Engineered dnase enzymes and use in therapy

Abstract

The present invention provides engineered DNase proteins (including DNase1-like 3 and DNase1) that are useful for treating conditions characterized by neutrophil extracellular trap (NET) accumulation and/or release. In some aspects, the invention provides compositions and methods for preventing or treating vascular occlusion involving NETs. As demonstrated herein, NETs participate in a non-canonical mechanism for vascular occlusion, which is not dependent on fibrin or platelets.

1 . A DNase 1-like 3 (D1L3) variant comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 2, with one or more modifications as compared to SEQ ID NO:2 selected from: a mutated DNA binding site or mutated chromatin binding site, a glycosylation site, inactivation of a nuclear localization signal, and deletion of all or part of a C-terminal tail. 
     
     
         2 - 5 . (canceled) 
     
     
         6 . The D1L3 variant of  claim 1 , wherein the variant comprises an inactivated nuclear localization signal (NLS). 
     
     
         7 . The D1L3 variant of  claim 6 , wherein an NLS is inactivated by deletion of all or part of NLS1 and/or NLS2 of SEQ ID NO:2. 
     
     
         8 . The D1L3 variant of  claim 6 , wherein an NLS is inactivated by substitution and/or deletion of amino acids within NLS1 and/or NLS2. 
     
     
         9 - 23 . (canceled) 
     
     
         24 . A pharmaceutical composition comprising the D1L3 variant of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         25 - 32 . (canceled) 
     
     
         33 . A DNase1 (D1) variant comprising an amino acid sequence that is at least 80% identical to the enzyme of SEQ ID NO:1, and having one or more modifications with respect to SEQ ID NO:1 that are selected from: one or more mutated DNA binding sites or mutated chromatin binding sites, a mutated actin binding site, a mutated glycosylation site, addition of a nuclear localization signal, addition of a C-terminal tail having at least 50% sequence identity to the C-terminal tail of wild-type D1L3 protein of SEQ ID NO:2; and/or an N-terminal or C-terminal fusion to a half-life extending moiety. 
     
     
         34 - 40 . (canceled) 
     
     
         41 . A DNase enzyme comprising an amino acid sequence that is at least 80% identical to the enzyme of SEQ ID NO:1, and comprising the substitution, insertion, or addition of one or more arginine and/or lysine residues, wherein the DNase enzyme has increased chromatin-degrading activity as compared to the enzyme of SEQ ID NO:1. 
     
     
         42 - 77 . (canceled) 
     
     
         78 . A pharmaceutical composition comprising the enzyme of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         79 - 85 . (canceled) 
     
     
         86 . A method for treating a subject in need of extracellular DNA degradation, extracellular chromatin degradation, extracellular trap (ET) degradation and/or neutrophil extracellular trap (NET) degradation, the method comprising administering a therapeutically effective amount of the D1L3 variant according to  claim 1 . 
     
     
         87 . The method of  claim 86 , wherein upon treatment the subject exhibits extracellular DNA degradation, extracellular chromatin degradation, extracellular trap (ET) degradation and/or neutrophil extracellular trap (NET) degradation. 
     
     
         88 . The method of  claim 86 , wherein the subject has a chronic or acute inflammatory disorder. 
     
     
         89 . (canceled) 
     
     
         90 . The method of  claim 86 , wherein subject has or is at risk of a vascular occlusion comprising NETs. 
     
     
         91 . The method of  claim 86 , wherein the subject has chronic neutrophilia (e.g., an increase in the number of neutrophils), neutrophil aggregation and leukostasis, thrombosis and vascular occlusion (e.g. sickle cell disease), ischemia-reperfusion injury (e.g. midgut volvulus, testicular torsion, limb ischemia reperfusion, vital organ ischemia-reperfusion, organ transplantation), surgical and traumatic tissue injury, an acute or chronic inflammatory reaction or disease, an autoimmune disease (e.g. systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, vasculitis, systemic sclerosis), cardiovascular disease (e.g., myocardial infarction, stroke, atherosclerosis, venous thromboembolism, including thrombolytic therapy), metabolic disease (e.g., diabetes), systemic inflammation (e.g., systemic inflammatory response syndrome (SIRS), sepsis, septic shock, disseminated intravascular coagulation (DIC), and thrombotic microangiopathy (TMA)), inflammatory diseases of the respiratory tract (e.g. cystic fibrosis, chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), smoke induced lung injury, transfusion induced lung injury (TRALI), acute respiratory distress syndrome (ARDS), and asthma, atelectasis, bronchitis, empyema), renal inflammatory diseases (acute and chronic kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), inflammatory diseases related to transplated tissue (e.g. graft-versus-host disease) and cancer (e.g. leukemia, tumor metastasis, and solid tumors). 
     
     
         92 . (canceled) 
     
     
         93 . The method of  claim 86 , wherein the subject has or is at risk of a ductal occlusion in a ductal system. 
     
     
         94 - 97 . (canceled) 
     
     
         98 . The method of  claim 86 , wherein the subject has or is at risk of NETs accumulating on endothelial surfaces (e.g., surgical adhesions), the skin (e.g., wounds/scarring, uclers), or in synovial joints (e.g., gout, arthritis). 
     
     
         99 - 102 . (canceled) 
     
     
         103 . The method of  claim 86 , wherein the subject has thrombosis, stroke, sepsis, lung injury, atherosclerosis, viral infection, sickle cell disease, myocardial infarction, ear infection, wound healing, liver injury, endocarditis, liver infection, pancreatitis, primary graft dysfunction, limb ischemia reperfusion, kidney injury, blood clotting, alum-induced inflammation, hepatorenal injury, pleural exudations, hemotorax, intrabiliary blood clots, post pneumatic anemia, ulcers, otolaryngological conditions, oral infections, minor injuries, sinusitis, post-operative rhinoplasties, infertility, bladder catheter, wound cleaning, skin reaction test, pneumococcal meningitis, gout, leg ulcers, cystic fibrosis, Kartegener's syndrome, asthma, lobar atelectasis, chronic bronchitis, bronchiectasis, lupus, primary cilliary dyskinesia, bronchiolitis, empyema, pleural infections, cancer, dry eyes disease, lower respiratory tract infections, chronic hematomas, Alzheimer's disease, and obstructive pulmonary disease. 
     
     
         104 . The method of  claim 86 , wherein the subject further exhibits a coronary artery disease. 
     
     
         105 . (canceled) 
     
     
         106 . The method of  claim 86 , comprising monitoring the NET-degrading activity of blood, plasma, or serum from the subject. 
     
     
         107 . A method for treating a subject exhibiting or at risk of intravascular occlusion involving NETs, the method comprising administering a therapeutically effective amount of a D1L3 or variant thereof, and/or a therapeutically effective amount of a D1 or a variant thereof. 
     
     
         108 - 109 . (canceled) 
     
     
         110 . A method for treating a subject that has or is at risk of a ductal occlusion in a ductal system, comprising administering a therapeutically effective amount of a D1L3 or variant thereof, and/or a therapeutically effective amount of D1 or a variant thereof. 
     
     
         111 - 155 . (canceled)