Combining deoxyribonuclease enzymes and cell therapy for the treatment of cancer

Abstract

The present invention relates to a method of treating cancer utilizing a combination of adoptive cellular immunotherapy comprising a DNase enzyme and cells expressing a chimeric antigen receptor (CAR) or a T-cell receptor (TCR). In certain embodiments, the DNase enzyme can be given parenterally or encoded by a vector or secreted by cells containing the TCR or CAR.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 135,438, filed January 8, 2021, and U.S. Provisional Patent Application No. 63 / 148,304, filed February 11, 2021, the disclosures of which are incorporated by reference in their entireties herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 7, 2022, is named 252732_000023_SL.txt and is 117,776 bytes in size.

[0003] FIELD OF THEINVENTION The present invention relates to methods of treating cancer utilizing a combination of desoxyribonuclease enzymes and adoptive cellular immunotherapy involving cells expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Background technology]

[0004] Summary of the Invention

[0005] The present invention addresses a great need in the art for new and more effective treatments for cancer. Effect of the Invention

[0006] In one aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a deoxyribonuclease (DNase) enzyme and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the DNase enzyme is used in an amount effective to reduce expression of an immunosuppressive protein in tumor tissue and / or increase the content of CAR-expressing or TCR-expressing cells in the tumor tissue.

[0007] In another aspect, the invention provides a method of preventing one or more side effects of CAR-T cell therapy in a subject in need thereof, comprising administering a deoxyribonuclease (DNase) enzyme to the subject, wherein the DNase enzyme is administered simultaneously or sequentially with the CAR-T cell therapy. In some embodiments, the side effects of CAR-T cell therapy are selected from neurological side effects, cytokine release syndrome (CRS), neutropenia, anemia, fever, febrile neutropenia, and thrombocytopenia.

[0008] In a further aspect, the present invention provides a method for preventing recurrence of a disease following treatment of said disease with CAR-T cell therapy in a subject, comprising administering a deoxyribonuclease (DNase) enzyme to the subject, wherein said DNase enzyme is administered simultaneously or sequentially with said CAR-T cell therapy.

[0009] In yet another aspect, the present invention provides a method of increasing the persistence of CAR-T cells in the blood of a subject receiving CAR-T cell therapy, comprising administering to the subject a deoxyribonuclease (DNase) enzyme, wherein said DNase enzyme is administered simultaneously or sequentially with said CAR-T cell therapy.

[0010] In a further aspect, the present invention provides a method of increasing the efficacy of CAR-T cell therapy in a subject in need thereof, comprising administering a deoxyribonuclease (DNase) enzyme to the subject, wherein said DNase enzyme is administered simultaneously or sequentially with said CAR-T cell therapy.

[0011] In some embodiments of any of the above methods, the DNase enzyme is administered as a DNase enzyme protein.

[0012] In some embodiments of any of the above methods, the DNase enzyme is encoded by a vector. In some embodiments, the vector is a gene therapy vector. In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding the DNase enzyme. In some embodiments, the promoter is a liver-specific promoter. In some embodiments, the promoter is specific to a tumor-originating tissue or a metastatic target tissue.

[0013] In some embodiments of any of the above methods, the DNase enzyme is encoded by an mRNA molecule.

[0014] In some embodiments of any of the above methods, the DNase enzyme is administered parenterally or to the site of the tumor.

[0015] In some embodiments of any of the above methods, the DNase enzyme is co-expressed by a cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0016] In some embodiments of any of the above methods, the CAR-expressing or TCR-expressing cells are administered parenterally or to the site of the tumor. In some embodiments of any of the above methods, the CAR-expressing or TCR-expressing cells are single targets or multiple targets.

[0017] In some embodiments of any of the above methods, the CAR is selected from the group consisting of: (i) CD5, CD7, CD19, CD28, mesothelin, CD123, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, FR-1, c-MET, EGFR / CD133, IL13Ra2, HER2, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV a tumor antigen selected from E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, and mut hsp70-2; (ii) an antigen associated with a solid tumor;(iii) mesothelin, EGFRvIII, GD2, CLDN6, Tn Ag, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, legumain, CD171, PSCA, TARP, MAD-CT-1, Lewis Y, CD24, folate receptor alpha, folate receptor beta, ERBBs, MUC1, EGFR, NCAM, PDGFR-beta, MAD-CT-2, Fos-related antigen, SSEA-4, neutrophil elastase, CAIX, HPV E6 E7, ML-IAP, NA17, ALK, androgen receptor polysialic acid acid), TRP-2, CYP1B1, PLAC1, GloboH, NY-BR-1, sperm protein 17, HMWMAA, beta human chorionic gonadotropin, AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, and mut hsp 70-2; (iv) solid tumor associated antigens present in / on mesothelioma, lung cancer, pancreatic cancer, esophageal adenocarcinoma, ovarian cancer, breast cancer, colorectal cancer, bladder cancer, or any combination thereof; (v) tumor antigens associated with blood cancer; (vi) tumors present in diseases selected from acute leukemia (including, but not limited to, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL)), and chronic leukemia (including, but not limited to, chronic myelogenous leukemia (CIVIL) and chronic lymphocytic leukemia (CLL)). (vii) an antigen binding domain capable of specifically binding to one or more antigens selected from tumor antigens present in therapy-resistant cancers, including, but not limited to, myeloperoxidase and neutrophil elastase, including any protein or peptide that is a non-physiological variant, mutation, fragment or derivative of ScFv, Fab, CDR loop, CDR graft, dAb and nanobody fragments, whether produced by cell line, hybridoma expression, in vitro expression by bacteria, yeast or other host cells, or any other synthetic method;

[0018] In some embodiments of any of the above methods, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4.

[0019] In some embodiments of any of the above methods, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:53.

[0020] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously once on the same day as or the day before administration of the CAR-expressing or TCR-expressing cells.

[0021] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously once a week starting after administration of the CAR-expressing or TCR-expressing cells.

[0022] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously once a month starting after administration of the CAR-expressing or TCR-expressing cells.

[0023] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously one week prior to administration of the CAR-expressing or TCR-expressing cells.

[0024] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously one month prior to administration of the CAR-expressing or TCR-expressing cells.

[0025] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously once starting from 21 days prior to administration of the CAR-expressing cells or TCR-expressing cells and continuing until 14 days after administration.

[0026] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously for at least 14 days following administration of the CAR-expressing cell or TCR-expressing cell.

[0027] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously for at least 16 days following administration of the CAR-expressing cell or TCR-expressing cell.

[0028] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously for at least 3 days before, during, or after administration of the CAR-expressing or TCR-expressing cells.

[0029] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously for at least 7 days following administration of the CAR-expressing or TCR-expressing cells.

[0030] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously for at least 7 days prior to administration of the CAR-expressing or TCR-expressing cells.

[0031] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously for at least 3 days prior to or after administration of the CAR-expressing cell or TCR-expressing cell.

[0032] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously at a dose of at least 250 μg / kg / day.

[0033] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously at a dose of at least 600 μg / kg / day.

[0034] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously at a dose of at least 900 μg / kg / day.

[0035] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously at a dose of at least 2.5 mg / kg / day.

[0036] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously at a dose of at least 5 mg / kg / day.

[0037] In some embodiments of any of the above methods using a DNase enzyme protein, the DNase enzyme protein is injected intravenously at at least 7.5 mg / kg / day.

[0038] In some embodiments of any of the above methods, the CAR-expressing cell or TCR-expressing cell is further modified to express an immune checkpoint inhibitor molecule.

[0039] In some embodiments of any of the above methods, the DNase enzyme is used in an amount that increases the effectiveness of the CAR-expressing cells in a hypoxic environment.

[0040] In some embodiments of any of the above methods using a vector encoding a DNase enzyme, the vector encoding the DNase enzyme is injected intravenously and / or at the tumor site at least 14 days prior to administration of the CAR-expressing or TCR-expressing cells.

[0041] In some embodiments of any of the above methods using a vector encoding a DNase enzyme, the vector encoding the DNase enzyme is injected intravenously and / or at the tumor site at least 3 days prior to administration of the CAR-expressing or TCR-expressing cells.

[0042] In some embodiments of any of the above methods using a vector encoding a DNase enzyme, the vector encoding the DNase enzyme is injected intravenously and / or at the tumor site simultaneously with administration of the CAR-expressing or TCR-expressing cells.

[0043] In some embodiments of any of the above methods using a vector encoding a DNase enzyme, the vector encoding the DNase enzyme is injected intravenously and / or at the tumor site three days after administration of the CAR-expressing or TCR-expressing cells.

[0044] In some embodiments of any of the above methods, the CAR-expressing cell is a CAR T cell.

[0045] In separate aspects, the invention provides cells expressing (i) a deoxyribonuclease (DNase) enzyme and (ii) a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR). In some embodiments, the cells further express an immune checkpoint inhibitor molecule. In some embodiments, the CAR or TCR is single target or multi-target. In some embodiments, the CAR is (i) CD5, CD7, CD19, CD28, mesothelin, CD123, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, FR-1, c-MET, EGFR / CD133, IL13Ra2, HER2, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG(TMPRSS2(ii) a tumor antigen selected from among an antigen associated with a solid tumor; (iii) an antigen associated with a tumor, selected from the group consisting of mesothelin, EGFRvIII, GD2, CLDN6, Tn Ag, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, legumain, CD171, PSCA, TARP, MAD-CT-1, Lewis Y, CD24, folate receptor alpha, folate receptor beta, ERBBs, MUC1, EGFR, NCAM, PDGFR-beta, MAD-CT-2, Fos-related antigen, SSEA-4, neutrophil elastase, CAIX, HPV E6 E7, ML-IAP, NA17, ALK, androgen receptor polysialic acid, TRP-2, CYP1B1, PLAC1, GloboH, NY-BR-1, sperm protein 17, HMWMAA, beta human chorionic gonadotropin, AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, and mut hsp70-2; (iv) solid tumor associated antigens present in / on mesothelioma, lung cancer, pancreatic cancer, esophageal adenocarcinoma, ovarian cancer, breast cancer, colorectal cancer, bladder cancer, or any combination thereof; (v) tumor antigens associated with blood cancer; (vi) tumors present in diseases selected from acute leukemia (including, but not limited to, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL)), and chronic leukemia (including, but not limited to, chronic myelogenous leukemia (CIVIL) and chronic lymphocytic leukemia (CLL)). and (vii) an antigen binding domain capable of specifically binding to one or more antigens selected from tumor antigens present in therapy-resistant cancers, including, but not limited to, myeloperoxidase and neutrophil elastase, including any protein or peptide that is a non-physical variant, mutation, fragment or derivative of ScFv, Fab, CDR loop, CDR graft, dAb and Nanobody fragments, whether produced by cell line, hybridoma expression, in vitro expression by bacteria, yeast or other host cells, or any other synthetic method. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4. In some embodiments, the deoxyribonuclease enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:53.

[0046] These and other aspects of the invention will become apparent to those skilled in the art in the following description, claims and drawings. [Brief description of the drawings]

[0047] [Figure 1] Figure 1 shows the tumor volumes (mm2) measured with calipers and estimated using the ellipsoid formula for the control, CAR19 plus DNase I, CAR19, and DNase I experimental groups. Intravenous (IV) injection of human recombinant DNase I enzyme provided substantial efficacy enhancement to CD19 CAR T cells. [Diagram 2]Figure 2 shows survival graphs for the control, CAR19 plus DNase I, CAR19, and DNase I experimental groups, demonstrating the survival benefit of the combination of CAR T cells and AAV DNase I gene transfer. [Figure 3A-C] Figures 3A-3C show flow cytometry analysis showing the percentage of metastatic MC32a cells expressing PD-L1. Compared to controls (Figure 3), PD-L1 expression was suppressed in metastatic MC32a cells from metastatic lesions of mice treated with AAV-DNase I gene transfer (Figure 3B) and in combination with CAR T cells (Figure 3C). [Figure 4] Figure 4 shows a graph of the percentage of carcinoembryonic antigen (CEA)-targeted CAR T cells in the parenchyma of MC32a metastatic lesions. The data show an increase in the percentage of CAR T cells in metastatic lesions from mice treated with a combination of CAR T cells and AAV DNase I gene transfer. [Diagram 5] Figures 5A-5B show schematics of the construction of exemplary viral vectors. Figure 5A shows a diagram of mDNaseI mut P2A 4D5 IgG4 CAR (4,237bp) containing the EF-1α promoter and CD3ζ domain. Figure 5B shows the complete sequence map of the viral vector containing mDNase I P2A IgG4 CAR (12,146bp). [Figure 6] Figure 6 shows a graph of DNase activity analyzed using a fluorescent probe in the culture medium of DNase I FL1-CAR-T, FL1-CAR-T, and non-transduced control T cells. Activity was measured as pM / s (picometers per second). The data shows a gradual increase in deoxyribonuclease activity in the culture medium of DNase I FL1-CAR-T. [Figure 7]Figure 7 shows the evaluation of cytotoxicity by lactase dehydrogenase (LDH) release assay for FL1-CAR T, DNase I FL1-CAR-T and non-transduced (mock) control T cells co-incubated with Raji-FL1 cells. Reprogramming T cells to simultaneously express CAR and DNase I enzyme resulted in superior cytotoxicity against target lymphoma cells. E:T ratio, effector to target cell ratio. [Figure 8] Figure 8 shows the tumor volumes (mm2) measured with calipers and estimated using the ellipsoid formula for the experimental groups of placebo control (group 1), FL1 CAR T cells (group 2) and FL1 CAR T cells expressing a highly active actin-resistant mutant DNase I (SEQ ID NO: 5) (group 3). Reprogramming T cells to simultaneously express CAR and DNase enzyme provided superior efficacy for such dual-reprogrammed T cells. [Figure 9] Figure 9 shows the number of CAR-T cells in peripheral blood (determined by flow cytometric detection using biotinylated protein L and streptavidin conjugated with FITC). The data show that the combination of DNase and CAR-T cell therapy significantly (p<0.05 for all) increases the persistence of CAR-T cells in the blood. [Figure 10] FIG. 10 demonstrates that the combination of DNase and CAR-T cell therapy enhances animal survival. [Figure 11] FIG. 11 shows the percentage of animals with cytokine release syndrome (CRS), demonstrating that the use of DNase enhances the safety of CAR-T therapy and protects against CAR-T-associated side effects. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Detailed Description of the Invention The present invention relates to a method of treating cancer utilizing a combination of a desoxyribonuclease enzyme and adoptive cellular immunotherapy comprising cells expressing a chimeric antigen receptor (CAR) or a T-cell receptor (TCR). In certain embodiments, the desoxyribonuclease enzyme can be given parenterally or encoded by a vector or secreted by cells comprising the TCR or CAR.

[0049] definition Unless otherwise defined, all 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.

[0050] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0051] The term "about" or "approximately" includes being within a statistically meaningful range of a value. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The permissible variations encompassed by the term "about" or "approximately" depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0052] DNase enzyme In some embodiments, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a DNase enzyme and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In a related embodiment, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a DNase enzyme and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the DNase enzyme is effective to reduce expression of an immunosuppressive protein in tumor tissue and / or to increase the content of cells comprising a chimeric antigen receptor or a T cell receptor in tumor tissue. In some embodiments, the DNase enzyme is administered parenterally as a DNase enzyme protein. In some embodiments, the DNase enzyme is encoded by a vector. In some embodiments, the DNase enzyme is co-expressed by cells comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the DNase enzyme is used to increase the effectiveness of CAR-expressing cells in a hypoxic environment.

[0053] Non-limiting examples of DNase enzymes that may be useful in any of the methods of the invention include, for example, DNase I, DNase X, DNase gamma, DNase1L1, DNase1L2, DNase1L3, DNase II, DNase II alpha, DNase II beta, caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives, variants or analogs thereof.

[0054] In some embodiments, the DNase may be DNase I or a variant or derivative thereof.

[0055] In some embodiments, DNase I can be human DNase I or a variant or derivative thereof. In some embodiments, DNase I can be non-human DNase I or a variant or derivative thereof, such as, but not limited to, rodent (e.g., mouse) DNase I or a variant or derivative thereof.

[0056] In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to human DNase 1 enzyme. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to human DNase I enzyme.

[0057] In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 21-305 of the DNase 1-like 3 (D1L3) enzyme (SEQ ID NO: 51). In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of the DNase 1-like 3 (D1L3) enzyme (SEQ ID NO: 51).

[0058] In some embodiments, the DNase I comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 1. In some embodiments, the DNase I amino acid sequence is encoded by a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22. In some embodiments, the DNase I comprises the amino acid sequence of SEQ ID NO: 1 encoded by the nucleotide sequence of SEQ ID NO: 22.

[0059] In some embodiments, the DNase I comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 4. In some embodiments, the DNase I amino acid sequence is encoded by a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 23. In some embodiments, the DNase I comprises the amino acid sequence of SEQ ID NO: 4 encoded by the nucleotide sequence of SEQ ID NO: 23.

[0060] In some embodiments, the DNase I mutant may comprise one or more mutations in the actin binding site. In some embodiments, the one or more mutations in the actin binding site are selected from mutations at Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combination thereof. In some embodiments, one of the mutations in the actin binding site is a mutation at Ala-114.

[0061] In some embodiments, the DNase I mutant comprises one or more mutations that increase DNase activity. In some embodiments, the one or more mutations that increase DNase activity are selected from the group consisting of Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combination thereof. In some embodiments, the one or more mutations that increase DNase activity are selected from the group consisting of Q9R, E13R, N74K, and A114F.

[0062] In some embodiments, the DNase I mutants are H44C, H44N, L45C, V48C, G49C, L52C, D53C, D53R, D53K, D53Y, D53A, N56C, D58S, D58T, Y65A, Y65E, Y65R, Y65C, V66N, V67E, V67K, V67C, E69R, E69C, A114C, A114R, H44N:T46S, D53R:Y65A, D53R:E69R, H44A :D53R:Y65A, H44A:Y65A:E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T, and any combination thereof. In some embodiments, the DNase I variant is a long-acting form of DNase. In some embodiments, the DNase I variant is a high activity variant form of DNase. In some embodiments, the DNase I variant comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the DNase I variant comprises the amino acid sequence of SEQ ID NO:2.

[0063] In some embodiments, the DNase I variant comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 5. In some embodiments, the DNase I variant amino acid sequence is encoded by a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:21. In some embodiments, the DNase I variant amino acid sequence is encoded by a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the DNase I variant comprises the mutations Q9R, E13R, N74K and A114F. In some embodiments, the DNase I variant comprises the sequence of SEQ ID NO: 5. In some embodiments, the DNase I variant comprises the amino acid sequence of SEQ ID NO: 5 encoded by the nucleotide sequence of SEQ ID NO: 21.

[0064] In some embodiments, the DNase I variant comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 2. In some embodiments, the DNase I variant amino acid sequence is encoded by a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the DNase I variant comprises the mutations Q9R, E13R, N74K and A114F. In some embodiments, the DNase I variant comprises the sequence of SEQ ID NO: 2. In some embodiments, the DNase I variant comprises the amino acid sequence of SEQ ID NO: 2, which is encoded by the nucleotide sequence of SEQ ID NO:19.

[0065] In some embodiments, the DNase I variant consists of the sequence of SEQ ID NO:2 or SEQ ID NO:5.

[0066] In some embodiments of any of the methods of the invention, the sequence encoding the DNase comprises a secretory signal sequence that mediates efficient secretion of the enzyme into the hepatic porto-sinusoidal circulation upon administration of the vector to a subject. In some embodiments, the secretory signal sequence is selected from the group consisting of a DNase I secretory signal sequence, an IL2 secretory signal sequence, an albumin secretory signal sequence, a β-glucuronidase secretory signal sequence, an alkaline protease secretory signal sequence, and a fibronectin secretory signal sequence. In some embodiments, the secretory signal sequence comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In some embodiments, the secretory signal sequence comprises the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO:6). In some embodiments, the secretory signal sequence consists of the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO:6). In some embodiments, the secretory signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:7). In some embodiments, the secretory signal sequence comprises the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:7). In some embodiments, the secretory signal sequence consists of the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:7).

[0067] In some embodiments, the DNase enzyme is selected from the group consisting of DNase I, DNase X, DNase gamma, DNase1L1, DNase1L2, DNase1L3, DNase II, DNase II alpha, DNase II beta, caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives, variants or analogs thereof. In some embodiments, the DNase enzyme is DNase I or a mutant or derivative thereof. In some embodiments, the DNase I is human DNase I or a mutant or derivative thereof. In some embodiments, the DNase I mutant comprises one or more mutations in the actin binding site. In some embodiments, the one or more mutations in the actin binding site are selected from mutations at Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combination thereof. In some embodiments, one of the mutations in the actin binding site is a mutation at Ala-114. In some embodiments, the DNase I mutant comprises one or more mutations that increase DNase activity. In some embodiments, the one or more mutations that increase DNase activity are selected from the group consisting of Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combination thereof. In some embodiments, the one or more mutations that increase DNase activity are selected from the group consisting of Q9R, E13R, N74K, and A114F, and any combination thereof. In some embodiments, the DNase I variant comprises a sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments, the DNase I variant comprises the mutations Q9R, E13R, N74K, and A114F. In some embodiments, the DNase I variant comprises the sequence of SEQ ID NO: 5.In some embodiments, the DNase I variant consists of the sequence of SEQ ID NO:5. In some embodiments, the DNase I variant comprises a sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity with the sequence of SEQ ID NO:2. In some embodiments, the DNase I variant comprises the mutations Q9R, E13R, N74K, and A114F. In some embodiments, the DNase I variant comprises the sequence of SEQ ID NO:2. In some embodiments, the DNase I variant consists of ... The I mutants are H44C, H44N, L45C, V48C, G49C, L52C, D53C, D53R, D53K, D53Y, D53A, N56C, D58S, D58T, Y65A, Y65E, Y65R, Y65C, V66N, V67E, V67K, V67C, E69R, E69C, A114C, A114R, H44N:T46S, D53R:Y65A, D53R:E69R, H44A:T46S, :D53R:Y65A, H44A:Y65A:E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T, and any combination thereof. In some embodiments, the nucleic acid encodes a DNase I comprising the sequence of SEQ ID NO:4. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 85% or at least 90% or at least 95% identical to SEQ ID NO:23. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:23. In some embodiments, the nucleic acid encodes a DNase I comprising the sequence of SEQ ID NO:1. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 85%, or at least 90%, or at least 95% identical to SEQ ID NO: 22. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 85%, or at least 90%, or at least 95% identical to SEQ ID NO: 32.In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleic acid encodes a DNase I comprising the sequence of SEQ ID NO:24. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 85% or at least 90% or at least 95% identical to SEQ ID NO:29. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:29. In some embodiments, the nucleic acid encodes a DNase I comprising the sequence of SEQ ID NO:26. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 85% or at least 90% or at least 95% identical to SEQ ID NO:28. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:28. In some embodiments, the nucleic acid encodes a DNase I variant comprising the sequence of SEQ ID NO:5. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 85% or at least 90% or at least 95% identical to SEQ ID NO:21. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleic acid encodes a DNase I variant comprising the sequence of SEQ ID NO:2. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 85% or at least 90% or at least 95% identical to SEQ ID NO:19. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the DNase enzyme is a fusion protein comprising (i) the DNase enzyme or a fragment thereof linked to (ii) albumin or Fc or a fragment thereof. In some embodiments, the sequence encoding the DNase enzyme comprises a sequence encoding a secretory signal sequence, said secretory signal sequence mediating efficient secretion of the enzyme. In some embodiments, the secretory signal sequence is selected from the group consisting of a DNase I secretory signal sequence, an IL2 secretory signal sequence, an albumin secretory signal sequence, a β-glucuronidase secretory signal sequence, an alkaline protease secretory signal sequence, and a fibronectin secretory signal sequence.In some embodiments, the secretory signal sequence comprises the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO:6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO:7). In some embodiments, the secretory signal sequence consists of the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO:6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO:7). In some embodiments, the secretory signal sequence comprises a sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO:6) or a sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO:7). In some embodiments, the sequence encoding the secretory signal sequence comprises a nucleotide sequence that is at least 85%, or at least 90%, or at least 95% identical to SEQ ID NO:20. In some embodiments, the sequence encoding the secretory signal sequence comprises the nucleotide sequence of SEQ ID NO:20. In some embodiments, the secretory signal sequence comprises the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In some embodiments, the secretory signal sequence consists of the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In some embodiments, the secretory signal sequence comprises a sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In some embodiments, the sequence encoding the secretory signal sequence comprises the nucleotide sequence of SEQ ID NO: 27. In another embodiment, the nucleic acid of the invention comprises the sequence of SEQ ID NO: 30 or SEQ ID NO: 31.

[0068] AAV vectors In some embodiments, the DNase enzyme disclosed herein may be encoded by a vector (e.g., a gene therapy vector). In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a deoxyribonuclease (DNase) enzyme.

[0069] In some embodiments, the vector is a viral vector. Non-limiting examples of viral vectors include, for example, adeno-associated viral (AAV) vectors, adenoviral vectors, retroviral vectors (e.g., lentiviral vectors), and hepatotropic viral vectors (e.g., Hepatitis B virus (HBV) vectors).

[0070] In one embodiment, the vector of the present invention is a parvovirus vector, such as an adeno-associated virus (AAV) vector. The term "parvovirus" as used herein encompasses the Parvoviridae family, which includes autonomously replicating parvoviruses and dependoviruses. Autonomous parvoviruses include members of the Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus genera. Autonomous parvoviruses include, but are not limited to, minute virus of mice, bovine parvovirus, canine parvovirus, chicken parvovirus, feline pancytopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, B19 virus, and any other autonomous parvovirus now known or later discovered. Other autonomous parvoviruses are known to those of skill in the art. See, for example, Bernard N. Fields et al., Virology, Vol. 2, Chapter 69 (4th Edition, Lippincott-Raven Publishers).

[0071] In some embodiments, the parvovirus vector is a single stranded parvovirus vector, such as an AAV vector. AAV (Dependovirus genus) typically infects humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6) or primates (e.g., serotypes 1 and 4), or other warm-blooded animals (e.g., bovine, canine, equine, and ovine AAV). Further information on parvoviruses and other members of the Parvoviridae family is provided, for example, in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69, Fields Virology (3rd ed., 1996).

[0072] The AAV vectors disclosed herein can be derived from any AAV serotype or various genomes (e.g., single-stranded or self-complementary), including combinations of serotypes (e.g., "pseudotype" AAV). "Serotypes" are traditionally defined based on the lack of cross-reactivity between antibodies to one virus and another. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the sequences of VP1, VP2, and / or VP3). Non-limiting examples of AAV serotypes that can be used to develop the AAV expression vectors of the invention include, for example, AAV serotype 1 (AAV1), AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO (e.g., as disclosed in U.S. Pat. No. 9,790,472, International Patent Publication Nos. WO2017180857 and WO2017 / 180861), AAV-LK03, AAV-LK06, AAV-LK12 ... Examples of AAV vectors that can be used include AAV-KP1 (e.g., as disclosed in Wang et al., Mol. Ther., 2015, 23(12):1877-1887), AAV-KP1 (e.g., as disclosed in International Patent Application Publication No. WO2019191701A1), AAVhu37 (e.g., as disclosed in International Patent Application Publication No. WO2017180857), AAVrh64R1 (e.g., as disclosed in International Patent Application Publication No. WO2017180857), Anc80 (based on the predicted ancestry of serotypes AAV1, AAV2, AAV8, and AAV9; see Zinn et al., Cell Rep., 2015, 12(67):1056-1068), avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any chimeras thereof. See, e.g., Fields et al., Virology, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers).

[0073] A number of putative new AAV serotypes and clades have been identified (e.g., Gao et al., (2004) J. Virology 78:6381-6388; Morris et al., (2004) Virology 33:375-383). The genomic sequences of the various serotypes of AAV and autonomous parvoviruses, as well as the sequences of the terminal repeats, Rep proteins, and capsid subunits, are known in the art and are described, for example, in Srivistava et al., (1983) J. Virology 45:555; Chiorini et al., (1998) J. Virology 71:6823; Chiorini et al., (1999) J. Virology 73:1309; Bantel-Schaal et al., (1999) J. Virology 73:939; Xiao et al., (1999) J. Virology 73:3994; Muramatsu et al., (1996) Virology 221:208; Shade et al., (1986) J. Virology 58:921; Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854; Morris et al., (2004) Virology 33-:375-383; GenBank Accession No. U89790; GenBank Accession No. J01901; GenBank Accession No. AF043303; GenBank Accession No. AF085716; GenBank Accession No. NC006152; GenBank Accession No. Y18065; GenBank Accession No. NC 006260; GenBank Accession No. NC_006261; International Patent Publications WO00 / 28061, WO99 / 61601, WO98 / 11244; and U.S. Patent No. 6,156,303, the disclosures of which are incorporated by reference herein for teaching nucleic acid and amino acid sequences of parvoviruses and AAV.

[0074] The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank unique coding nucleotide sequences for nonstructural replication (Rep) and structural (VP) proteins. The VP proteins (VP1, VP2, and VP3) form the capsid. The terminal 145 nucleotides are self-complementary and organized in such a way that an energetically stable intramolecular duplex can form, forming a T-shaped hairpin. These hairpin structures serve as the origin of viral DNA replication and act as a primer for the cellular DNA polymerase complex. After wtAAV infection in mammalian cells, the Rep genes (i.e., Rep78 and Rep52) are expressed from the P5 and P19 promoters, respectively, and both Rep proteins have a function in the replication of the viral genome.

[0075] In some embodiments, a recombinant AAV (rAAV) vector comprises one or more nucleotide sequences of interest flanked by at least one parvovirus or AAV inverted terminal repeat (ITR). Such rAAV vectors can be replicated and packaged into viral particles when produced in packaging cells expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). The term "AAV Cap protein" or "AAV capsid protein" as used herein refers to a polypeptide having at least one functional activity of a native AAV Cap protein (e.g., VP1, VP2, VP3). Examples of functional activities of Cap proteins (e.g., VP1, VP2, VP3) include the ability to induce capsid formation, promote accumulation of single-stranded DNA, promote AAV DNA packaging into capsids (i.e., encapsidation), bind to a cellular receptor, and promote entry of virions into host cells.

[0076] In some embodiments, the AAV vector may contain a desired protein or protein variant. As used herein, "variant" refers to an amino acid sequence in which one or more amino acids are changed. A variant may have "conservative" changes, where the substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have "non-conservative" changes, e.g., replacement of glycine with tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both.

[0077] AAV vectors are packaged in AAV viral capsids. The sequence of AAV viral capsid proteins defines many characteristics of a particular AAV vector. For example, capsid proteins affect, among others, capsid structure and assembly, interaction with AAV nonstructural proteins such as Rep and AAP proteins, interaction with host body fluids and extracellular matrix, clearance of virus from blood, vascular permeability, antigenicity, reactivity to neutralizing antibodies, tissue / organ / cell type tropism, cell adhesion and internalization efficiency, intracellular trafficking pathway, virion uncoating rate. The sequence of capsid proteins (e.g., VP3) can be altered to enhance delivery to the liver.

[0078] AAV constructs may include sequences encoding one or more capsid proteins (VP1 and / or VP2, and / or VP3 capsid proteins, preferably simply the VP3 capsid protein) that package the polynucleotide sequences disclosed herein. Sequences encoding the capsid protein(s) for use in the context of the present invention may be obtained from any of the 42 known serotypes, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, Anc80, or from newly developed AAV-like particles obtained, e.g., by capsid shuffling techniques and / or the use of AAV capsid libraries. For some non-limiting examples of capsid protein sequences, see, e.g., U.S. Pat. Nos. 9,790,472; 9,677,089; 7,282,199; International Patent Publication WO2015 / 054653, WO2017 / 180857 (AAV8, AAV9, AAVrhlO, AAVhu37, AAVrh64R1), WO2017 / 180861 (AAVrhlO), WO2017 / 180854 (AAV8 variants), and Wang et al., Mol. Ther., 2015,23(12):1877-1887 (AAV8, AAVrhlO, AAV3B, and AAV-LK03). When the sequence encoding the capsid protein is derived from an AAV serotype different from the ITRs, the AAV construct is known as a "hybrid" parvovirus genome (i.e., the AAV capsid and the AAV terminal repeat(s) are from different AAVs) as described in International Patent Application Publication WO 00 / 28004 and Chao et al., (2000) Molecular Therapy 2:619.

[0079] In some embodiments, the capsid protein(s) mediate efficient targeting of the AAV vector to the liver. In some embodiments, the capsid protein(s) mediate preferential targeting of the AAV vector to the liver. Some capsid proteins (e.g., Anc80, VP3 of AAV8 and AAV3B) are naturally liver-specific. The present invention also encompasses the use of AAV capsid mutants that enhance liver targeting and / or liver specificity. Non-limiting examples of such point mutations to the AAV8 capsid sequence include, for example, S279A, S671A, K137R, and T252A, as well as the AAV8 capsid mutations disclosed in International Patent Application Publication WO2017 / 180854 (e.g., AAV3G1, AAVT20, or AAVTR1, amino acids 263-267 [e.g., 263NGTSG267→SGTH or 263NGTSG267→SDTH ("NGTSG" as SEQ ID NO: 54)). disclosed as SEQ ID NO:55; "SGTH" is disclosed as SEQ ID NO:56), and / or amino acids 457 to 459 [e.g., 457TAN459→SRP], and / or amino acids 455 to 459 [e.g., 455GGTAN459→DGSGL] ("GGTAN" is disclosed as SEQ ID NO:57; "DGSGL" is disclosed as SEQ ID NO:58), and / or a VP3 mutation at amino acids 583 to 597).

[0080] In some embodiments, the capsid protein(s) mediate efficient targeting of AAV vectors to the nervous system. In some embodiments, the capsid protein(s) mediate preferential targeting of AAV vectors to both the liver and the nervous system. Some capsid proteins naturally target both the liver and the nervous system. The present invention also encompasses the use of AAV capsid mutants that enhance targeting to the liver and / or nervous system.

[0081] In some embodiments, the capsid protein comprises one or more mutations that improve the efficiency and / or specificity of delivery of the vector to the liver and / or nervous system compared to the corresponding wild-type capsid protein. In one embodiment, the improved efficiency and / or specificity of delivery of the vector to the liver substantially enhances expression of an enzyme in the liver compared to other tissues and organs. In one embodiment, the improved efficiency and / or specificity of delivery of the vector to the nervous system substantially enhances expression of an enzyme in the nervous system compared to other tissues and organs. In one embodiment, the capsid protein comprises VP3. In one embodiment, the one or more mutations in the capsid protein are selected from the group consisting of S279A, S671A, K137R, T252A, and any combination thereof. In one embodiment, the one or more mutations in the capsid protein include the mutation K137R. In one embodiment, the capsid protein comprises the sequence of SEQ ID NO:34. In one embodiment, the capsid protein consists of the sequence of SEQ ID NO:34.

[0082] In some embodiments, the capsid protein comprises one or more mutations selected from the group consisting of S279A, S671A, K137R, T252A, and any combination thereof. In one embodiment, the one or more mutations in the capsid protein comprise the mutation K137R. In one embodiment, the capsid protein comprises the sequence of SEQ ID NO:3 [Anc80]. In one embodiment, the capsid protein consists of the sequence of SEQ ID NO:3 [Anc80]. In one embodiment, the capsid protein comprises the sequence of SEQ ID NO:9 [Anc80]. In one embodiment, the capsid protein consists of the sequence of SEQ ID NO:9 [Anc80]. In one embodiment, the capsid protein comprises the sequence of SEQ ID NO:34 [Anc80L65]. In one embodiment, the capsid protein consists of the sequence of SEQ ID NO:34 [Anc80L65]. In one embodiment, the capsid protein comprises the sequence of SEQ ID NO:35 [Anc80L65 variant]. In one embodiment, the capsid protein consists of the sequence of SEQ ID NO: 35 [Anc80L65 variant]. In one embodiment, the capsid protein is a mutant AAV8 capsid protein, such as AAV3G1, AAVT20 or AAVTR1, or another mutant capsid protein disclosed in International Patent Application Publication WO2017 / 180854 (e.g., comprising amino acids 263-267 [e.g., 263NGTSG267→SGTH or 263NGTSG267→SDTH ("NGTSG" is disclosed as SEQ ID NO: 54: "SGTH" is disclosed as SEQ ID NO: 55 and "SDTH" is disclosed as SEQ ID NO: 56)], and / or amino acids 457-459 [e.g., 457TAN459→SRP], and / or amino acids 455-459 [e.g., 455GGTAN459→DGSGL] ("GGTAN" is disclosed as SEQ ID NO: 57 and "DGSGL" is disclosed as SEQ ID NO: 58), and / or a VP3 mutation at amino acids 583-597). In one embodiment, the capsid protein comprises the sequence of SEQ ID NO: 47 [AAV-LK03]. In one embodiment, the capsid protein consists of the sequence of SEQ ID NO: 47 [AAV-LK03].In one embodiment, the capsid protein comprises the sequence [AAV-KP1] of SEQ ID NO: 49. In one embodiment, the capsid protein consists of the sequence [AAV-KP1] of SEQ ID NO: 49.

[0083] The AAV vectors disclosed herein include a nucleic acid encoding a deoxyribonuclease (DNase) enzyme. In various embodiments, the nucleic acid may also include one or more regulatory sequences that allow for expression and secretion of the encoded enzyme, such as a promoter, an enhancer, a polyadenylation signal, an internal ribosome entry site (IRES), a sequence encoding a protein transduction domain (PTD), a secretion signal sequence, and the like. Thus, in some embodiments, the nucleic acid may include a promoter region operably linked to the coding sequence to cause or improve expression of the protein of interest in the transfected cell. Such promoters may be ubiquitous, cell or tissue specific, strong, weak, regulated, chimeric, etc. promoters to allow efficient and stable production of the protein in, for example, the liver and / or nervous system. The promoter may be homologous or heterologous to the encoded protein, but generally, the promoters used in the disclosed methods are functional in human cells. Examples of regulated promoters include, but are not limited to, Tet on / off element-containing promoters, rapamycin-inducible promoters, tamoxifen-inducible promoters, and metallothionein promoters. Other promoters that can be used include, for example, promoters that are tissue-specific for the liver, nervous system, and / or intestine. Non-limiting examples of liver-specific promoters include, for example, albumin promoter (Alb), human alpha-1 antitrypsin (hAAT) promoter, thyroxine-binding globulin (TBG), apolipoprotein E hepatic control region promoter, apolipoprotein A-II (APOA2) promoter, serpin peptidase inhibitor, clade A, member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3A4) promoter, microRNA 122 (miR-122) promoter, liver-specific IGF-II promoter P1, mouse transthyretin (MTTR) promoter, and alpha-fetoprotein (AFP) promoter.Non-limiting examples of nervous system specific promoters include, for example, microglia specific promoters (e.g., F4 / 80, CD68, TMEM119, CX3CR1, CMV, Iba1), myeloid specific promoters (e.g., TTR, CD11b, c-fes), neuron specific promoters (e.g., CMV, NSE, synapsin [SynI, SynII], CaMKII, alpha-CaMKII, VGLUT1), and other neuronal and glial cell (e.g., oligodendrocyte astrocyte) type specific promoters (e.g., GFAP). Non-limiting examples of intestinal specific promoters include MUC2, villin, T3b, CB / CMV, GFAP, miCMV, CMV+I, tetO-CMV, and β-actin-CMV. Non-limiting examples of ubiquitous promoters include viral promoters such as the CMV promoter, the RSV promoter, the SV40 promoter, and cellular promoters such as the phosphoglycerate kinase (PGK) promoter, the EF1a promoter, the CMVE / CAG promoter system, and the β-actin promoter. In some embodiments, the promoter is specific to a tumor-originating tissue or a metastatic target tissue.

[0084] In some embodiments, any AAV vector disclosed herein may include one or more enhancers located upstream or downstream of the promoter. In some embodiments, the one or more enhancers may be selected from the group consisting of an apolipoprotein E (ApoE) enhancer, an alpha fetoprotein enhancer, a TTR enhancer, an LSP enhancer, an alpha 1-microglobulin / bikunin enhancer, an albumin gene enhancer (Ealb), an nPE2 enhancer, a Gal4 enhancer, a foxP2 enhancer, an aMef2 enhancer, a CMV enhancer, and any combination thereof. In one embodiment, the enhancer is an apolipoprotein E (ApoE) enhancer. In some embodiments, the enhancer is a hepatic control region (HCR) enhancer. In some embodiments, the enhancer comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity with SEQ ID NO: 17. In one embodiment, the enhancer comprises the sequence of SEQ ID NO: 17. In one embodiment, the enhancer consists of the sequence of SEQ ID NO:17.

[0085] In one embodiment of any of the above vectors, the nucleic acid further comprises a polyadenylation signal operably linked to the nucleotide sequence encoding the DNase enzyme.

[0086] In some embodiments, the nucleic acid further comprises a Kozak sequence. In one specific embodiment, the Kozak sequence comprises the sequence 5'-GCCGCCACC-3' (SEQ ID NO:33). In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:30. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:30. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:30. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:30. In one embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:30.

[0087] In one embodiment of any of the above vectors, the nucleic acid further comprises a post-transcriptional regulatory element. In one embodiment, the post-transcriptional regulatory element is a Woodchuck Hepatitis Post-transcriptional Regulatory Element (WPRE). In one embodiment, the WPRE does not encode a functional X protein. In one embodiment, the post-transcriptional regulatory element comprises a sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 16. In one embodiment, the post-transcriptional regulatory element comprises the sequence of SEQ ID NO: 16. In one embodiment, the post-transcriptional regulatory element consists of the sequence of SEQ ID NO: 16.

[0088] The AAV vector of the present invention may also comprise a non-degradable terminal repeat. As used herein, the expression "non-degradable terminal repeat" refers to a terminal repeat that is not recognized and degraded (i.e., "nicked") by the AAV Rep protein, such that the resolution of the terminal repeat is substantially reduced (e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, 98% or more compared to a degradable terminal repeat) or eliminated. Such a non-degradable terminal repeat may be a naturally occurring terminal repeat sequence (including modified versions thereof), e.g., derived from a parvovirus, including AAV, or derived from another virus, or, as a further alternative, may be partially or completely synthetic. The non-degradable terminal repeat may be a non-AAV viral sequence that is not recognized by the AAV Rep protein, or may be an AAV terminal repeat that has been modified (e.g., by insertion, substitution and / or deletion) so that it is no longer recognized by the AAV Rep protein. Furthermore, the non-degradable terminal repeat may be any terminal repeat that is not degradable under the conditions used to generate the viral vector. Additionally, the AAV terminal repeats can be modified such that their degradability by the AAV Rep protein is substantially reduced or eliminated. The non-degradable terminal repeats can be any inverted repeat sequence that forms a hairpin structure and cannot be nicked by the AAV Rep protein.

[0089] Inverted terminal repeats (ITRs) are typically present in at least two copies in an AAV vector and are typically adjacent to an expression cassette containing the nucleotide sequence(s) encoding a DNase enzyme. ITRs are usually present at the 5' and 3' ends of the nucleotide sequence(s) encoding the DNase enzyme, but do not have to be adjacent to it. The ITRs may be the same as or different from each other. The term "terminal repeat" includes any viral terminal repeat and / or partial or complete synthetic sequences that form a hairpin structure and function as an inverted terminal sequence, such as the "double D sequence" described in U.S. Patent No. 5,478,745 to Samulski et al. An "AAV terminal repeat" may be from any AAV, including, but not limited to, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, Anc80, or any other AAV now known or later discovered. The AAV terminal repeats do not need to have a wild-type sequence (e.g., the wild-type sequence can be modified by insertion, deletion, truncation, or missense mutation), so long as the terminal repeats mediate the desired function, such as replication, nicking, viral packaging, integration, and / or proviral rescue. The vector construct can include one or more (e.g., two) AAV terminal repeats, which can be the same or different. Furthermore, the one or more AAV terminal repeats can be from the same AAV serotype as the AAV capsid, or can be different. In certain embodiments, the vector construct includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and / or Anc80 terminal repeats.

[0090] Parvovirus ITR nucleotide sequences are typically palindromic sequences that contain mostly complementary, symmetrically arranged sequences, also referred to as "A", "B", and "C" regions. ITRs function as origins of replication that are sites with a "cis" role in replication, i.e., recognition sites for trans-acting replication proteins, such as Rep78 (or Rep68), that recognize the palindrome and specific sequences within the palindrome. One exception to the symmetry of ITR sequences is the "D" region of the ITRs. It is unique (has no complement within one ITR). Nicking of single-stranded DNA occurs at the junction between the A and D regions. It is the region where new DNA synthesis begins. The D region is usually located on one side of the palindrome and provides directionality to the nucleic acid replication process. Parvoviruses that replicate in mammalian cells typically have two ITR sequences. However, it is possible to engineer the ITRs so that the binding sites are on both strands of the A region and the D regions are symmetrically located, one on each side of the palindrome. On a double-stranded circular DNA template (such as a plasmid), Rep78- or Rep68-assisted nucleic acid replication then proceeds in both directions, and a single ITR is sufficient for parvoviral replication of the circular vector. Therefore, one ITR nucleotide sequence can be used in the context of the present invention. Two or another even number of conventional ITRs can be used.

[0091] Functional ITR sequences are necessary for replication, rescue and packaging of AAV virions. ITR sequences may be wild-type sequences or may have at least 80%, 85%, 90%, 95%, or 100% sequence identity with the wild-type sequence. ITR sequences may be altered, for example, by insertion, mutation, deletion or substitution of nucleotides, so long as they remain functional. In this context, functionality refers to the ability to direct packaging of the genome into a capsid shell and subsequently allow expression in transduced host or target cells.

[0092] AAV vectors can contain single-stranded or double-stranded (self-complementary) DNA. Single-stranded nucleic acid molecules are either sense or antisense strands, since both polarities allow gene expression equally. AAV vectors can further contain marker or reporter genes, such as antibiotic resistance genes, genes encoding fluorescent proteins (e.g., GFP), or genes encoding chemically, enzymatically, or otherwise detectable and / or selectable products (e.g., lacZ, aph, etc.) known in the art.

[0093] The AAV expression vector may include a nucleic acid that may include a secretory signal sequence that allows for secretion of the encoded DNase enzyme from the transduced cell. Non-limiting examples of such secretory signal sequences include, for example, the DNase I secretory signal sequence, the IL2 secretory signal sequence, the albumin secretory signal sequence, the β-glucuronidase secretory signal sequence, the alkaline protease secretory signal sequence, and the fibronectin secretory signal sequence.

[0094] In some embodiments, AAV8-based or Anc80-based vectors are used as expression vectors.AAV8 and Anc80 vectors are particularly suitable for liver targeting and expression.For example, both AAV8 and Anc80 vectors can transfect hepatocytes with higher efficiency compared to AAV2 vectors.Both AAV8 and Anc80 induce less neutralizing antibody than some other AAV vectors.

[0095] AAV8 or Anc80 vectors containing nucleotides encoding DNase enzymes can be transfected into hepatocytes and administered intrahepatically (e.g., via direct organ injection) or systemically, e.g., by intravenous injection, with an AAV8 or Anc80 vector effective to mediate efficient production of the encoded DNase enzyme and its secretion in the periendothelial space and the space of Disse. In some embodiments, the Anc80 capsid protein (e.g., Anc80 VP1 capsid protein comprising the sequence of SEQ ID NO:3 or SEQ ID NO:9, or Anc80L65 VP1 capsid protein comprising the sequence of SEQ ID NO:34, or variant Anc80L65 VP1 capsid protein comprising the sequence of SEQ ID NO:35) is encoded by a nucleotide sequence in an expression vector. In some embodiments, the AAV8 capsid protein (e.g., AAV8 VP1 or AAV8 VP3) is encoded by a nucleotide sequence in an expression vector. Peripheral administration of the AAV vectors of the invention can include systemic injection, for example, intramuscular, intravenous, intraperitoneal, and intra-arterial injection.

[0096] In some embodiments, AAV3-based, or AAV-LK03, or AAV-KP1 vectors are used as expression vectors. AAV3, AAV-LK03 and AAV-KP1 vectors are particularly suitable for liver targeting and expression. For example, AAV3, AAV-LK03 and AAV-KP1 vectors can transfect hepatocytes with higher efficiency compared to AAV2 vectors. Both AAV-LK03 and AAV-KP1 induce less neutralizing antibodies than some other AAV vectors.

[0097] The desired dose of the AAV vector encoding the DNase enzyme of the present invention can be readily adapted by one of skill in the art depending, for example, on the disease state, the subject, the treatment schedule, etc. In some embodiments, 10e5-101e4, e.g., 10e6-10e11, 10e7-10e11, or 108-1014 recombinant viral particles are administered per dose. In other embodiments, exemplary doses to achieve a therapeutic effect may include titers of at least about 10e5, 10e6, 10e7, 10e8, 10e9, 10e10, or 10e11 recombinant viral particles or more.

[0098] The exogenous targeting sequence(s) may replace or replace part or all of the major capsid subunit (e.g., VP3). As a further alternative, more than one exogenous targeting sequence may be introduced into the virion capsid, e.g., 2, 3, 4, 5 or more sequences. In alternative embodiments, insertions and substitutions may be made within the minor capsid subunits (e.g., VP1 and VP2). In the case of AAV capsids, insertions or substitutions may be made into VP2 or VP3.

[0099] The tropism of the native virion may be reduced or abolished by the insertion or substitution of an amino acid sequence. Alternatively, the insertion or substitution of an exogenous amino acid sequence may target the virion to a particular cell type(s). The exogenous targeting sequence may be any amino acid sequence that encodes a protein or peptide that alters the tropism of the virion. In certain embodiments, the targeting peptide or protein may be naturally occurring or alternatively, fully or partially synthetic. Exemplary peptides and proteins include ligands and other peptides that bind to cell surface receptors present on hepatocytes, ligands that can bind to the Sr-B1 receptor for apolipoprotein E, galactose and lactose specific lectins, low density lipoprotein receptor ligands, asialoglycoprotein (galactose terminal) ligands, and the like.

[0100] Alternatively, the exogenous targeting sequence may be an antibody or an antigen-recognizing portion thereof. As used herein, the term "antibody" refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Antibodies may be monoclonal or polyclonal, and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. The term "antibody" also encompasses bispecific or "cross-linked" antibodies, which are known to those of skill in the art. Antibody fragments within the scope of the present invention include, for example, Fab, F(ab')2, and Fc fragments, as well as corresponding fragments obtained from antibodies other than IgG. Such fragments may be generated by known techniques. The exogenous amino acid sequence inserted into the virion capsid may be a sequence that facilitates purification or detection of the virion. For example, the exogenous amino acid sequence may include a polyhistidine sequence, which is useful for purifying virions on a nickel column, or an antigenic peptide or protein that may be used to purify virions by standard immunopurification techniques, as known to those of skill in the art. Alternatively, the amino acid sequence may encode a receptor ligand or any other peptide or protein that can be used to purify the modified virions by affinity purification or any other technique known in the art (e.g., purification techniques based on different size, density, charge, or isoelectric point, ion exchange chromatography, or peptide chromatography).

[0101] Alternatively, the exogenous targeting sequence may be an antibody or an antigen-recognizing portion thereof. As used herein, the term "antibody" refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Antibodies may be monoclonal or polyclonal, and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. The term "antibody" also encompasses bispecific or "cross-linked" antibodies, as known to those skilled in the art. Antibody fragments within the scope of the present invention include, for example, Fab, F(ab')2, and Fc fragments, as well as corresponding fragments obtained from antibodies other than IgG. Such fragments may be generated by known techniques.

[0102] The exogenous amino acid sequence inserted into the virion capsid may be one that facilitates purification or detection of the virion. According to this aspect of the invention, the exogenous amino acid sequence need not also modify the virion of the modified parvovirus. For example, the exogenous amino acid sequence may include a polyhistidine sequence useful for purifying virions on a nickel column, or an antigenic peptide or protein that can be used to purify virions by standard immunopurification techniques, as known to those skilled in the art. Alternatively, the amino acid sequence may code for a receptor ligand, or any other peptide or protein that can be used to purify modified virions by affinity purification or any other technique known in the art (e.g., purification techniques based on different sizes, densities, charges, or isoelectric points, ion exchange chromatography, or peptide chromatography).

[0103] In some embodiments, the AAV vectors disclosed herein may be useful for treating and / or preventing a neurodegenerative, neurodevelopmental, psychiatric, oncological, or autoimmune disease or an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the above-described rAAV vectors or pharmaceutical compositions comprising such vectors.

[0104] promoter In some embodiments, the vector disclosed herein may be a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a deoxyribonuclease (DNase) enzyme. In some embodiments, the promoter is a liver-specific promoter. In some embodiments, the promoter is specific to a tumor-originating tissue or a metastasis target tissue. In some embodiments, the liver-specific promoter is specific to a tumor-originating tissue or a metastasis target tissue.

[0105] Non-limiting examples of promoters that can be used in the vectors of the present invention include, for example, albumin promoter, alpha1-antitrypsin (AAT) promoter, thyroid hormone-binding globulin promoter, alpha-fetoprotein promoter, alcohol dehydrogenase promoter, factor VIII (FVIII) promoter, HBV basic core promoter (BCP), HBV PreS2 promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter, thyroxine-binding globulin (TBG) promoter, hepatic control region (HCR)-ApoCII hybrid promoter, HCR-hAAT hybrid promoter, apolipoprotein E (ApoE) promoter, low density lipoprotein promoter, pyruvate kinase promoter, phosphophenolpyruvate carboxykinase promoter, phenylalanine hydroxylase promoter, lecithin-cholesterol acyltransferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, apolipoprotein A-II promoter (APOA2), transferrin promoter, transthyretin promoter, alpha-fibrinogen activator (AFAC) promoter, and the like. These include the linogen promoter, beta-fibrinogen promoter, alpha1-antichymotrypsin promoter, alpha2-HS glycoprotein promoter, haptoglobin promoter, ceruloplasmin promoter, plasminogen promoter, promoters of complement proteins, alpha1-acid glycoprotein promoter, LSP1 promoter, serpin peptidase inhibitor promoter, clade A member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, microRNA 122 (miR-122) promoter, liver-specific IGF-II promoter P1, transthyretin (MTTR) promoter, and alpha-fetoprotein (AFP) promoter.

[0106] In some embodiments, the promoter is a liver-specific / nervous system-specific tandem promoter, in some embodiments, the liver-specific / nervous system-specific tandem promoter mediates substantially increased expression of the enzyme in the liver and / or nervous system compared to other tissues and organs.In some embodiments, the liver-specific / nervous system-specific tandem promoter is an albumin promoter, an alpha 1-antitrypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, an HBV basic core promoter (BCP), an HBV PreS2 promoter, a phosphoenolpyruvate carboxykinase (PEPCK) promoter, a thyroxine-binding globulin (TBG) promoter, a hepatic control region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphophenolpyruvate carboxykinase promoter, a phenylalanine hydroxylase promoter, a lecithin-cholesterol acyltransferase (LCAT) promoter, an apolipoprotein H (ApoH) promoter, an apolipoprotein A-II promoter (APOA2), a transferrin promoter, a transthyretin promoter, an alpha-fibrinogen promoter. The liver-specific promoter may be selected from the group consisting of a beta-fibrinogen promoter, an alpha1-antichymotrypsin promoter, an alpha2-HS glycoprotein promoter, a haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a promoter of a complement protein, an alpha1-acid glycoprotein promoter, an LSP1 promoter, a serpin peptidase inhibitor promoter, a clade A member 1 (SERPINA1) (hAAT) promoter, a cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, a microRNA 122 (miR-122) promoter, a liver-specific IGF-II promoter P1, a transthyretin (MTTR) promoter, and an alpha fetoprotein (AFP) promoter. In some embodiments, the liver-specific promoter is an antitrypsin (AAT) promoter.In some embodiments, the AAT promoter is a human alpha 1-antitrypsin (hAAT) promoter. In some embodiments, the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity with the sequence of SEQ ID NO: 15. In some embodiments, the liver-specific promoter comprises a sequence of SEQ ID NO: 15. In some embodiments, the liver-specific promoter consists of the sequence of SEQ ID NO: 15. In some embodiments, the liver-specific promoter is an albumin promoter. In some embodiments, the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity with the sequence of SEQ ID NO: 8. In some embodiments, the liver-specific promoter comprises a sequence of SEQ ID NO: 8. In some embodiments, the liver-specific promoter consists of the sequence of SEQ ID NO: 8. In some embodiments, the liver-specific / nervous system-specific tandem promoter comprises a nervous system-specific promoter selected from the group consisting of F4 / 80 promoter, CD68 promoter, TMEM119 promoter, CX3CR1 promoter, CMV promoter, MEF2 promoter, FoxP2 promoter, Iba1 promoter, TTR promoter, CD11b promoter, c-fes promoter, NSE promoter, synapsin promoter, CamKII promoter, α-CamKII promoter, VGLUT1 promoter, and glial fibrillary acidic protein (GFAP) promoter. In some embodiments, the nervous system-specific promoter is a synapsin promoter. In some embodiments, the synapsin promoter is a human synapsin promoter. In one embodiment, the nervous system-specific promoter is an F4 / 80 promoter. In some embodiments, the nervous system-specific promoter is a CMV promoter. In some embodiments, the nervous system-specific promoter is a TMEM119 promoter. In some embodiments, the nervous system-specific promoter is a MEF2 promoter.In some embodiments, the nervous system specific promoter is a FoxP2 promoter. In some embodiments, the liver-specific / nervous system specific tandem promoter comprises a neural specific promoter comprising a sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 36 and 38-42. In one embodiment, the nervous system specific promoter comprises any one of the sequences of SEQ ID NOs: 36 and 38-42. In one embodiment, the nervous system specific promoter consists of any one of the sequences of SEQ ID NOs: 36 and 38-42.

[0107] In some embodiments of any of the methods of the invention, the promoter is a liver-specific promoter. In some embodiments of any of the methods of the invention, the liver-specific promoter mediates substantially increased expression of the enzyme in the liver compared to other tissues and organs.Non-limiting examples of liver-specific promoters include, for example, albumin promoter (Alb), human alpha-1 antitrypsin (hAAT) promoter, thyroxine-binding globulin (TBG) promoter, apolipoprotein E hepatic control region promoter, apolipoprotein A-II (APOA2) promoter, serpin peptidase inhibitor, clade A, member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3A4) promoter, microRNA 122 (miR-122) promoter, liver-specific IGF-II promoter P1, mouse transthyretin (MTTR) promoter, alpha-fetoprotein (AFP) promoter, thyroid hormone-binding globulin promoter, alcohol dehydrogenase promoter, factor VIII (FVIII) promoter, HBV basic core promoter (BCP) and PreS2 promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoters, hepatic control region (HCR)-ApoCII hybrid promoter, AAT promoter in combination with mouse albumin gene enhancer (Ealb) element, low density lipoprotein promoter, pyruvate kinase promoter, phosphenolpyruvate carboxykinase promoter, lecithin-cholesterol acyltransferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, transferrin promoter, transthyretin promoter, alpha-fibrinogen and beta-fibrinogen promoters, alpha1-antichymotrypsin promoter, alpha2-HS glycoprotein promoter, haptoglobin promoter, ceruloplasmin promoter, plasminogen promoter, promoters of complement proteins (e.g., C1q, C1r, C2, C3, C4, C5, C6, C8, C9, complement factor I, and complement factor H), C3 complement activator, and [alpha]1-acid glycoprotein promoter.Further tissue-specific promoters can be found in the tissue-specific promoter database TiProD (Nucleic Acids Research, J4:DI04-DI07 (2006)).

[0108] In some embodiments, for example, when liver targeting is mediated by a capsid protein, the nucleic acid encoding the DNase enzyme can be operably linked to a promoter that allows for efficient systemic expression (e.g., a CMV promoter, a chicken beta actin promoter (CBA), an EF1a promoter).

[0109] In some embodiments, the liver-specific promoter is an alpha 1-antitrypsin (AAT) promoter. In one specific embodiment, the antitrypsin promoter is a human alpha 1-antitrypsin (AAT) promoter. In some embodiments, the promoter is an alpha 1-antitrypsin (AAT) promoter. In some embodiments, the promoter is a human alpha 1-antitrypsin (hAAT) promoter. In some embodiments, the antitrypsin promoter comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 15. In some embodiments, the AAT promoter comprises the sequence of SEQ ID NO: 15. In some embodiments, the AAT promoter consists of the sequence of SEQ ID NO: 15.

[0110] In some embodiments, a promoter region may be operably linked to the coding sequence to cause or improve the expression of the protein of interest in transfected cells. Such promoters may be ubiquitous, cell or tissue specific, strong, weak, regulated, chimeric promoters, etc., to allow efficient and stable production of proteins in the liver and / or nervous system. The promoter may be homologous or heterologous to the encoded protein, but the promoters used in the disclosed methods are generally functional in human cells. Examples of regulated promoters include, but are not limited to, Tet on / off element-containing promoters, rapamycin-inducible promoters, tamoxifen-inducible promoters, and metallothionein promoters.

[0111] In some embodiments, the promoter that can be used in the present invention can include, for example, promoters that are tissue-specific for the liver and / or nervous system.Non-limiting examples of liver-specific promoters include, for example, albumin promoter (Alb), human alpha-1 antitrypsin (hAAT) promoter, thyroxine-binding globulin (TBG), apolipoprotein E liver control region promoter, apolipoprotein A-II (APOA2) promoter, serpin peptidase inhibitor, clade A, member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3A4) promoter, microRNA 122 (miR-122) promoter, liver-specific IGF-II promoter P1, mouse transthyretin (MTTR) promoter, and alpha-fetoprotein (AFP) promoter. Non-limiting examples of nervous system specific promoters include, for example, microglia specific promoters (e.g., F4 / 80, CD68, TMEM119, CX3CR1, CMV, Iba1), myeloid specific promoters (e.g., TTR, CD11b, c-fes), neuron specific promoters (e.g., CMV, NSE, synapsin [SynI, SynII], CamKII, α-CaMKII, VGLUT1), and other neuronal and glial cell (e.g., oligodendrocyte astrocyte) type specific promoters (e.g., GFAP). Non-limiting examples of intestinal specific promoters include MUC2, villin, T3b, CB / CMV, GFAP, miCMV, CMV+I, tetO-CMV, and β-actin-CMV.

[0112] In some embodiments, the promoter that can be used in the present invention can include a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include viral promoters such as CMV promoter, RSV promoter, SV40 promoter, and cellular promoters such as phosphoglycerate kinase (PGK) promoter, EF1a promoter, CMVE / CAG promoter system, and β-actin promoter.

[0113] Administration of DNase enzyme In one aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a DNase enzyme and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0114] In a related aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a DNase enzyme and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the DNase enzyme is effective to reduce expression of an immunosuppressive protein in tumor tissue and / or increase the content of cells comprising a chimeric antigen receptor or a T cell receptor in the tumor tissue. Non-limiting examples of immunosuppressive proteins include, for example, PD-L1, PD-L2, CD80, CD86, and MHC.

[0115] Administration of DNase enzyme according to the methods of the present invention can be by any suitable route, including systemic administration and direct administration to the site of disease (e.g., the primary tumor). Specific non-limiting examples of useful administration routes include intravenous (IV), subcutaneous (SC), intraperitoneal (IP), and intramuscular.

[0116] In some embodiments of any of the above methods, the DNase enzyme protein is administered via injection. In some embodiments, the DNase enzyme protein is administered via intravenous injection. In some embodiments, the DNase enzyme protein is injected intravenously following infusion of the CAR-expressing or TCR-expressing cells disclosed herein. In some embodiments, the DNase enzyme protein is injected intravenously prior to infusion of the CAR-expressing or TCR-expressing cells disclosed herein. In some embodiments, the DNase enzyme protein is injected intravenously together with infusion of the CAR-expressing or TCR-expressing cells disclosed herein. In some embodiments, the DNase enzyme protein is injected intravenously at least 14 days after infusion of the CAR-expressing or TCR-expressing cells. In some embodiments, the DNase enzyme protein is injected intravenously at least 16 days after infusion of the CAR-expressing or TCR-expressing cells. In some embodiments, the DNase enzyme protein is intravenously injected at least 3 days before, with, or after the infusion of the CAR-expressing or TCR-expressing cells. In some embodiments, the DNase enzyme protein is intravenously injected at least 7 days after the infusion of the CAR-expressing or TCR-expressing cells. In some embodiments, the DNase enzyme protein is intravenously injected at least 7 days before the infusion of the CAR-expressing or TCR-expressing cells. In some embodiments, the DNase enzyme protein is intravenously injected at least 3 days before or after the infusion of the CAR-expressing or TCR-expressing cells. In some embodiments, the DNase enzyme protein is intravenously injected for at least 14 days.

[0117] The DNase dose useful in the methods of the present invention depends on the type of CAR / TCR therapy, the patient's medical history and response to DNase, and the discretion of the attending physician. Non-limiting examples of useful dosage ranges include 0.5-100 mg / kg / day or 1000-200,000 KU / kg / day, preferably 0.5-50 mg / kg / day or 1000-100,000 Kunitz Units (KU) / kg / day, more preferably 1.5-50 mg / kg / day or 3000-100,000 KU / kg / day, and most preferably 10-50 mg / kg / day or 20,000-100,000 KU / kg / day.

[0118] In some aspects, the DNase enzyme protein is injected intravenously at 250 μg / kg / day. In some embodiments, the DNase enzyme protein is injected intravenously at 250 μg / kg / day for at least 14 days.

[0119] In some embodiments, the CAR is selected from the group consisting of BCMA, CD44v6, CAIX (carbonic anhydrase IX), CEA (carcinoembryonic antigen), CD5, CD7, CD19, CD20, CD22, CD30, CD36, CD37, CD70, CD123, CD133, c-Met (hepatocyte growth factor receptor), EGFR (epidermal growth factor), EGFRvIII (epidermal growth factor type III variant), EGFRB3, Epcam (epithelial cell adhesion molecule), EphA2 (erythropoietin producing hepatocellular carcinoma A2) , fetal acetylcholine receptor, FLT3, GD2 (ganglioside GD2), GPC3 (glypican-3), GUCY2C (guanylyl cyclase C), HER1 (human epidermal growth factor receptor 1), HER2 (human epidermal growth factor receptor 2), ICAM-1 (intercellular adhesion molecule 1), IL3R-alpha, IL13Rα2 (interleukin-13 receptor α2), IL11Rα (interleukin-11 receptor α), Kras (Kristen rat sarcoma viral oncogene homolog) Kras The solid tumor associated target antigen binding domain is capable of specifically binding to one or more antigens selected from G12D, L1CAM (L1-cell adhesion molecule), MAGE, MET, mesothelin, MUC (mucin 1), MUC16ecto (mucin 16), NKG2D (natural killer group 2 member D), NY-ESO-1, PSCA (prostate stem cell antigen), PSMA, ROR1 and WT-1 (Wilms tumor 1).

[0120] As used herein, an "effective amount" includes a dose adequate to treat a mammal having a clinically defined disorder.

[0121] An "analog," "variant," or "derivative" is a compound that is substantially similar in structure and has the same biological activity as a naturally occurring molecule, but possibly with a different degree of activity. For example, a polypeptide variant refers to a polypeptide that shares a substantially similar structure and has the same biological activity as a reference polypeptide. A variant or analog differs in its amino acid sequence composition compared to the naturally occurring polypeptide from which it is derived based on one or more mutations involving (i) a deletion of one or more amino acid residues at one or more termini of the polypeptide and / or at one or more internal regions (e.g., fragments) of the naturally occurring polypeptide, (ii) an insertion or addition (typically an "addition" or "fusion") of one or more amino acids at one or more termini of the polypeptide and / or an insertion or addition (typically an "insertion") of one or more amino acids at one or more internal regions of the naturally occurring polypeptide sequence, or (iii) a substitution of one or more amino acids for other amino acids in the naturally occurring polypeptide sequence. By way of example, a "derivative" refers to a polypeptide that shares the same or substantially similar structure as a reference polypeptide, e.g., that has been chemically modified.

[0122] Variant or analog polypeptides include insertion variants in which one or more amino acid residues are added to the protein amino acid sequence of the invention. The insertion may be located at either or both termini of the protein and / or may be located within an internal region of the protein amino acid sequence. Insertion variants with additional residues at either or both termini include, for example, fusion proteins and proteins containing amino acid tags or other amino acid labels. In one embodiment, the protein molecule optionally contains a Met at the N-terminus, especially when the molecule is recombinantly expressed in, for example, yeast, insect cells or bacterial cells such as E. coli.

[0123] In deletion variants, one or more amino acid residues are removed in the proteins or polypeptides described herein. Deletions may be effected at one or both termini of the protein or polypeptide and / or by removal of one or more residues within the protein amino acid sequence. Thus, deletion variants include fragments of the protein or polypeptide sequence.

[0124] In substitution variants, one or more amino acid residues of a protein or polypeptide are removed and replaced with alternative residues. In one embodiment, the substitutions are conservative in nature, and conservative substitutions of this type are well known in the art. Alternatively, the present invention encompasses substitutions that are also non-conservative. Exemplary conservative substitutions are described in Lehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc., New York (1975), pp. 71-77], which is incorporated herein by reference.

[0125] The DNase enzyme protein and / or its derivatives, variants or analogs and / or at least one cell comprising a chimeric antigen receptor (CAR) or T cell receptor (TCR) may be administered orally, topically, transdermally, parenterally, by inhalation spray, intravaginally, rectally or intracranially. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intracisternal injection or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and / or surgical implantation at a specific site is also contemplated. In general, the DNase enzyme protein and / or its derivatives, variants or analogs and / or at least one cell comprising a chimeric antigen receptor (CAR) or T cell receptor (TCR) is essentially free of pyrogens and other impurities that may be harmful to the recipient.

[0126] Single or multiple administrations of at least one cell comprising a deoxyribonuclease enzyme protein and / or its derivative, variant or analogue and / or chimeric antigen receptor (CAR) or T cell receptor (TCR) can be performed with the dosage level and pattern selected by the treating physician. For the prevention or treatment of cancer, the appropriate dosage depends on the type of disease being treated as described above, the severity and course of the cancer, whether the drug is administered for prevention or treatment, previous treatments, the patient's medical history and response to the drug, and the discretion of the attending physician.

[0127] The present invention also relates to at least one cell comprising a deoxyribonuclease enzyme protein and / or its derivative, variant or analogue and / or a chimeric antigen receptor (CAR) or a T cell receptor (TCR), comprising a pharma- ceutically acceptable carrier, diluent, salt, buffer or excipient. The pharmaceutical composition can be used to treat clinically defined disorders. The pharmaceutical composition of the present invention can be a solution or a lyophilisate. The solution of the pharmaceutical composition can be subjected to any suitable lyophilisation process.

[0128] In other aspects of this embodiment, the desoxyribonuclease enzyme proteins and / or derivatives, variants or analogs thereof disclosed herein reduce the severity of cancer by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. In still other aspects of this embodiment, the deoxyribonuclease enzyme protein disclosed herein reduces tumor size by, for example, about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 9 ... % to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[0129] In aspects of this embodiment, the desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof disclosed herein may be administered at a concentration of, for example, at least 25 μg / kg, at least 50 μg / kg, at least 75 μg / kg, at least 100 μg / kg, at least 125 μg / kg, at least 150 μg / kg, at least 175 μg / kg, at least 200 μg / kg, at least 225 μg / kg, at least 250 μg / kg, at least 275 μg / kg, at least 300 μg / kg, at least 325 μg / kg, at least 340 μg / kg, at least 360 μg / kg, at least 380 μg / kg, at least 390 μg / kg, at least 400 μg / kg, at least 410 μg / kg, at least 425 μg / kg, at least 430 μg / kg, at least 440 μg / kg, at least 450 μg / kg, at least 460 μg / kg, at least 470 μg / kg, at least 480 μg / kg, at least 490 μg / kg, at least 500 μg / kg, at least 510 μg / kg, at least 520 μg / kg, at least 530 μg / kg, at least 540 μg / kg, at least 550 μg / kg, at least 560 μg / kg, at least 570 μg / kg, at least 580 μg / kg, at least 590 μg / kg, at least 600 μg / kg, at least 610 μg / kg, at least 620 μg / kg, at least 630 μg / kg, at least 640 μg / kg, at least 650 μg / kg / kg, at least 350μg / kg, at least 375μg / kg, at least 400μg / kg, at least 425μg / kg, at least 450μg / kg, at least 475μg / kg, at least 500μg / kg, at least 525μg / kg, at least 550μg / kg, at least 575μg / kg, at least 600μg / kg, at least 625μg / kg, at least 650μg / kg, at least 675μg / kg, at least 700μg / kg, at least 725μg / kg, at least 750μg / kg, at least 775μg / kg, at least 800μg / kg, at least 825μg / kg, at least 850μg / kg, at least 875μg / kg, at least 900μg / kg, at least 925μg / kg, at least 950μg / kg, at least 975μg / kg, at least 1mg / kg, at least 1.25mg / kg, at least 1.5mg / kg, at least 2mg / kg, at least 2.5mg / kg, at least 3mg / kg, at least 3.5mg / kg, at least 4mg / kg, at least 4.5mg / kg, at least 5mg g / kg, at least 5.5 mg / kg, at least 6 mg / kg, at least 6.5 mg / kg, at least 7 mg / kg, at least 7.5 mg / kg, at least 8 mg / kg, at least 8.5 mg / kg, at least 9 mg / kg, at least 9.5 mg / kg, at least 10 mg / kg, at least 10.5 mg / kg, at least 11 mg / kg, at least 11.5 mg / kg, at least 12 mg / kg, at least 12.5 mg / kg, at least 13 mg / kg, at least 13.5 mg / kg, at least 14 mg / kg,at least 14.5mg / kg, at least 15mg / kg, at least 15.5mg / kg, at least 16mg / kg, at least 16.5mg / kg, at least 17mg / kg, at least 17.5mg / kg, at least 18mg / kg, at least 18.5mg / kg, at least 19mg / kg, at least 19.5mg / kg, at least 20mg / kg, at least 20.5mg / kg, at least 21mg / kg, at least 21.5mg / kg, at least 22mg / kg, at least 22.5mg / kg, at least 23mg / kg, at least 23.5mg / kg, at least 24mg / kg, at least 24.5mg / kg, at least 25mg / kg, at least 25.5mg / kg, at least 26mg / kg, at least 26.5mg / kg, at least 27mg / kg, at least 27.5mg / kg, at least 28mg / kg, at least 28.5mg / kg, at least 29mg / kg, at least 29.5mg / kg, at least 30mg / kg, at least 31mg / kg, at least 32mg / kg, at least 33mg / kg, at least 34mg / kg, kg, at least 35mg / kg, 36mg / kg, at least 37mg / kg, at least 38mg / kg, at least 39mg / kg, at least 40mg / kg, 41mg / kg, at least 42mg / kg, at least 43mg / kg, at least 44mg / kg, at least 45mg / kg, at least 46mg / kg, at least mg / kg, at least 48mg / kg, at least 49mg / kg at least 50mg / kg, at least 51mg / kg, at least 52mg / kg, at least 53mg / kg, at least 54mg / kg g, at least 55 mg / kg, at least 56 mg / kg, at least 57 mg / kg, at least 58 mg / kg, at least 59 mg / kg, at least 60 mg / kg, at least 61 mg / kg, at least 62 mg / kg, at least 63 mg / kg, at least 64 mg / kg, at least 65 mg / kg, at least 66 mg / kg, at least 67 mg / kg, at least 68 mg / kg, at least 69 mg / kg, at least 70 mg / kg, at least 71 mg / kg, at least 72 mg / kg, at least 73 mg / kg,at least 74mg / kg, at least 75mg / kg, at least 76mg / kg, at least 77mg / kg, at least 78mg / kg, at least 79mg / kg, at least 80mg / kg, at least 81mg / kg, at least 82mg / kg, at least 83mg / kg, at least 84mg / kg, at least 85mg / kg, at least 86mg / kg, at least 87mg / kg, at least 88mg / kg, at least 89mg / kg, at least 90mg / kg, at least 91mg / kg, at least 92mg / kg, at least 93mg / kg, at least 94mg / kg, at least 95mg / kg, at least 96mg / kg, at least 97mg / kg, at least 98mg / kg, at least 99mg / kg, at least 100mg / kg, at least 150mg / kg, at least 200mg / kg, at least The dose may be at least 250 mg / kg, at least 300 mg / kg, at least 350 mg / kg, at least 400 mg / kg, at least 450 mg / kg, at least 500 mg / kg, at least 550 mg / kg, at least 600 mg / kg, at least 650 mg / kg, at least 700 mg / kg, at least 750 mg / kg, at least 800 mg / kg, at least 850 mg / kg, at least 900 mg / kg, at least 950 mg / kg, at least 1 g / kg, at least 2 g / kg, at least 3 g / kg, at least 4 g / kg, at least 5 g / kg, at least 6 g / kg, at least 7 g / kg, at least 8 g / kg, at least 9 g / kg, at least 10 g / kg, at least 15 g / kg, at least 20 g / kg, at least 25 g / kg, at least 50 g / kg, at least 100 g / kg.

[0130] In aspects of this embodiment, the desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof disclosed herein may be administered at a concentration of, for example, about 25 μg / kg, about 50 μg / kg, about 75 μg / kg, about 100 μg / kg, about 125 μg / kg, about 150 μg / kg, about 175 μg / kg, about 200 μg / kg, about 225 μg / kg, about 250 μg / kg, about 275 μg / kg, about 300 μg / kg, about 325 μg / kg, about 350 μg / kg, about 375 μg / kg, about 400 μg / kg, about 425 μg / kg, about 450 μg / kg, about 460 μg / kg, about 470 μg / kg, about 480 μg / kg, about 490 μg / kg, about 500 μg / kg, about 510 μg / kg, about 520 μg / kg, about 530 μg / kg, about 540 μg / kg, about 550 μg / kg, about 560 μg / kg, about 570 μg / kg, about 580 μg / kg, about 590 μg / kg, about 600 μg / kg, about 610 μg / kg, about 620 μg / kg, about 630 μg / kg, about 640 μg / kg, about 650 μg / kg, about 660 μg / kg, about 670 μg / kg, about 680 μg / kg, about 690 μg / kg, about 700 μg / kg, about 710 μg / kg, about 720 μg / kg, about 750 μg / kg, about 760 μg / kg, about kg, approximately 475μg / kg, approximately 500μg / kg, approximately 525μg / kg, approximately 550μg / kg, approximately 575μg / kg, approximately 600μg / kg, approximately 625μg / kg, approximately 650μg / kg, approximately 675μg / kg, approximately 700μg / kg, approximately 725μg / kg, approximately 750μg / kg, Approximately 775μg / kg, approximately 800μg / kg, approximately 825μg / kg, approximately 850μg / kg, approximately 875μg / kg, approximately 900μg / kg, approximately 925μg / kg, approximately 950μg / kg, approximately 975μg / kg, approximately 1mg / kg, approximately 1.25mg / kg, approximately 1.5mg / kg, approximately 2mg / kg, about 2.5mg / kg, about 3mg / kg, about 3.5mg / kg, about 4mg / kg, about 4.5mg / kg, about 5mg / kg, about 5.5mg / kg, about 6mg / kg, about 6.5mg / kg, about 7mg / kg, about 7.5mg / kg, about 8mg / kg, about 8.5mg / kg, about 9mg / kg, about 9.5mg / kg, about 10mg / kg, about 10.5mg / kg, about 11mg / kg, about 11.5mg / kg, about 12mg / kg, about 12.5mg / kg, about 13mg / kg, about 13.5mg / kg, about 14mg / kg, about 14.5mg / kg, about 15mg / kg kg, about 15.5mg / kg, about 16mg / kg, about 16.5mg / kg, about 17mg / kg, about 17.5mg / kg, about 18mg / kg, about 18.5mg / kg, about 19mg / kg, about 19.5mg / kg, about 20mg / kg, about 20.5mg / kg, about 21mg / kg, About 21.5mg / kg, about 22mg / kg, about 22.5mg / kg, about 23mg / kg, about 23.5mg / kg, about 24mg / kg, about 24.5mg / kg, about 25mg / kg, about 25.5mg / kg, about 26mg / kg, about 26.5mg / kg, about 27mg / kg, about 27.5mg / kg, about 28mg / kg, about 28.5mg / kg, about 29mg / kg, about 29.5mg / kg, about 30mg / kg, about 31mg / kg, about 32mg / kg, about 33mg / kg, about 34mg / kg, about 35mg / kg, 36mg / kg, about 37mg / kg, about 38 mg / kg, approximately 39 mg / kg, approximately 40 mg / kg, 41 mg / kg, approximately 42 mg / kg, approximately 43 mg / kg, approximately 44 mg / kg, approximately 45 mg / kg, approximately 46 mg / kg, approximately mg / kg, approximately 48 mg / kg, approximately 49 mg / kg, approximately 50 mg / kg, approximately 51 mg / kg, approximately 52 mg / kg, about 53 mg / kg, about 54 mg / kg, about 55 mg / kg, about 56 mg / kg, about 57 mg / kg, about 58 mg / kg, about 59 mg / kg, about 60 mg / kg, about 61 mg / kg, about 62 mg / kg, about 63 mg / kg, about 64 mg / kg, about 65 mg / k g, about 66 mg / kg, about 67 mg / kg, about 68 mg / kg, about 69 mg / kg, about 70 mg / kg, about 71 mg / kg, about 72 mg / kg, about 73 mg / kg, about 74 mg / kg, about 75 mg / kg, about 76 mg / kg, about 77 mg / kg, about 78 mg / kg, about 79 mg / kg, approximately 80 mg / kg, approximately 81 mg / kg, approximately 82 mg / kg, approximately 83 mg / kg, approximately 84 mg / kg, approximately 85 mg / kg, approximately 86 mg / kg, approximately 87 mg / kg, approximately 88 mg / kg, approximately 89 mg / kg, approximately 90 mg / kg, approximately 91 mg / kg, approximately 92 mg / k g, about 93mg / kg, about 94mg / kg, about 95mg / kg, about 96mg / kg, about 97mg / kg, about 98mg / kg, about 99mg / kg, about 100mg / kg, about 150mg / kg, about 200mg / kg, about 250mg / kg, about 300mg / kg, about 350mg / kg The dose may be about 1 g / kg, about 2 g / kg, about 3 g / kg, about 4 g / kg, about 5 g / kg, about 6 g / kg, about 7 g / kg, about 8 g / kg, about 9 g / kg, about 10 g / kg, about 15 g / kg, about 20 g / kg, about 25 g / kg, about 50 g / kg, about 100 g / kg.

[0131] In aspects of this embodiment, the desoxyribonuclease enzymes and / or derivatives, variants, or analogs thereof disclosed herein may be administered at a concentration of, for example, 25 μg / kg or less, 50 μg / kg or less, 75 μg / kg or less, 100 μg / kg or less, 125 μg / kg or less, 150 μg / kg or less, 175 μg / kg or less, 200 μg / kg or less, 225 μg / kg or less, 250 μg / kg or less, 275 μg / kg or less, 300 μg / kg or less, 325 μg / kg or less, 350 μg / kg or less, 375 μg / kg or less, 400 μg / kg or less, 425 μg / kg or less, Below, 450μg / kg or less, 475μg / kg or less, 500μg / kg or less, 525μg / kg or less, 550μg / kg or less, 575μg / kg or less, 600μg / kg or less, 625μg / kg or less, 650μg / kg or less, 675μg / kg or less, 700μg / kg or less, 725μg / kg or less, 750μg / kg or less, 775μg / kg or less, 800μg / kg or less, 825μg / kg or less, 850μg / kg or less, 875μg / kg or less, 900μg / kg or less, 925μg / kg or less, 950μg / kg or less, 975μg / kg or less, 1mg / kg or less, 1.25mg / kg or less, 1.5mg / kg or less, 2mg / kg or less, 2.5mg / kg or less, 3mg / kg or less, 3.5mg / kg or less, 4mg / kg or less, 4.5mg / kg or less, 5mg / kg or less, 5.5mg / kg or less, 6mg / kg or less, 6.5mg / kg or less, 7mg / kg or less, 7.5mg / kg or less, 8mg / kg or less, 8.5mg / kg or less, 9mg / kg or less, 9.5mg / kg or less, 10mg / kg or less, 10.5mg / kg or less, 11mg / kg or less, 11.5mg / kg or less, 12mg / kg or less, 12.5m g / kg or less, 13mg / kg or less, 13.5mg / kg or less, 14mg / kg or less, 14.5mg / kg or less, 15mg / kg or less, 15.5mg / kg or less, 16mg / kg or less, 16.5mg / kg or less, 17mg / kg or less, 17.5mg / kg or less, 18mg / kg Below, 18.5mg / kg or less, 19mg / kg or less, 19.5mg / kg or less, 20mg / kg or less, 20.5mg / kg or less, 21mg / kg or less, 21.5mg / kg or less, 22mg / kg or less, 22.5mg / kg or less, 23mg / kg or less, 23.5mg / kg or less,24mg / kg or less, 24.5mg / kg or less, 25mg / kg or less, 25.5mg / kg or less, 26mg / kg or less, 26.5mg / kg or less, 27mg / kg or less, 27.5mg / kg or less, 28mg / kg or less, 28.5mg / kg or less, 29mg / kg or less, 29.5mg / kg or less, 30mg / kg or less, 31mg / kg or less, 32mg / kg or less, 33mg / kg or less, 34mg / kg or less, 35mg / kg or less, 36mg / kg, 37mg / kg or less, 38mg / kg or less, 39mg / kg or less, 40mg / kg or less, 41mg / kg, 42 mg / kg or less, 43mg / kg or less, 44mg / kg or less, 45mg / kg or less, 46mg / kg or less, mg / kg or less, 48mg / kg or less, 49mg / kg or less, 50mg / kg or less, 51mg / kg or less, 52mg / kg or less, 53mg / kg or less, 54mg / kg or less, 55mg / kg or less, 56mg / kg or less, 57mg / kg or less, 58mg / kg or less, 59mg / kg or less, 60mg / kg or less, 61mg / kg or less, 62mg / kg or less, 63mg / kg or less, 64mg / kg or less, 65mg / kg or less, 66mg / kg or less, 67mg / kg or less, 68mg / kg or less, 69mg / kg or less, 70mg / kg or less, 71mg / kg or less, 72mg / kg or less, 73mg / kg or less, 74mg / kg or less, 75mg / kg or less, 76mg / kg or less, 77mg / kg or less, 78mg / kg or less, 79mg / kg or less, 80mg / kg or less, 81mg / kg, 82mg / kg or less, 83mg / kg or less, 84mg / kg or less, 85mg / kg or less, 86mg / kg or less, 87mg / kg or less, 88mg / kg or less, 89mg / kg or less, 90mg / kg or less, 91mg / kg or less, 92mg / kg or less g or less, 93mg / kg or less, 94mg / kg or less, 95mg / kg or less, 96mg / kg or less, 97mg / kg or less, 98mg / kg or less, 99mg / kg or less, 100mg / kg or less, 150mg / kg or less, 200mg / kg or less, 250mg / kg or less, 300mg / kg or less, 350mg / kg or less, 400mg / kg or less, 450mg / kg or less, 500mg / kg or less, 550mg / kg or less, 600mg / kg or less, 650mg / kg or less, 700mg / kg or less, 750mg / kg or less, 800mg / kg or less, 850mg / kg or less,900mg / kg or less, 950mg / kg or less, 1g / kg or less, 2g / kg or less, 3g / kg or less, 4g / kg or less, 5g / kg or less, 6g / kg or less, 7g / kg or less, The dose can be up to 8g / kg, up to 9g / kg, up to 10g / kg, up to 15g / kg, up to 20g / kg, up to 25g / kg, up to 50g / kg, up to 100g / kg. ,

[0132] In yet another aspect of this embodiment, the deoxyribonuclease enzyme protein and / or derivatives, variants, or analogs thereof disclosed herein may be administered at a concentration of, for example, about 1 μg / kg to about 30 mg / kg, about 5 μg / kg to about 50 mg / kg, about 5 μg / kg to about 75 μg / kg, about 100 μg / kg to about 40 mg / kg, about 500 μg / kg to about 35 mg / kg, about 100 μg / kg to about 30 mg / kg, about 150 μg / kg to about 35mg / kg, about 250μg / kg to about 25mg / kg, about 350μg / kg to about 30mg / kg, about 500μg / kg to about 25mg / kg, about 150μg / kg to about 30mg / kg, about 750μg / kg to about 30m g / kg, about 500μg / kg to about 12mg / kg, about 200μg / kg to about 30mg / kg, about 250μg / kg to about 40mg / kg, about 100μg / kg to about 10mg / kg, about 200μg / kg to about 35mg / k g, about 500 μg / kg to about 30 mg / kg, or about 10 μg / kg to about 30 mg / kg, about 1 mg / kg to about 30 mg / kg, about 5 mg / kg to about 50 mg / kg, about 5 mg / kg to about 75 mg / kg, about 10 mg / kg to about 40 mg / kg, about 5 mg / kg to about 35 mg / kg, about 10 mg / kg to about 30 mg / kg, about 15 mg / kg to about 35 mg / kg, about 25 mg / kg to about 50 mg / kg, about 35 mg / kg to about 60 mg / kg The dosage range may be about 50 mg / kg to about 75 mg / kg, about 15 mg / kg to about 60 mg / kg, about 150 mg / kg to about 100 mg / kg, about 5 mg / kg to about 120 mg / kg, about 20 mg / kg to about 60 mg / kg, about 25 mg / kg to about 75 mg / kg, about 10 mg / kg to about 35 mg / kg, about 20 mg / kg to about 35 mg / kg, about 15 mg / kg to about 30 mg / kg, or about 10 mg / kg to about 30 mg / kg.

[0133] In aspects of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof disclosed herein reduces symptoms associated with cancer by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%.

[0134] In other aspects of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivatives, variants, or analogs thereof disclosed herein reduces symptoms associated with cancer by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 100%.

[0135] In yet other aspects of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivative, variant, or analog thereof disclosed herein may be administered to treat symptoms associated with cancer, for example, by about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about reduced by 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0136] In one embodiment, the desoxyribonuclease enzyme proteins and / or derivatives, variants or analogs thereof disclosed herein are capable of reducing the severity of disease in an individual suffering from cancer by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a patient not receiving the same treatment.

[0137] In other aspects of this embodiment, the deoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof may reduce disease severity in an individual suffering from cancer, as compared to a patient not receiving the same treatment, for example, by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about It is possible to reduce by 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[0138] In aspects of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof disclosed herein and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) reduces symptoms associated with cancer by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0139] In other aspects of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivatives, variants, or analogs thereof disclosed herein and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) reduces symptoms associated with cancer by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95% or 100%.

[0140] In yet another aspect of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivatives, variants, or analogs thereof disclosed herein and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) is administered to treat a symptom associated with cancer, e.g., about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 10 ... Reduced by about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0141] In one embodiment, the DNase enzyme protein and / or derivatives, variants or analogs thereof disclosed herein and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) are capable of reducing the severity of disease in an individual suffering from cancer by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a patient not receiving the same treatment.

[0142] In other aspects of this embodiment, the DNase enzyme protein and / or derivatives, variants, or analogs thereof, and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) are used to reduce disease severity in an individual suffering from cancer, as compared to a patient not receiving the same treatment, by, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about It is possible to reduce by 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[0143] In further embodiments, the DNase enzyme protein and / or derivatives, variants or analogs thereof are administered one or more times every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months or more after the initial administration of the DNase enzyme protein.

[0144] In further embodiments, the at least one cell comprising a DNase enzyme protein and / or derivative, variant or analog thereof and a chimeric antigen receptor (CAR) or a T cell receptor (TCR) is administered one or more times every 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months or more after the initial administration of the at least one cell comprising a DNase enzyme protein and / or derivative, variant or analog thereof and a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0145] In embodiments, the administration period of the desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof is 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0146] In embodiments, the administration period of the DNase enzyme protein and / or derivatives, variants, or analogs thereof, and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) is about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, or more.

[0147] In embodiments, the administration period of the desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof is at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more.

[0148] In embodiments, the administration period of the desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof is within 10 minutes, within 20 minutes, within 30 minutes, within 45 minutes, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, within 19 hours, within 20 hours, within 21 hours, Within 22 hours, within 23 hours, within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, within 11 days, within 12 days, within 13 days, within 14 days, within 3 weeks, within 4 weeks, within 5 weeks, within 6 weeks, within 7 weeks, within 8 weeks, within 9 weeks, within 10 weeks, within 11 weeks, within 12 weeks, within 4 months, within 5 months, within 6 months, within 7 months, within 8 months, within 9 months, within 10 months, within 11 months, within 12 months, or more.

[0149] In further embodiments, the period during which administration of the desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof is stopped is for 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0150] In further embodiments, the period during which administration of the desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof is stopped is about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, or more.

[0151] In further embodiments, administration of the desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof is stopped within 10 minutes, within 20 minutes, within 30 minutes, within 45 minutes, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, within 19 hours, within 20 hours, within 21 hours, within 22 hours, within 23 hours, within 24 hours, within 25 hours, within 26 hours, within 27 hours, within 28 hours, within 29 hours, within 30 hours, within 35 hours, within 36 hours, within 37 hours, within 38 hours, within 39 hours, within 40 hours, within 41 hours, within 42 hours, within 45 hours, within 46 hours, within 47 hours, within 48 hours, within 49 hours, within 50 hours, within 51 hours, within 52 hours, within 53 hours, within 54 hours, within 55 hours, within 56 hours, within 57 hours, within 58 hours, within 59 hours, within 60 hours, within 61 hours, within 62 hours, within 63 hours, within 64 hours, within 65 hours, within 66 hours, within 67 hours, within 68 hours, within 69 hours, within 72 hours, within 73 hours, within 74 hours, within 75 hours, within 76 hours, within 79 hours, within 80 hours, within 81 hours, within 82 hours, within 85 hours, within 86 hours, within 87 hours, within 88 hours, within 89 hours, within 9 within 1 hour, within 22 hours, within 23 hours, within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, within 11 days, within 12 days, within 13 days, within 14 days, within 3 weeks, within 4 weeks, within 5 weeks, within 6 weeks, within 7 weeks, within 8 weeks, within 9 weeks, within 10 weeks, within 11 weeks, within 12 weeks, within 4 months, within 5 months, within 6 months, within 7 months, within 8 months, within 9 months, within 10 months, within 11 months, within 12 months, or more.

[0152] In further embodiments, administration of the DNase enzyme protein and / or derivatives, variants or analogs thereof is halted for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more.

[0153] In embodiments, the administration period of the DNase enzyme protein and / or derivatives, variants or analogs thereof, and / or at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) is 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 18 hours, 20 hours, 25 ...26 hours, 27 hours, 28 hours, 29 hours, 30 minutes, 35 minutes, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 minutes, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 8 7 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0154] In embodiments, the administration period of the DNase enzyme protein and / or derivatives, variants or analogs thereof, and / or at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) is about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, or more.

[0155] In embodiments, the administration period of the DNase enzyme protein and / or derivatives, variants or analogs thereof and / or at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) is at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 24 hours, at least 26 hours, at least 28 hours, at least 29 hours, at least 30 hours, at least 35 minutes, at least 36 minutes, at least 37 minutes, at least 38 minutes, at least 39 minutes, at least 40 minutes, at least 41 minutes, at least 42 minutes, at least 43 minutes, at least 44 minutes, at least 45 minutes, at least 45 minutes, at least 46 minutes, at least 47 minutes, at least 48 minutes, at least 49 minutes, at least 50 minutes, at least 51 minutes, at least 52 minutes, at least 53 minutes, at least 54 minutes, at least 55 minutes, at least 56 minutes, at least 57 minutes, at least 58 minutes, at least 59 minutes, at least 60 minutes, at least 61 minutes, at least 62 minutes, at least 63 minutes, at least 64 minutes, at least 65 minutes, at least 66 minutes, at least 67 minutes, at least 68 minutes, at least 69 minutes, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more.

[0156] In embodiments, the administration period of the at least one cell comprising a DNase enzyme protein and / or a derivative, variant or analog thereof and / or a chimeric antigen receptor (CAR) or a T cell receptor (TCR) is within 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, Within, within 19 hours, within 20 hours, within 21 hours, within 22 hours, within 23 hours, within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, within 11 days, within 12 days, within 13 days, within 14 days, within 3 weeks, within 4 weeks, within 5 weeks, within 6 weeks, within 7 weeks, within 8 weeks, within 9 weeks, within 10 weeks, within 11 weeks, within 12 weeks, within 4 months, within 5 months, within 6 months, within 7 months, within 8 months, within 9 months, within 10 months, within 11 months, within 12 months, or more.

[0157] In further embodiments, the administration period of the DNase enzyme protein and / or derivatives, variants or analogs thereof and / or at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), during which administration is stopped, is 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, , 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0158] In embodiments, the administration period of the at least one cell comprising a DNase enzyme protein and / or a derivative, variant or analog thereof and / or a chimeric antigen receptor (CAR) or a T cell receptor (TCR) is within 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, Within, within 19 hours, within 20 hours, within 21 hours, within 22 hours, within 23 hours, within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, within 11 days, within 12 days, within 13 days, within 14 days, within 3 weeks, within 4 weeks, within 5 weeks, within 6 weeks, within 7 weeks, within 8 weeks, within 9 weeks, within 10 weeks, within 11 weeks, within 12 weeks, within 4 months, within 5 months, within 6 months, within 7 months, within 8 months, within 9 months, within 10 months, within 11 months, within 12 months, or more.

[0159] The period during which administration of the DNase enzyme protein and / or derivatives, variants or analogs thereof and / or at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) is suspended may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0160] The administration period of the deoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof and / or at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) can be at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 24 hours, at least 28 hours, at least 29 hours, at least 30 hours, at least 35 minutes, at least 36 minutes, at least 37 minutes, at least 38 minutes, at least 39 minutes, at least 40 minutes, at least 41 minutes, at least 42 minutes, at least 43 minutes, at least 44 minutes, at least 45 minutes, at least 46 minutes, at least 47 minutes, at least 48 minutes, at least 49 minutes, at least 50 minutes, at least 51 minutes, at least 52 minutes, at least 53 minutes, at least 54 minutes, at least 55 minutes, at least 56 minutes, at least 57 minutes, at least 58 minutes, at least 59 minutes, at least 60 minutes, at least 61 minutes, at least 62 minutes, at least 63 minutes, at least 64 minutes, at least 65 minutes, at least 66 minutes, at least 67 minutes, at least 68 minutes, at least 69 minutes, at least 70 minutes, at least 71 minutes, at least 72 2 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more.

[0161] In further embodiments, the administration period of the DNase enzyme protein and / or derivatives, variants or analogs thereof and / or at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), during which administration is stopped, is 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, , 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0162] In further embodiments, the administration period of the DNase enzyme protein and / or derivatives, variants or analogs thereof and / or at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), during which administration is stopped, is at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least at least 21 hours, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more.

[0163] In further embodiments, the administration period of the DNase enzyme protein and / or derivatives, variants or analogs thereof and / or at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), during which administration is stopped, is within 10 minutes, within 20 minutes, within 30 minutes, within 45 minutes, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, within 19 hours, within 21 hours, within 22 hours, within 23 hours, within 24 hours, within 25 hours, within 26 hours, within 27 hours, within 28 hours, within 29 hours, within 30 hours, within 35 hours, within 36 hours, within 37 hours, within 38 hours, within 39 hours, within 40 hours, within 41 hours, within 42 hours, within 43 hours, within 44 hours, within 45 hours, within 46 hours, within 47 hours, within 48 hours, within 49 hours, within 50 hours, within 51 hours, within 52 hours, within 53 hours, within 54 hours, within 55 hours, within 56 hours, within 57 hours, within 58 hours, within 59 hours, within 60 hours, within 61 hours, within 62 hours, within 63 hours, within 64 hours, within 65 hours, within 66 hours, within 67 hours, within 68 hours, within 69 hours, within 70 hours, within 71 hours, within 72 hours, within 73 hours, within 74 hours, within 75 hours, within 76 hours, within 77 hours, within 78 hours, within, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, within 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0164] In further embodiments, the administration period of the DNase enzyme protein and / or derivatives, variants, or analogs thereof and / or at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), during which administration is stopped, is about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, or more.

[0165] In aspects of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof disclosed herein reduces or maintains the severity of cancer in an individual, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%. In other aspects of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivatives, variants, or analogs thereof disclosed herein reduces or maintains the severity of cancer in an individual by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95% or up to 100%. In yet other aspects of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivative, variant, or analog thereof disclosed herein reduces disease severity in an individual by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, or about 10% to about 20%. %, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0166] In aspects of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof disclosed herein and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) reduces or maintains the severity of the cancer in an individual, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%. In other aspects of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivatives, variants or analogs thereof disclosed herein and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) reduces or maintains the severity of cancer in an individual, e.g., by up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95% or up to 100%. In yet another aspect of this embodiment, a therapeutically effective amount of a desoxyribonuclease enzyme protein and / or derivatives, variants, or analogs thereof disclosed herein, and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), can reduce the severity of disease in an individual by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 90%, about 10% to about 90%, about 10% to about 10 ... Reduced or maintained at 0%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0167] At least one cell comprising a deoxyribonuclease enzyme protein and / or a derivative, variant or analog thereof, and a chimeric antigen receptor (CAR) or a T cell receptor (TCR) is administered to an individual. Typically, the individual is a human, but may be an animal, including but not limited to dogs, cats, birds, cattle, horses, sheep, goats, reptiles and other animals, whether domesticated or not. Typically, any individual who is a candidate for treatment is a candidate for some form of therapy for the disease that the individual suffers from, whether that disease is benign or malignant. With regard to cancer, the most common types of cancer include, but are not limited to, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, kidney cancer, renal cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, gastric cancer and thyroid cancer. Typically, preoperative evaluation includes a routine medical history and physical examination, as well as full informed consent disclosing all relevant risks and benefits of the procedure.

[0168] In embodiments, the deoxyribonuclease enzyme protein disclosed herein, or compositions comprising such therapeutic compounds, can be in liquid formulations. In embodiments, liquid formulations suitable for enteral or parenteral administration include, but are not limited to, solutions, syrups, elixirs, dispersions, emulsions, and suspensions. In embodiments, the therapeutic compounds or compositions disclosed herein intended for such administration can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, including, but not limited to, the manufacture of pharmaceutical compositions. In embodiments, in such liquid dosage forms, the therapeutic compounds or compositions disclosed herein can be mixed with, but are not limited to, (a) suitable aqueous and non-aqueous carriers, (b) diluents, (c) solvents, including, but not limited to, water, ethanol, propylene glycol, polyethylene glycol, glycerol, vegetable oils, including, but not limited to, rapeseed oil and olive oil, and injectable organic esters, including, but not limited to, ethyl oleate; and / or flow agents, including, but not limited to, surfactants or coating agents such as lecithin. In the case of dispersions and suspensions, flowability can also be controlled by maintaining a specific particle size.In embodiments, in a liquid formulation, a therapeutically effective amount of a therapeutic compound disclosed herein is typically from about 0.0001% (w / v) to about 90% (w / v), 0.0001% (w / v) to about 80% (w / v), 0.0001% (w / v) to about 70% (w / v), 0.0001% (w / v) to about 60% (w / v), 0.0001% (w / v) to about 50% (w / v), 0.0001% (w / v) to about 70% (w / v), 0.0001% (w / v) to about 80% (w / v), 0.0001% (w / v) to about 90% (w / v), 0.0001% (w / v) to about 100% (w / v), 0.0001% (w / v) to about 15 ... / v) ~ approx. 40% (w / v), 0.0001% (w / v) ~ approx. 30% (w / v), 0.0001% (w / v) ~ approx. 20% (w / v), 0.0001% (w / v) ~ approx. 10% (w / v), approx. 0.0 01% (w / v) ~ approx. 90.0% (w / v), 0.001% (w / v) ~ approx. 80.0% (w / v), 0.001% (w / v) ~ approx. 70.0% (w / v), 0.001% (w / v) ~ approx. 60.0% (w / v), 0.001% (w / v) to about 0.0% (w / v), 0.001% (w / v) to about 40.0% (w / v), 0.001% (w / v) to about 30.0% (w / v), 0.001% (w / v) to about 20.0% (w / v), 0.001% (w / v) to about 10.0% (w / v), or about 0.01% (w / v) to about 90.0% (w / v), about 0.01% (w / v) to about 80.0% (w / v) , about 0.01% (w / v) to about 70.0% (w / v), about 0.01% (w / v) to about 60.0% (w / v), about 0.01% (w / v) to about 50.0% (w / v), about 0.01% (w / v) to about 40.0% (w / v), about 0.01% (w / v) to about 30.0% (w / v), about 0.01% (w / v) to about 20.0% (w / v), or about 0.01% (w / v) to about 10.0% (w / v).

[0169] In embodiments, liquid suspensions may be formulated by suspending the therapeutic compound, including but not limited to, the deoxyribonuclease enzyme protein disclosed herein, in admixture with excipients suitable for the manufacture of aqueous suspensions. In embodiments, such excipients are suspending agents, including but not limited to, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, pectin, polyvinylpyrrolidone, polyvinyl alcohol, natural gums, agar, tragacanth gum and acacia gum; dispersing or wetting agents may be natural phosphatides, such as lecithin, or condensation products of alkylene oxides with fatty acids, including but not limited to, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, including but not limited to, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids, including but not limited to, polyoxyethylene sorbitan monooleate.

[0170] As an additional aspect, the present invention includes a kit comprising a composition of the present invention packaged to facilitate its use for administration to a subject. In one embodiment, such a kit comprises a compound or composition described herein (e.g., a composition comprising a DNase enzyme protein and / or a derivative, variant or analog thereof, and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR)) packaged in a container such as a sealed bottle or container with a label attached to the container or included in the package that describes the use of the compound or composition in carrying out the method. In one embodiment, the kit contains a first container with a composition comprising the DNase enzyme protein and a second container with a physiologically acceptable reconstitution solution for the composition in the first container. In one aspect, the compound or composition is packaged in a unit dosage form. The kit may further comprise a device suitable for administering the composition according to a particular route of administration. Preferably, the kit contains a label that describes the use of the therapeutic protein or peptide composition.

[0171] In certain embodiments, the DNase enzyme is formulated in a pharmaceutical composition together with a pharma- ceutically acceptable carrier or excipient.

[0172] The preparations used in the method of the present invention can be conveniently presented in unit dosage form and can be prepared by methods known in the art.The amount of active ingredient that can be combined with carrier material to produce a single dosage form varies depending on the host treated and specific mode of administration.The amount of active ingredient that can be combined with carrier material to produce a single dosage form is generally the amount of compound that produces therapeutic effect.

[0173] In general, the formulations can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0174] Pharmaceutical compositions suitable for parenteral administration may contain one or more active ingredients (DNase and, optionally, another compound [e.g., anticancer compound]) in combination with one or more pharma- ceutically acceptable sterile, isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, which may include antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0175] These compositions may also contain preservatives, wetting agents, emulsifiers and dispersing agents. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0176] Injectable depot forms can be made by forming microencapsule matrices of one or more active ingredients in biodegradable polymers such as polylactide-polyglycolide. The release rate of the active ingredient can be controlled depending on the ratio of the active ingredient to the polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the active ingredient in liposomes or microemulsions that are compatible with body tissues.

[0177] Formulations for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid (for example, as a mouthwash, as a composition to be swallowed, or as an enema), or as an oil-in-water or water-in-oil liquid emulsion, each containing a predetermined amount of one or more of the active ingredients.

[0178] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more active ingredients are combined with one or more pharma- ceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerin; (4) disintegrants, such as agar, charcoal, or the like. The pharmaceutical composition may be mixed with any of the following: calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption enhancers, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical composition may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols.

[0179] Suspensions may contain, in addition to one or more active ingredients, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0180] Powders and sprays can contain, in addition to one or more active ingredients, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0181] Cancer and other treatments In one aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a DNase enzyme and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0182] In a related aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a DNase enzyme and at least one cell comprising a chimeric antigen receptor (CAR) or a T-cell receptor (TCR), wherein said DNase enzyme is effective to reduce expression of an immunosuppressive protein in tumor tissue and / or increase the content of cells comprising the chimeric antigen receptor or T-cell receptor in the tumor tissue.

[0183] The methods of the present invention can be used in subjects suffering from a wide range of cancers. Non-limiting examples of relevant cancers include, for example, breast cancer, prostate cancer, multiple myeloma, transitional cell carcinoma, lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer, thyroid cancer, leukemia (e.g., chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia), lymphoma (e.g., B cell lymphoma, T cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma), head and neck cancer, esophageal cancer, gastric cancer, colon cancer, intestinal cancer, colorectal cancer, rectal cancer, pancreatic cancer, liver cancer, bile duct cancer, gallbladder cancer, ovarian cancer, uterine cancer, vaginal cancer, cervical cancer, bladder cancer, neuroblastoma, sarcoma, osteosarcoma, malignant melanoma, squamous cell carcinoma, primary bone cancer (e.g., osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant fibrous histiocytoma, adamantine sarcoma ... Bone cancer, including both primary (cancerous, giant cell, and chordoma) and secondary (metastatic) bone cancer, soft tissue sarcoma, basal cell carcinoma, angiosarcoma, hemangiosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, testicular cancer, uterine cancer, gastrointestinal cancer, mesothelioma, leiomyosarcoma, These include rhabdomyosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Waldenstrom's macroglobulinemia, papillary adenocarcinoma, cystadenocarcinoma, bronchogenic carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, epithelial carcinoma, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, medullary carcinoma, thymoma, and sarcoma.

[0184] In some embodiments of any of the methods of the invention, the subject is a human.

[0185] Working Example The present invention is also described and demonstrated by the following examples. However, the use of these and other examples elsewhere in this specification is merely illustrative and in no way limits the scope and meaning of the present invention or any exemplified term. Similarly, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the present invention may be apparent to those skilled in the art upon reading this specification, and such modifications can be made without departing from the spirit or scope of the present invention. Therefore, the present invention should be limited only by the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0186] Example 1. Deoxyribonuclease enzyme enhances the therapeutic efficacy of chimeric antigen receptor (CAR)-bearing T cells Female NOD SCID mice (CB17_Prkdc) aged 6-8 weeks and weighing an average of 16-20 g were scid / NcrCrl) mice were used. 5 × 10 6 Lymphomas were engrafted in mice by subcutaneous inoculation of Raji cells into the left flank. Tumors were at least 50 mm 3 After reaching a palpable volume of 100 mm, mice were randomly assigned to four experimental groups: Group "control": 3 × 10 6 A single IV injection of non-transduced human CD8 T cells; Group "DNase I": 10 daily IV injections of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO: 4) at a dose of 4 mg / kg starting on day 10 after tumor inoculation; Group "CAR19": 10 days after tumor inoculation, CD19CAR [FMC63 scFv / IgG4 hinge / IgG4 CH2-CH3 / CD28TM / OX-40 / CD3z] (Stepanov AV, Markov OV, Chernikov IV, Gladkikh DV, Zhang H, Jones T, Sen'kova AV, Chernolovskaya EL, Zenkova MA, Kalinin RS, Rubtsova MP, Meleshko AN, Genkin DD, Belogurov AA Jr, Xie J, Gabibov AG, Lerner RA. "Autocrine-based selection of ligands for personalized CAR-T therapy of lymphoma" Sci Adv 2018 Nov 14;4(11):eaau4580.doi:10.1126 / sciadv.aau4580.PMID:30443597;PMCID:PMC6235538). 6 A single IV injection of human CD8 T cells; Group "CAR19 and DNase I": 3 × 10 mice transduced with a lentiviral vector encoding CD19 CAR [FMC63 scFv / IgG4 hinge / IgG4 CH2- CH3 / CD28TM / OX-40 / CD3z] on day 10 after tumor inoculation. 6 A single IV injection of untransduced human CD8 T cells followed by 10 daily IV injections of human recombinant DNase I enzyme at a dose of 4 mg / kg starting on day 10 after tumor inoculation.

[0187] Tumor volumes were measured with calipers and estimated using the formula for an ellipsoid. The data are shown in Figure 1. The data clearly demonstrate that IV injection of human recombinant DNase I enzyme provides a statistically significant (p<0.05) enhanced efficacy for CD19 CAR T cells.

[0188] Example 2. AAV gene delivery of deoxyribonuclease enzyme increases recruitment of chimeric antigen receptor (CAR)-bearing T cells to tumor tissue and reduces local immunosuppression AAV-DNase I vectors were generated as described in Xia Y, He J, Zhang H, et al. AAV-mediated gene transfer of DNase I in the liver of mice bearing colorectal cancer reduces liver metastasis and restores local innate and adaptive immune responses [Published online ahead of print, August 19, 2020] Mol Oncol. 2020;14(11):2920-2935. doi:10.1002 / 1878-0261.12787. Briefly, AAV-DNase I vectors were generated using polyethylenimine triple plasmid transfection of rep / cap plasmid encoding Anc80L65 capsid, pCLS-014, and adenovirus helper plasmid in HEK293 cells and further purified on an extensive scale with iodixanol gradient. Plasmid pAAV-ApoEHCR enhancer-hAAT promoter-hDNase I (high activity)-WPRE Xinact (pCLS-014) specifically contained a transgene expression cassette consisting of (a) the apolipoprotein E-hepatic control region (APOE-HCR enhancer) and human alpha-1-antitrypsin (hAAT) promoter, (b) a Kozak sequence, (c) a human high activity actin-resistant DNase I variant containing the native signal sequence, and (d) a woodchuck hepatitis virus posttranscriptional element (WPRE) with the X protein coding region inactivated by mutating the start codon. Purified AAV-DNase I vectors were reconstituted in PBS supplemented with 35 mM NaCl and 0.001% Pluronic F68, and titers (genome copies [GC] / mL) were determined by quantitative PCR. Analysis of the purified AAV vector by SDS / PAGE revealed three bands of 60, 72, and 90 kDa corresponding to the VP1-3 proteins in a ratio of approximately 1:1:10. AAV-null contained all components of the CLS-014 transgene expression cassette except for the absence of DNase I cDNA.

[0189] ScFv of monoclonal antibodies specific for CEA have been generated using a proprietary scfv phage display library. The scFv were cloned into the pMS3 retroviral vector together with the CD8α hinge, CD28 transmembrane domain, and CD3ζ and CD28 signaling domains. Murine CEA-targeted CAR T cells were generated as described in Wang L, Ma N, Okamoto S, et al. Efficient tumor regression by adoptively transferred CEA-specific CAR-T cells associated with symptoms of mild cytokine release syndrome. Oncoimmunoiogy. 2016;5(9):e1211218. Published July 25, 2016. doi:10.1080 / 2162402X.2016.1211218. Total splenocytes from C57BL / 6 (CD45.1) syngeneic mice (1.5 × 10 7 Cells / 5 mL) were stimulated with immobilized anti-CD3 (1 mg / mL; 145-2C11) and soluble anti-CD28 (1 mg / mL; 37.51) in one well of a 6-well plate. One day after stimulation (day 1), 5 × 10 5 Cells were transduced with viral vectors using the RetroNectin-bound virus infection method, in which viral solution was preloaded into RetroNectin (Takara Bio)-coated wells of a 24-well plate containing 1 mL of medium. On day 3, cells were transferred to a 50 mL flask containing 10 mL culture medium for expansion. On day 5, cells were harvested and used for experiments. 60 IU / mL of recombinant human IL-2 (Novartis) was added during culture.

[0190] To establish the liver metastasis model, anesthetized C57BL / 6 mice were placed in a supine position. After the skin of the surgical site was disinfected, a midline abdominal incision was made, followed by displacing the duodenum to identify the portal vein. One million CEA+MC32a cells were injected into the portal vein using a 30G needle. After removing the needle, bleeding was stopped by gently pressing the puncture site with a cotton swab. After injection, the intestine was repositioned, and the abdominal wall was closed with non-absorbable sutures.

[0191] Five days after tumor challenge, mice (7 per group) were divided into four groups: Group “control”: tumor-bearing control mice; Group "CAR19": 5×10 6 IV injection of CAR T cells; Group "AAV-DNase I": 1.5 × 10 per mouse 12 IV injection of AAV-DNase I vector in GCs; Group "CAR19 and AAV-DNase I": 1.5 × 10 per mouse 12 5 x 10 combined with AAV-DNase I vector at GC 6 IV injection of CAR T cells were randomly assigned to

[0192] A survival graph is presented in Figure 2. The data in Figure 2 clearly demonstrate a significant survival (p<0.05) benefit from the combination of CAR T cells and AAV-DNase I gene transfer.

[0193] On day 15, one animal from each of the groups “control,” “CAR19,” and “CAR19 and AAV-DNase I” was euthanized (one mouse per group) and the liver was removed for examination. Metastatic tissues were analyzed by flow cytometry for PD-L1 expression using an anti-PD-L1 antibody (clone 10F.9G2, Merck) and the presence of CEA-targeted CAR T cells using streptavidin conjugated with biotinylated protein L and FITC [as described in Xia Y, He J, Zhang H, et al. "AAV-mediated gene transfer of DNase I in the liver of mice with colorectal cancer reduces liver metastasis and restores local innate and adaptive immune response" Mol Oncol. 2020;14(11):2920-2935. doi:10.1002 / 1878-0261.12787].

[0194] Figure 3 shows the percentage of metastatic MC32a cells expressing PD-L1. The data show significant suppression (p<0.05) of PD-L1 expression in metastatic MC32a cells from metastatic lesions of mice treated with the combination of CAR T cells and AAV-DNase I gene transfer.

[0195] Figure 4 shows the percentage of CEA-targeted CAR T cells in the parenchyma of MC32a metastatic lesions. The data show a significant increase (p<0.05) in the number of CAR T cells in metastatic lesions of mice treated with the combination of CAR T cells and AAV-DNase I gene transfer.

[0196] Example 3. Development of chimeric antigen receptor (CAR)-bearing T cells expressing deoxyribonuclease enzyme Human peripheral blood T cells from healthy donors were engineered with the FL1 CAR, which contains the CILDLPKFC / IgG1 Fc spacer domain ("CILDLPKFC" disclosed as SEQ ID NO:59), a GGGS linker (SEQ ID NO:60), CD28 transmembrane and intracellular regions, and the intracellular domains of OX-40 and CD3 zeta, with or without the gene for full-length human highly active actin-resistant mutant DNase I (SEQ ID NO:5) via the self-cleaving peptide P2A (Stepanov et al., Autocrine-based selection of ligands for personalized CAR-T therapy of lymphoma Sci Adv. 2018,4(11):eaau4580;doi:10.1126 / sciadv.aau4580). See FIG. 5.

[0197] DNase activity in the culture medium of FL1-CAR-T cells, DNase I FL1-CAR-T cells and non-transduced control T cells was analyzed using a fluorescent probe (Terekhov et al., PNAS 2017,114(10):2550-2555) during 6 h incubation of the cells in complete RPMI 1640 medium supplemented with human IL-2 (40 U / mL). All experiments were performed in triplicate. The data are presented in Figure 6. The data show a gradual increase in DNase activity in the culture medium of DNase I FL1-CAR T cells.

[0198] Cytotoxicity and specificity of engineered T cells were assessed by a standard LDH release assay (CytoTox 96 non-radioactive cytotoxicity assay, Promega) according to the manufacturer's recommendations. FL1-CAR-T cells, DNase I FL1-CAR-T cells and non-transduced control T cells were cultured at 10 4 The cells were incubated with Raji-FL1 cells for 6 h. All experiments were performed in triplicate.

[0199] The data are shown in Figure 7. The data clearly demonstrate that reprogramming T cells to simultaneously express a CAR and a desoxyribonuclease enzyme provides superior cytotoxicity against target lymphoma cells (p<0.01).

[0200] Example 4. Biological activity of chimeric antigen receptor (CAR)-bearing T cells expressing deoxyribonuclease enzyme Female NOD SCID mice (CB17_Prkdc) aged 6-8 weeks and weighing an average of 16-20 g were scid / NcrCrl) mice were used. 5 × 10 6 Tumors were engrafted by subcutaneous inoculation of Raji-FL1 CAR cells into the left flank of mice. 3 After reaching a palpable volume of 100 μg / kg, mice were randomly assigned and treated as follows: on days 10 and 15, tumor-bearing mice received 3 × 10 6 aFL1 CAR T cells (group 2; 6 mice), 3 × 10 expressing highly active actin-resistant mutant DNase I (SEQ ID NO: 5) 6 aFL1 CAR T cells (group 3; 6 mice) and placebo control (group 1; 6 mice) were injected intravenously. Tumor volumes were measured with calipers and estimated using the ellipsoid formula. Animals were euthanized on day 20.

[0201] The data are shown in Figure 8. The data clearly demonstrate that reprogramming T cells to simultaneously express CAR and DNase enzyme provides superior efficacy to such dual-reprogrammed T cells (p<0.05).

[0202] Example 5. Treatment with DNase enhances the efficacy of CAR-T cell therapy Human peripheral blood T cells from healthy donors were engineered with CD19 CAR containing FMC63 scFv / IgG4 hinge / IgG4 CH2-CH3 / CD28TM / OX-40 / CD3z (as described in Stepanov et al., Autocrine-based selection of ligands for personalized CAR-T therapy of lymphoma. Sci Adv. 2018,4(11):eaau4580.doi:10.1126 / sciadv.aau4580). Female NOD SCID mice (CB17_Prkdc) aged 6-8 weeks and weighing an average of 16-20 g were used. scid / NcrCrl) mice were used. 5 × 10 6 Tumors were engrafted by subcutaneous inoculation of Raji-CD19 CAR cells into the left flank of mice. 3 After reaching a palpable volume of 100 μg / kg, mice were randomly assigned and treated as follows: on days 10 and 15, tumor-bearing mice received 0.5 × 10 6 Tumor-bearing mice were injected intravenously with CD19 chimeric antigen receptor (CAR) T cells at a dose of CAR-T cells / kg alone or in combination with different DNase I regimens (human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO: 4) administered by IV injection). The animals were randomized into different groups: Group 1 (n = 5 mice)-Treatment with CD19 CAR-T cells; Group 2 (n = 5 mice) - one treatment with CD19 CAR-T cells + 500 μg / kg DNase I 24 hours before the start of CAR-T cell therapy; Group 3 (n=5 mice)-two treatments with CD19 CAR-T cells + 1000μg / kg DNase I 24 hours before and 24 hours after the initiation of CAR-T cell therapy; Group 4 (n=5 mice)-two treatments with CD19 CAR-T cells + 1000μg / kg DNase I 72 hours before and 30 days after the start of CAR-T cell therapy; Group 5 (n = 5 mice) - treatment with CD19 CAR-T cells + 250 μg / kg DNase I 3 days before the start of CAR-T cell therapy; Group 6 (n = 5 mice) - treatment with CD19 CAR-T cells + 2500 μg / kg DNase I 1 day before and daily for 6 days after initiation of CAR-T cell therapy; Group 7 (n = 5 mice) - one treatment with CD19 CAR-T cells + 500 μg / kg DNase I 7 days before the start of CAR-T cell therapy; Group 8 (n=5 mice) - one treatment with CD19 CAR-T cells + 500μg / kg DNase I 7 days before initiation of CAR-T cell therapy and weekly for the next 8 weeks after initiation of CAR T cell therapy; Group 9 (n=5 mice) - One treatment with CD19 CAR-T cells + 1500 μg / kg DNase I 28 days prior to initiation of CAR-T cell therapy. Clinical efficacy and side effects were monitored.

[0203] Figure 9 shows flow cytometric detection of CAR-T cells in peripheral blood using streptavidin conjugated with biotinylated protein L and FITC (Stepanov et al., Autocrine-based selection of ligands for personalized CAR-T therapy of lymphoma, Sci Adv. 2018, 4(11):eaau4580; doi:10.1126 / sciadv.aau4580). The data demonstrate that the combination of DNase and CAR-T cell therapy significantly (p<0.05 for all) increases the persistence of CAR T cells in the blood.

[0204] Figure 10 demonstrates that the combination of DNase and CAR-T cell therapy enhances animal survival. All tested combinations of DNase I and CD19 CAR-T cell treatment are shown to be significantly superior to CD19 CAR-T cell treatment alone. Pretreatment of mice with high dose DNase I in group 9 provided a particular advantage that may be related to more extensive preconditioning of the tumor microenvironment prior to CD19 CAR-T cell treatment.

[0205] Example 6. Treatment with DNase reduces cytokine release syndrome associated with CAR T cell therapy To model cytokine release syndrome (CRS), 6- to 8-week-old female CBIgh-1b / GbmsTac-Prkdc mice were transfected with scid Lyst bg N7 (SCID) mice (Charles River) were used, and 3 × 10 6 3 × 10 Raji cells were injected intraperitoneally. 7 CD19 CAR-T cells (same as in Example 5) were injected intraperitoneally on day 25 after tumor growth. CAR-T cells were administered alone or in combination with different DNase I regimens (human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO: 4), administered by IV injection). The animals were randomized into different groups: Group 1 (n = 5 mice) - treatment with CAR-T cells; Group 2 (n = 5 mice) - one treatment with CAR-T cells + 500 μg / kg DNase I 24 hours before the start of CAR-T cell therapy; Group 3 (n = 5 mice) - treatment with CAR-T cells + 1000 μg / kg DNase I 24 hours after initiation of CAR-T cell therapy; Group 4 (n = 5 mice) - two treatments with CAR-T cells + 1000 μg / kg DNase I 72 hours before the start of CAR-T cell therapy; Group 5 (n = 5 mice) - treatment with CAR-T cells + 500 μg / kg DNase I 7 days before the start of CAR-T cell therapy; Group 6 (n=5 mice) - Treatment with CAR-T cells + 500 μg / kg DNase I 28 days prior to the start of CAR-T cell therapy.

[0206] The data on the % of animals with CRS shown in FIG. 11 demonstrate that the use of DNase enhances the safety of CAR-T therapy and protects against CAR-T-associated side effects.

[0207] array SEQ ID NO:1 - Human DNase I, wild type (WT), precursor; Genbank accession number NP_005214.2; secretory signal sequence is underlined. [ka] SEQ ID NO:2 - Human DNAse I mutant, precursor; mutated residues compared to SEQ ID NO:1 are shown in bold and underlined; secretory signal sequence is underlined. [ka] SEQ ID NO:3 - Anc80 VP1 capsid protein: [ka] X 1 = K or R; X 2 = A or S; X 3 = A or G; X 4 = R or K; X 5 = E or Q; X 6 = T or E; X 7 = A or T; X 8 = S or N; X 1 = Q or E; X 10 = S or A, and X 11 =N or D. SEQ ID NO:4 - Mature wild type (WT) human DNase I (without secretory signal sequence; GenBank Accession No. 4AWN_A: [ka] SEQ ID NO:5 - Mature human DNAse I variant (without secretory signal sequence); mutated residues compared to SEQ ID NO:4 are shown in bold and underlined: [ka] SEQ ID NO:6 - Secretory signal sequence of human DNase I: [ka] SEQ ID NO:7 - Secretory signal sequence of IL2: [ka] SEQ ID NO:8 - Human albumin promoter: [ka] SEQ ID NO:9 - Anc80 VP1 capsid protein: [ka] X 1 = K or R; X 2 = A or S; X 3 = A or G; X 4 = R or K; X 5 = E or Q; X 6 = T or E; X 7 = A or T; X 8 = S or N; X 1 = Q or E; X 10 = S or A, and X 11 =N or D. SEQ ID NO:10 - Human beta globin primer: [ka] SEQ ID NO:11 - Forward NLRP3 primer: [ka] SEQ ID NO:12 - Reverse NLRP3 primer: [ka] SEQ ID NO:13 - Forward 16S universal bacterial RNA gene primer: [ka] SEQ ID NO:14 - Reverse 16S universal bacterial RNA gene primer: [ka] SEQ ID NO:15 - Human antitrypsin promoter sequence: [ka] SEQ ID NO:16 - Woodchuck hepatitis virus post-transcriptional regulatory element that does not encode a functional protein X: [ka] SEQ ID NO:17 - Apolipoprotein E (ApoE) enhancer, hepatic control region (HCR): [ka] Polynucleotides encoding human DNase I hyperactive variants of SEQ ID NO: 18-SEQ ID NO: 5: [ka] SEQ ID NO:19 - Polynucleotide encoding the precursor of the human DNAse I variant of SEQ ID NO:2 (secretory signal sequence is underlined): [ka] SEQ ID NO:20 - Polynucleotide encoding the secretory signal sequence of human DNase I (SEQ ID NO:6): [ka] SEQ ID NO:21 - Polynucleotide encoding the mature human DNAse I variant (without secretory signal sequence) of SEQ ID NO:5: [ka] SEQ ID NO:22 - Polynucleotide encoding human DNase I wild-type (WT) precursor of SEQ ID NO:1: [ka] Polynucleotides encoding mature wild-type (WT) human DNase I of SEQ ID NO:23-SEQ ID NO:4: [ka] SEQ ID NO:24 - Mus musculus wild-type DNase I, precursor; GenBank Accession No. NP_034191.3; secretion signal sequence is underlined: [ka] SEQ ID NO:25 - Secretory signal sequence of Mus musculus wild-type DNase I [ka] SEQ ID NO: 26 - Mature wild type (WT) Mus musculus wild type DNase I [ka] SEQ ID NO:27 - Polynucleotide encoding the secretory signal sequence of Mus musculus wild-type DNase I [ka] SEQ ID NO:28 - Polynucleotide encoding mature wild type (WT) Mus musculus wild type DNase I [ka] SEQ ID NO: 29 - Polynucleotide encoding the precursor of Mus musculus wild-type DNase I [ka] SEQ ID NO:30 - Complete sequence of ApoEHCR enhancer-hAAT promoter-hDNase I (high activity) precise leader-WPRE Xinact (VR-18013AD) [ka] (APOE HCR enhancer: bases 1-320; human alpha-1-antitrypsin promoter: bases 321-717; Kozak sequence: bases 718-726; native human DNase I hyperactive variant with a perfectly accurate leader sequence: bases 727-1575; inactivated WPRE X protein: bases 1576-2212) SEQ ID NO:31 - Complete sequence of ApoEHCR enhancer-hAAT promoter-hDNase I wild type-WPRE Xinact (VR-18014AD) [ka] (APOE HCR enhancer: bases 1-320; human alpha-1-antitrypsin promoter: bases 321-717; Kozak sequence: bases 718-726; human DNase I wild type with native and perfectly accurate leader sequence: bases 727-1575; inactivated WPRE X protein: bases 1576-2212) SEQ ID NO:32 - Polynucleotide encoding human DNase I wild type (WT) precursor of SEQ ID NO:1 with a stop codon: [ka] SEQ ID NO:33 - Kozak sequence [ka] SEQ ID NO:34 - Anc80L65 VP1 capsid protein [ka] SEQ ID NO:35 - Variant Anc80L65 VP1 capsid protein [ka] SEQ ID NO:36 - Human synapsin promoter [ka] SEQ ID NO:37 - CMV promoter [ka] SEQ ID NO:38-F4 / 80 promoter [ka] SEQ ID NO:39-TMEM119 promoter [ka] SEQ ID NO:40 - MEF2 promoter [ka] SEQ ID NO:41 - FoxP2 promoter [ka] SEQ ID NO:42 - hDNase I (high activity) precise leader - WPRE.bGH [ka] SEQ ID NO:43 - hDNase I (wild type) WPRE.bGH [ka] SEQ ID NO:44 - Human DNase I mutant [ka] SEQ ID NO:45 - AAV5 capsid sequence (VP1) [ka] SEQ ID NO:46-AAV9 capsid sequence (VP1) [ka] SEQ ID NO:47 - AAVLK03 VP1 capsid protein: [ka] SEQ ID NO:48 - Polynucleotide encoding AAV-LK03 VP1 capsid protein: [ka] SEQ ID NO:49 - AAV-KP1 VP1 capsid protein: [ka] SEQ ID NO:50 - Polynucleotide encoding AAV-KP1 VP1 capsid protein: [ka] SEQ ID NO:51 - Precursor of human DNase1L3 (Q13609) (signal peptide underlined): [ka] SEQ ID NO:52 - Polynucleotide encoding the precursor of human DNase1L3 (Q13609): [ka] SEQ ID NO:53 - Mature DNase1L3: [ka] NGTSG (SEQ ID NO:54) SGTH (SEQ ID NO:55) SDTH (SEQ ID NO:56) GGTAN (SEQ ID NO:57) DGSGL (SEQ ID NO:58) CILDLPKFC (SEQ ID NO:59), IgG1 Fc spacer domain GGGS linker (SEQ ID NO:60)

[0208] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

[0209] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein.

Claims

1. A method for treating cancer in a subject in need thereof, comprising administering to said subject a therapeutic agent comprising: a deoxyribonuclease (DNase) enzyme; and at least one cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR). composition.

2. 2. The composition of claim 1, wherein the DNase enzyme is used in an amount effective to increase the content of CAR-expressing cells or TCR-expressing cells in tumor tissue.

3. 1. A composition comprising a deoxyribonuclease (DNase) enzyme for use in preventing one or more side effects of a CAR-T cell therapy in a subject in need thereof, wherein the DNase enzyme is administered simultaneously or sequentially with the CAR-T cell therapy. composition.

4. 4. The composition of claim 3, wherein the side effects of the CAR-T cell therapy are selected from neurological side effects, cytokine release syndrome (CRS), neutropenia, anemia, fever, febrile neutropenia, and thrombocytopenia.

5. The composition of any one of claims 1 to 3, wherein the DNase enzyme is administered as a DNase enzyme protein.

6. The composition of any one of claims 1 to 3, wherein the DNase enzyme is encoded by a vector.

7. 7. The composition of claim 6, wherein the vector is a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding the DNase enzyme.

8. The composition of claim 7 , wherein the promoter is specific to a tumor-initiating tissue or a metastatic target tissue.

9. The composition of any one of claims 1 to 3, wherein the DNase enzyme is encoded by an mRNA molecule.

10. The composition of any one of claims 1 to 9, wherein the DNase enzyme is administered parenterally or to the site of a tumor.

11. 11. The composition of any one of claims 1 to 10, wherein the DNase enzyme is co-expressed by a cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

12. The composition of any one of claims 1 to 11, wherein the CAR-expressing cell or TCR-expressing cell is administered parenterally or to the site of a tumor.

13. The composition of any one of claims 1 to 12, wherein the CAR-expressing cell or the TCR-expressing cell is a single target or multiple targets.

14. The CAR is (i) CD5, CD7, CD19, CD28, mesothelin, CD123, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, FR-1, c-MET, EGFR / CD133, IL13Ra2, HER2, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, and mut hsp70-2; (ii) an antigen associated with a solid tumor;(iii) mesothelin, EGFRvIII, GD2, CLDN6, Tn Ag, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, legumain, CD171, PSCA, TARP, MAD-CT-1, Lewis Y, CD24, folate receptor alpha, folate receptor beta, ERBBs, MUC1, EGFR, NCAM, PDGFR-beta, MAD-CT-2, Fos-related antigen, SSEA-4, neutrophil elastase, CAIX, HPV E6 E7, ML-IAP, NA17, ALK, androgen receptor polysialic acid, TRP-2, CYP1B1, PLAC1, GloboH, NY-BR-1, sperm protein 17, HMWMAA, beta human chorionic gonadotropin, AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, and mut hsp 70-2; (iv) a solid tumor associated antigen present in / on mesothelioma, lung cancer, pancreatic cancer, esophageal adenocarcinoma, ovarian cancer, breast cancer, colorectal cancer, bladder cancer, or any combination thereof; (v) a tumor antigen associated with a blood cancer; (vi) a tumor antigen present in a disease selected from acute leukemia and chronic leukemia, and (vii) a tumor antigen present in a treatment-resistant cancer.

15. 15. The composition of any one of claims 1 to 14, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:53.

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