acid vector of the invention (e.g., a circular DNA vector) and an electrode.

[0248] [Table 1] TIFF2024509084000003.tif250170TIFF2024509084000004.tif250170TIFF2024509084000005.tif250170TIFF2024509084000006.tif250170TIFF202 4509084000007.tif250170TIFF2024509084000008.tif245170TIFF2024509 084000009.tif251170TIFF2024509084000010.tif251170TIFF20245090840 00011.tif250170TIFF2024509084000012.tif250170TIFF2024509084000013.tif251170TIFF2024509084000014.tif250170TIFF2024509084000015.t if250170TIFF2024509084000016.tif250170TIFF2024509084000017.tif250170TIFF2024509084000018.tif250170TIFF2024509084000019.tif212170 EXAMPLES

[0249] Below are non-limiting examples of how the above compositions can be made and tested, and how such compositions can be used to treat individuals.

[0250] Example 1. Cell-free production of synthetic circular DNA vectors encoding therapeutic triplets This example describes the production and characterization of two synthetic circular DNA vectors encoding therapeutic triplets: (1) a single transcription unit vector with a single promoter driving expression of all three therapeutic sequences (shown in FIG. 1), and (2) a multiple transcription unit vector with three promoters, each driving expression of one of the three therapeutic sequences (shown in FIG. 2A). In this example, the therapeutic sequences are sFLT3L, IL-12, and XCL1 (operably linked in the 5'→3' orientation).

[0251] The following reagents were mixed in 1× Phi29 buffer (New England Biolabs) to prepare a rolling circle amplification (RCA) solution: plasmid DNA (5 μg / mL final concentration) containing the therapeutic triplet in either a single transcription unit or multiple transcription unit configuration, random primers (50 μM final concentration), NaOH (10 mM final concentration), dNTPs (2 mM final concentration), bovine serum albumin (BSA) (0.2 mg / mL final concentration), Phi29 DNA polymerase (200 U / mL final concentration), and pyrophosphatase stock (New England Biolabs, 0.4 mU / mL final concentration). The RCA solution was mixed continuously for 18 h at 30° C.

[0252] After incubation, the RCA solution was heat inactivated by increasing the temperature to 65° C. for 45 minutes. The temperature of the inactivated RCA solution was then reduced to 37° C.

[0253] To generate the BsaI solution, the inactivated RCA solution (final concentration of 0.2 mg DNA per mL) was added to CUTSMART™ buffer (final concentration of 1×) containing BsaI (final concentration of 2.5 U per μg DNA). The BsaI solution was mixed continuously for 2 hours at 37° C. No heat inactivation was performed on the BsaI solution. The temperature of the digested BsaI solution was reduced to 25° C.

[0254] To generate the ligation solution, the digested BsaI solution (final concentration of 40 μg DNA per mL) was added to CUTSMART™ buffer containing T4 ligase (10 U T4 ligase per μg DNA) and riboATP (final concentration of 1 mM). The ligation solution was incubated at 25° C. for 2 hours.

[0255] After incubation, the ligation solution was heat inactivated by increasing the temperature to 65° C. for 45 minutes. The temperature of the inactivated ligation solution was then reduced to 37° C.

[0256] To generate a supercoiled solution, the ligation solution was added to gyrase buffer containing DNA gyrase (1.5 U of gyrase per μg of DNA). Gyrase buffer contained 35 mM Tris-HCl, 24 mM KCl, 4 mM MgCl2, 1 mM ATP, 2 mM DTT, 1.8 mM spermidine, 32% glycerol (wt / vol), and 100 μg / mL and 500 μg / mL BSA. The supercoiled solution was mixed continuously for 2 hours at 37°C. The supercoiled solution was not heat inactivated.

[0257] The supercoiled solution was then added to potassium acetate buffer (final concentration of 50 mM potassium acetate) containing T5 exonuclease (2.5 U T5 exonuclease per μg DNA) to generate the cleanup solution. The cleanup solution was mixed continuously for at least 2 hours at 37°C. The cleanup solution was not heat inactivated.

[0258] The cleanup solution was then sterile filtered through a 0.22 μm filter and diluted 1:1 in a buffer containing 1.5 M NaCl, 100 mM MOPS, 30% isopropyl alcohol (IPA), and 0.3% Triton X-100 (vol / vol) to achieve a final concentration of 760 mM NaCl, 50 mM MOPS, 15% isopropyl alcohol (IPA), and 0.15% Triton X-100 (vol / vol). The diluted cleanup solution was applied to a Qiagen plasmid prep column and DNA was eluted with QN buffer. The eluate was diluted with IPA (40% (vol / vol)) and centrifuged at 15000 g for 30 min at 4° C. The pellet was washed with 70% EtOH and centrifuged again at 15000 g for 30 min at 4° C. After the second centrifugation, the pellet was resuspended in PBS at 1.0 mg / mL.

[0259] Supercoiled monomers were calculated by densitometric analysis of gel electrophoresis preparations using Image Lab software (BIO-RAD™). 200 ng of synthetic circular DNA vector samples were loaded onto a Tris-Acetate-EDTA gel, and electrophoresis was performed at 100V for 40 minutes, followed by staining with 1% EtBr for 20 minutes. For each synthetic circular DNA vector sample, target bands were identified according to their size, and the "Band detection sensitivity" was set as "Custom sensitivity" with a value of 50 in "Detection settings".

[0260] The above method yielded 1.7 mg of the single transcription unit vector (4537 bp) of FIG. 1A from 0.42 mg of plasmid (approximately a 4-fold increase), compared to 4.0 mg of the multiple transcription unit vector (8035 bp) of FIG. 2A from 0.75 mg of plasmid (a 5.3-fold increase). The single transcription unit vector was calculated to be 78% supercoiled as measured by the densitometric analysis discussed above. The multiple transcription unit vector was calculated to be 85% supercoiled by the same analysis. In comparison, a sample of a synthetic circular DNA vector (3466 bp) encoding a single gene (IL-12) driven by a CAG promoter was produced by the same method, yielding 2.5 mg of vector from 0.32 mg of plasmid (a 7.8-fold increase). The single gene vector was calculated to be 84% supercoiled as measured by the densitometric analysis discussed above. This data indicates that the cell-free production methods described herein can be used to effectively produce relatively large synthetic circular DNA vectors containing multiple genes, as well as smaller synthetic circular DNA vectors containing a single gene.

[0261] Example 2. Protein expression for each of the three therapeutic sequences of synthetic circular DNA vectors The 8035 bp multiple transcription unit synthetic circular DNA vector of SEQ ID NO: 37 (c3-Tx) was characterized for in vitro protein expression. HEK293T cells were seeded at a density of 200K cells per well in a 12-well plate with a clear flat bottom and incubated with vector at a concentration of 1 μg per well + 3 μL of lipofetamine 3000 per well + 2 μL of P3000 per well for 48 hours. Protein expression of sFlt3L, XCL1, and IL-12 by c3-Tx w...

Claims

1. A nucleic acid vector comprising a gene encoding a chemoattractant for dendritic cells, a gene encoding a growth factor or activator for dendritic cells, and a gene encoding a signaling protein for lymphocytes, Optionally, the gene encoding a dendritic cell chemoattractant is XCL1, XCL2, CCL5, or CCL4; Optionally, the dendritic cell growth factor or activation factor is FLT3L, GMCSF, or CD40; Optionally, the lymphocyte signaling protein is IL-12, IL-15, CXCL9, or CXCL10; Optionally, the nucleic acid vector comprises a first promoter, a second promoter, and a third promoter driving expression of a gene encoding a chemoattractant for the dendritic cells, a gene encoding a growth factor or activator for the dendritic cells, and a gene encoding a signaling protein for the lymphocytes, respectively, or the nucleic acid vector comprises a single promoter driving expression of a gene encoding a chemoattractant for the dendritic cells, a gene encoding a growth factor or activator for the dendritic cells, and a gene encoding a signaling protein for the lymphocytes. Nucleic acid vectors.

2. A nucleic acid vector comprising a gene encoding XCL1, a gene encoding FLT3L, and a gene encoding IL-12.

3. A nucleic acid vector comprising a gene encoding XCL1, a gene encoding FLT3L, and a gene encoding IL-15.

4. A nucleic acid vector comprising a gene encoding XCL1, a gene encoding GMCSF, and a gene encoding IL-15.

5. 5. The nucleic acid vector of claim 1, wherein the nucleic acid vector is a DNA vector, optionally wherein the DNA vector is a circular DNA vector, optionally wherein the circular DNA vector is a synthetic circular DNA vector lacking a bacterial origin of replication, a drug resistance gene, and a recombination site.

6. 6. The nucleic acid vector of any one of claims 1 to 5, wherein the nucleic acid vector is 2.5 kb to 20 kb in length, optionally the nucleic acid vector is 3.5 kb to 10 kb in length, and / or optionally the nucleic acid vector comprises, in 5'→3' orientation, a gene encoding a growth factor or activator of the dendritic cells, a gene encoding a signaling protein of the lymphocytes, and a gene encoding a chemoattractant of the dendritic cells.

7. In the 5'→3' direction, (a) a promoter; (b) a sequence encoding a self-replicating RNA molecule, the sequence comprising (i) a sequence encoding a replicase that transcribes RNA from the self-replicating RNA molecule, and (ii) a sequence encoding one or more heterologous proteins; (c) a polyadenylation sequence; and A circular DNA vector comprising: The circular DNA vector lacks a bacterial origin of replication and / or a drug resistance gene; Optionally, the circular DNA vector is a synthetic circular DNA vector lacking recombination sites; and / or Optionally, the sequence encoding the one or more heterologous proteins comprises a gene encoding one or more immunomodulatory proteins, and optionally, the gene encoding the one or more immunomodulatory proteins comprises a gene encoding a chemoattractant for dendritic cells, a gene encoding a growth factor or activator for dendritic cells, and / or a gene encoding a lymphocyte signaling protein; Optionally, the gene encoding a dendritic cell chemoattractant is XCL1, XCL2, CCL5, or CCL4; and / or Optionally, said dendritic cell growth factor or activator is FLT3L, GMCSF, or CD40, and / or said lymphocyte signaling protein is IL-12, IL-15, CXCL9, or CXCL10. Circular DNA vector.

8. 8. The circular DNA vector of claim 7, wherein the nucleic acid vector is 10 kb to 13 kb in length, optionally the nucleic acid vector is about 11.5 kb in length, and / or optionally the circular DNA vector comprises, in 5' to 3' orientation, a gene encoding a growth factor or activator of the dendritic cells, a gene encoding a signaling protein of the lymphocytes, and a gene encoding a chemoattractant of the dendritic cells.

9. A pharmaceutical composition comprising: (a) the nucleic acid vector according to any one of claims 1 to 6, or the circular DNA vector according to claim 7 or 8; and (b) a pharma- ceutically acceptable carrier.

10. A nucleic acid vector according to any one of claims 1 to 6, a circular DNA vector according to claim 7 or 8, or a pharmaceutical composition according to claim 9 for treating cancer, comprising: Optionally, the nucleic acid vector, circular DNA vector, or pharmaceutical composition is administered intratumorally; and / or Optionally, the nucleic acid vector, circular DNA vector, or pharmaceutical composition is administered systemically and / or the nucleic acid vector, circular DNA vector, or pharmaceutical composition is administered in combination with an additional anti-cancer therapy. The nucleic acid vector, the circular DNA vector, or the pharmaceutical composition.

11. A nucleic acid vector according to any one of claims 1 to 6, a circular DNA vector according to claim 7 or 8, or a pharmaceutical composition according to claim 9 for modulating a tumor microenvironment, comprising: The method is characterized in that it is used in combination with an electric field to promote the introduction of the nucleic acid molecule or the circular DNA vector into tumor cells; Optionally, the electric field is a pulsed electric field; Optionally, the pulsed electric field is delivered via an electrode placed within the tumor; and / or Optionally, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered intratumorally; and / or Optionally, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered systemically; and / or Optionally, the nucleic acid vector, the circular DNA vector, or the pharmaceutical composition is administered in combination with an additional anti-cancer therapy. The nucleic acid vector, the circular DNA vector, or the pharmaceutical composition.

12. A non-viral nucleic acid vector comprising a gene encoding a chemoattractant for dendritic cells for modulating a tumor microenvironment, The method is characterized in that it is used in combination with an electric field to promote the introduction of the non-viral nucleic acid vector into tumor cells, Optionally, the electric field is a pulsed electric field; Optionally, the pulsed electric field is delivered via an electrode placed within the tumor; and / or Optionally, the non-viral nucleic acid vector is administered intratumorally; and / or Optionally, the non-viral nucleic acid vector is administered systemically; and / or Optionally, the non-viral nucleic acid vector is administered in combination with an additional anti-cancer therapy. Non-viral nucleic acid vectors.

13. A synthetic circular DNA vector encoding a protein for expression in a tumor microenvironment, comprising: A synthetic circular DNA vector, characterized in that it is used in combination with an electric field to promote the introduction of said synthetic circular DNA vector into tumor cells.

14. The electrolysis is a pulsed electric field, Optionally, the pulsed electric field is delivered via an electrode placed within the tumor; and / or Optionally, the synthetic circular DNA vector is administered intratumorally; and / or Optionally, the synthetic circular DNA vector is administered systemically; and / or Optionally, the synthetic circular DNA vector is administered in combination with an additional anti-cancer therapy. The synthetic circular DNA vector of claim 13.

15. the protein is selected from the group consisting of a dendritic cell chemoattractant, a dendritic cell growth factor or activator, and a lymphocyte signaling protein, or the synthetic circular DNA vector encodes a gene encoding a dendritic cell chemoattractant, a gene encoding a dendritic cell growth factor or activator, and a gene encoding a lymphocyte signaling protein; and / or Optionally, the synthetic circular DNA vector does not contain any origin of replication, drug resistance gene, or recombination site; and / or Optionally, the synthetic circular DNA vector is free of a bacterial backbone.

15. A synthetic circular DNA vector according to claim 13 or 14.

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