Methods of using dnase1-like 3 in therapy

Abstract

The present disclosure provides D1L3 enzymes having complete or partial C-terminal deletions of the basic domain (BD), which have substantially enhanced chromatin-degrading activity. In various aspects, the invention provides chromatinase enzyme therapy, which is optionally provided by delivering polynucleotides encoding chromatinases such as D1L3, or by delivering host cells expressing and secreting the same.

What is claimed is: 
     
         1 . An isolated human host cell comprising a heterologous polynucleotide encoding a chromatinase enzyme operably linked to a promoter, wherein the host cell expresses and secretes the chromatinase enzyme. 
     
     
         2 . The isolated host cell of  claim 1 , wherein the chromatinase is DNASE1-LIKE 3 (D1L3). 
     
     
         3 . The isolated host cell of  claim 2 , wherein the secreted D1L3 enzyme has at least a partial deletion of the C-terminal basic domain. 
     
     
         4 . The isolated host cell of  claim 3 , wherein the secreted D1L3 enzyme has a deletion of at least 12 amino acids of the C-terminal basic domain. 
     
     
         5 . The isolated host cell of  claim 4 , wherein the secreted D1L3 enzyme includes enzymes having deletions of one or more of: K291_S305 del, K292_S305 del, K293_S305 del, with respect to SEQ ID NO:4. 
     
     
         6 . The isolated host cell of any one of  claims 1  to  5 , wherein the host cell is a white blood cell, an endothelial cell, an epithelial cell, a hepatocyte, or a stem cell. 
     
     
         7 . The isolated host cell of  claim 6 , wherein the host cell is a white blood cell. 
     
     
         8 . The isolated host cell of  claim 7 , wherein the host cell is a macrophage. 
     
     
         9 . The isolated host cell of  claim 7 , wherein the host cell is a T cell. 
     
     
         10 . The isolated host cell of  claim 9 , wherein the T cell is CD8+ or CD4+. 
     
     
         11 . The isolated host cell of  claim 9 , wherein the host cell is a Chimeric Antigen Receptor (CAR) T cell. 
     
     
         12 . The isolated host cell of any one of  claims 9  to  11 , wherein D1L3 C-terminal basic domain processing is induced when the T cell is activated. 
     
     
         13 . The isolated host cell of any one of  claims 9  to  12 , wherein the T cell recognizes a cancer-associated antigen. 
     
     
         14 . The isolated host cell of  claim 13 , wherein the T cell recognizes a leukemia antigen. 
     
     
         15 . The isolated host cell of any one of  claims 9  to  12 , wherein the T cell recognizes a viral antigen. 
     
     
         16 . The isolated host cell of  claim 15 , wherein the T cell recognizes a coronavirus antigen. 
     
     
         17 . The isolated host cell of  claim 16 , wherein the coronavirus is SARS-CoV-2. 
     
     
         18 . The isolated host cell of any one of  claims 1  to  17 , wherein the heterologous polynucleotide is an mRNA or a modified mRNA (mmRNA). 
     
     
         19 . The isolated host cell of any one of  claims 1  to  17 , wherein the heterologous polynucleotide is DNA. 
     
     
         20 . The isolated host cell of any one of  claims 1  to  19 , wherein the D1L3 enzyme comprises an amino acid sequence that has at least 70% sequence identity to the enzyme of SEQ ID NO:4 or SEQ ID NO:5 lacking the C-terminal Basic Domain (BD). 
     
     
         21 . The isolated host cell of  claim 10 , wherein the amino acid sequence has at least 80% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD. 
     
     
         22 . The isolated host cell of  claim 21 , wherein the amino acid sequence has at least 85% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD. 
     
     
         23 . The isolated host cell of  claim 21 , wherein the amino acid sequence has at least 90% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD. 
     
     
         24 . The isolated host cell of  claim 21 , wherein the amino acid sequence has at least 95% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD. 
     
     
         25 . The isolated host cell of  claim 21 , wherein the amino acid sequence is the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5. 
     
     
         26 . The isolated host cell of any one of  claims 1  to  25 , wherein the polynucleotide encodes a D1L3 enzyme having a deletion of one or more amino acids of the C-terminal basic domain, and optionally at least five amino acids of the C-terminal basic domain. 
     
     
         27 . The isolated host cell of any one of  claims 1  to  26 , wherein the polynucleotide encodes a D1L3 enzyme having an inactivation of one or more of the NLS1 and the NLS2. 
     
     
         28 . The isolated host cell of  claim 27 , wherein the polynucleotide encodes a D1L3 enzyme having an inactivation of the NLS1. 
     
     
         29 . The isolated host cell of  claim 27 , wherein the polynucleotide encodes a D1L3 enzyme having an inactivation of NLS2. 
     
     
         30 . The isolated host cell of  claim 27 , wherein the polynucleotide encodes a D1L3 having an inactivation of both NLS1 and NLS2. 
     
     
         31 . The isolated host cell of any one of  claims 26  to  30 , wherein the D1L3 enzyme includes one or more amino acid substitutions and/or deletions within the NLS1 and/or NLS2. 
     
     
         32 . The isolated host cell of any one of  claims 1  to  31 , wherein the D1L3 enzyme is fused to a carrier protein. 
     
     
         33 . The isolated host cell of  claim 32 , wherein the carrier protein is albumin, which is optionally linked at the N-terminus of the D1L3 protein through a flexible or cleavable linker. 
     
     
         34 . A method for preparing the host cell of any one of  claims 1  to  33 , comprising introducing the polynucleotide encoding the chromatinase to a host cell in vitro. 
     
     
         35 . The method of  claim 34 , wherein the polynucleotide is introduced to the cell using a viral vector. 
     
     
         36 . The method of  claim 34 , wherein the polynucleotide is introduced to the cell using a gene-editing system selected from CRISPR-Cas9, zinc finger nuclease, or TALENS. 
     
     
         37 . A method for treating a subject in need of extracellular chromatin degradation, the method comprising administering the isolated host cell of any one of  claims 1  to  33 , or the host cell prepared by the method of any one of  claims 34  to  36 , to the subject. 
     
     
         38 . The method of  claim 37 , wherein the subject is in need of extracellular trap (ET) degradation, neutrophil extracellular trap (NET) degradation. 
     
     
         39 . The method of  claim 37  or  38 , wherein the subject is at risk of vascular occlusion involving extracellular chromatin. 
     
     
         40 . The method of any one of  claims 37  to  39 , wherein the extracellular chromatin includes chromatin released by cancer cells or injured endothelial cells. 
     
     
         41 . The method of any one of  claims 37  to  40 , wherein the subject has a loss of function mutation in one or both D1L3 genes. 
     
     
         42 . The method of any one of  claims 37  to  41 , wherein the subject has a condition selected from chronic neutrophilia, neutrophil aggregation or leukostasis, thrombosis or vascular occlusion, ischemia-reperfusion injury, surgical or traumatic tissue injury, an acute or chronic inflammatory reaction or disease, an autoimmune disease, inflammatory disease of the respiratory tract, renal inflammatory disease, inflammatory disease related to transplanted tissue, and cancer. 
     
     
         43 . The method of  claim 42 , wherein the subject has cancer. 
     
     
         44 . The method of  claim 43 , wherein the cancer is leukemia. 
     
     
         45 . The method of  claim 43 , wherein the cancer is a solid tumor. 
     
     
         46 . The method of  claim 45 , wherein the tumor is metastatic. 
     
     
         47 . The method of any one of  claims 43  to  46 , wherein the subject has or is at risk of tumor lysis syndrome. 
     
     
         48 . The method of  claim 47 , wherein the subject has an inflammatory disease of the respiratory tract. 
     
     
         49 . The method of  claim 48 , wherein the subject has an infection of the lower respiratory tract. 
     
     
         50 . The method of  claim 48  or  49 , wherein the subject has a viral infection. 
     
     
         51 . The method of any one of  claims 48  to  50 , wherein the subject has Acute Respiratory Distress Syndrome (ARDS) or Acute Lung Injury (ALI). 
     
     
         52 . The method of any one of  claims 48  to  51 , wherein the subject has pneumonia. 
     
     
         53 . The method of  claim 52 , wherein the subject has a coronavirus infection. 
     
     
         54 . The method of  claim 53 , wherein the coronavirus is SARS-CoV-2 or COVID-19. 
     
     
         55 . A method for treating a subject in need of extracellular chromatin degradation, the method comprising administering a composition comprising isolated T cells to the subject, the isolated T cells being modified in vitro to express a heterologous polynucleotide encoding a D1L3 enzyme operably linked to a promoter, so as to express and secrete the D1L3 enzyme. 
     
     
         56 . The method of  claim 55 , wherein the secreted D1L3 enzyme has at least a partial deletion of the C-terminal basic domain. 
     
     
         57 . The method of  claim 56 , wherein the secreted D1L3 enzyme has a deletion of at least 12 amino acids of the C-terminal basic domain. 
     
     
         58 . The method of  claim 57 , wherein the secreted D1L3 enzyme includes enzymes having deletions of one or more of: K291_S305 del, K292_S305 del, K293_S305 del, with respect to SEQ ID NO:4. 
     
     
         59 . The method of  claim 55 , wherein the heterologous polynucleotide is DNA. 
     
     
         60 . The method of  claim 55 , wherein the heterologous polynucleotide is an mRNA or a modified mRNA (mmRNA). 
     
     
         61 . The method of  claim 55 , wherein the polynucleotide is introduced to the cell using a viral vector. 
     
     
         62 . The method of  claim 55 , wherein the polynucleotide is introduced to the cell using a gene-editing system selected from CRISPR-Cas9, zinc finger nuclease, or TALENS. 
     
     
         63 . The method of  claim 55 , wherein the D1L3 enzyme comprises an amino acid sequence that has at least 90% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the C-terminal basic domain. 
     
     
         64 . The method of  claim 55 , wherein the polynucleotide encodes a D1L3 enzyme having a deletion of one or more amino acids of the C-terminal basic domain. 
     
     
         65 . The method of  claim 64 , wherein the polynucleotide encodes a D1L3 enzyme having a deletion of at least five amino acids of the C-terminal basic domain. 
     
     
         66 . The method of  claim 55 , wherein the polynucleotide encodes a D1L3 enzyme having an inactivation of one or more of the NLS1 and the NLS2. 
     
     
         67 . The method of  claim 55 , wherein the D1L3 enzyme includes one or more amino acid substitutions and/or deletions within the NLS1 and/or NLS2. 
     
     
         68 . The method of  claim 55 , wherein the D1L3 enzyme is fused to a carrier protein. 
     
     
         69 . The method of  claim 68 , wherein the carrier protein is albumin. 
     
     
         70 . The method of  claim 68 , wherein the D1L3 is fused to the carrier protein at the N-terminus of the D1L3 protein through a flexible or cleavable linker. 
     
     
         71 . The method of  claim 55 , wherein the subject is in need of extracellular trap (ET) degradation, neutrophil extracellular trap (NET) degradation. 
     
     
         72 . The method of  claim 55 , wherein the subject is at risk of occlusions, including vascular occlusions, involving extracellular chromatin. 
     
     
         73 . The method of  claim 55 , wherein the extracellular chromatin includes chromatin released by cancer cells or injured endothelial cells. 
     
     
         74 . The method of  claim 55 , wherein the subject has a loss of function mutation in one or both D1L3 genes. 
     
     
         75 . The method of  claim 55 , wherein the subject has a condition selected from chronic neutrophilia, neutrophil aggregation or leukostasis, thrombosis or vascular occlusion, ischemia-reperfusion injury, surgical or traumatic tissue injury, an acute or chronic inflammatory reaction or disease, an autoimmune disease, inflammatory disease of the respiratory tract, renal inflammatory disease, inflammatory disease related to transplanted tissue, and cancer. 
     
     
         76 . The method of  claim 75 , wherein the subject has cancer. 
     
     
         77 . The method of  claim 76 , wherein the cancer is leukemia. 
     
     
         78 . The method of  claim 76 , wherein the cancer is a solid tumor. 
     
     
         79 . The method of  claim 78 , wherein the tumor is metastatic. 
     
     
         80 . The method of  claim 75 , wherein the subject has an inflammatory disease of the respiratory tract. 
     
     
         81 . The method of  claim 80 , wherein the subject has an infection of the lower respiratory tract. 
     
     
         82 . The method of  claim 81 , wherein the subject has a viral infection. 
     
     
         83 . The method of  claim 75 , wherein the subject has Acute Respiratory Distress Syndrome (ARDS) or Acute Lung Injury (ALI). 
     
     
         84 . The method of  claim 75 , wherein the subject has pneumonia.