The present invention relates to methods for the manufacture, purification, formulation, and use of biologically active recombinant elastase proteins. Described are recombinant methods for producing therapeutically useful elastase proteins, as are pharmaceutical compositions comprising said elastase proteins. Novel recombinant elastase proteins and protein preparations are also disclosed. Methods are described for treating and preventing diseases of biological conduits using pharmaceutical compositions containing the elastase proteins of the invention.

The present invention relates to methods for the manufacture, purification, formulation, and use of biologically active recombinant elastase proteins. Described are recombinant methods for producing therapeutically useful elastase proteins, as are pharmaceutical compositions comprising said elastase proteins. Novel recombinant elastase proteins and protein preparations are also disclosed. Methods are described for treating and preventing diseases of biological conduits using pharmaceutical compositions containing the elastase proteins of the invention.

Provided herein are methods of determining a subject's risk of developing a cardiac arrhythmia, methods of treating a subject having or at risk of developing a cardiac arrhythmia, methods of selecting a treatment for a subject having or at risk of developing a cardiac arrhythmia, methods of selecting a subject for administration of a treatment for reducing risk of developing a cardiac arrhythmia, methods of determining the efficacy of a treatment in a subject having or at risk of developing a cardiac arrhythmia, and methods of monitoring a subject having or at increased risk of developing a cardiac arrhythmia based on a level of one or more of glucocorticoid (GC), corticosteroid-binding globulin (CBG), and neutrophil elastase (NE). Also provided are kits that include two or more of an antibody that binds specifically to GC, an antibody that binds specifically to GBC, and an antibody that binds specifically to NE.

Peptide compounds derived from cathepsin G (CG), neutrophil elastase (NE), and cationic antimicrobial protein of 37 kDa (CAP37), and methods of their use in inhibiting and/or reversing polymerization of amyloid peptide into oligomers and/or fibrils, and/or disaggregating amyloid plaques, and methods of use of the peptide compounds in treatments of, for example, Alzheimer's disease, cerebral amyloid angiopathy, dementia, and/or neuroinflammation, and/or symptoms thereof are disclosed.

The disclosed methods are directed to preparing polypeptides for multi-attribute analysis. The polypeptides are optionally denatured, reduced, and/or alkylated before being subjected to a first digestion. Following the first digestion the large and small fragments resulting from the digestion are separated from each other. A second digestion is then performed on the larger of the fragments. All of the fragments from the two digestions are then analyzed chromatographically, electrophoretically, or spectrometrically, or a combination of these methods. The methods are especially useful for the preparation of therapeutic polypeptides for analysis, especially those that are not easily cleaved.

Methods for inactivating in a cell a mutant allele of the elastase, neutrophil expressed gene (ELANE gene) gene having a mutation associated with severe congenital neutropenia (SCN) or cyclic neutropenia (CyN) and which cell is heterozygous at one or more polymorphic sites selected from the group consisting of: rs10424470, rs4807932, rs10414837, rs376107533, rs3761010, rs10413889, rs3761007, rs10409474, rs3761005, rs351107, rs3761001, rs740021, rs781452480, rs371057361, rs570466264, rs1041904080, rs7250194, rs17216649, rs199720952, rs6510983, rs17223066, rs7255385, rs112639467, rs141213775, rs28591229, rs10469327, rs3834645, rs1683564, rs71335276, and rs8107095, the method comprising introducing to the cell a composition comprising: a CRISPR nuclease or a sequence encoding the CRISPR nuclease; and a first RNA molecule comprising a guide sequence portion having 17-30 nucleotides, wherein a complex of the CRISPR nuclease and the first RNA molecule affects a double strand break in the mutant allele of the ELANE gene
the method optionally further comprising introduction of a second RNA molecule comprising a guide sequence portion capable of complexing with a CRISPR nuclease, wherein the complex of the second RNA molecule and CRISPR nuclease affects a second double strand break in the ELANE gene.

Described herein are methods and compositions relating to methods of inhibiting neutrophils, e.g., inhibiting NET release or NETosis, by means of a DEspR inhibitor, e.g., an anti-DEspR antibody reagent. In some embodiments, the methods can relate to the treatment of a disease, e.g., cancer or a disease wherein neutrophils; NETs; or NETosing or NETting neutrophils contribute to pathogenesis, chronicity, or worsening of disease. In some embodiments, the DEspR inhibitor can be a bi-specific reagent or an antibody-drug conjugate.

Embodiments are directed to: (i) neutrophil secreted factors that have the capacity to kill a broad range of cancer cells without affecting the viability of non-cancer cells. Two neutrophil killing factors have been identified by the inventors: (1) eosinophil cationic protein (ECP) and (2) neutrophil elastase (ELANE); or (ii) therapeutic compositions that include CD95 degrading polypeptide components and methods of treating cancer with the same.

Described herein are methods and compositions relating to methods of inhibiting neutrophils, e.g., inhibiting NET release or NETosis, by means of a DEspR inhibitor, e.g., an anti-DEspR antibody reagent. In some embodiments, the methods can relate to the treatment of a disease, e.g., cancer or a disease wherein neutrophils; NETs; or NETosing or NETting neutrophils contribute to pathogenesis, chronicity, or worsening of disease. In some embodiments, the DEspR inhibitor can be a bi-specific reagent or an antibody-drug conjugate.

The disclosed methods are directed to preparing polypeptides for multi-attribute analysis. The polypeptides are optionally denatured, reduced, and/or alkylated before being subjected to a first digestion. Following the first digestion the large and small fragments resulting from the digestion are separated from each other. A second digestion is then performed on the larger of the fragments. All of the fragments from the two digestions are then analyzed chromatographically, electrophoretically, or spectrometrically, or a combination of these methods. The methods are especially useful for the preparation of therapeutic polypeptides for analysis, especially those that are not easily cleaved.