Compositions and methods for using engineered deubiquitinases for probing ubiquitin-dependent cellular processes
Inventors
Colecraft, Henry M. • Kanner, Scott
Assignees
Columbia University in the City of New York
Publication Number
US-12312618-B2
Publication Date
2025-05-27
Expiration Date
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Abstract
The present disclosure provides, inter alia, a recombinant engineered deubiquitinase (DUB) and methods for treating or ameliorating an inherited ion channelopathy, such as long QT syndrome, Brugada syndrome, or cystic fibrosis, in a subject. Further provided are methods for screening mutations causing such inherited ion channelopathies for a trafficking-deficient mutation that is treatable by the recombinant engineered DUB disclosed herein.
Core Innovation
The present disclosure provides a recombinant engineered deubiquitinase (DUB) comprising: a catalytic unit; a protein binder; and a variable linker between the catalytic unit and the protein binder. The disclosure also provides a nucleic acid encoding the recombinant engineered DUB, methods of treating or ameliorating the effects of an inherited ion channelopathy in a subject by administering to the subject a nucleic acid encoding the recombinant engineered DUB, and a recombinant expression vector comprising a nucleic acid that encodes the recombinant engineered DUB and a cell transformed with said vector.
The background identifies that impaired surface trafficking of membrane proteins underlies diverse diseases including cystic fibrosis and cardiac arrhythmias and that ubiquitination regulates membrane protein trafficking, stability, and function. The disclosure addresses the need to better understand mechanisms controlling membrane protein surface density and provides a platform for high-throughput screening of disease-causing mutations to diagnose underlying pathological mechanisms and inform treatment options.
The disclosure further provides methods for screening mutations causing an inherited ion channelopathy for a trafficking-deficient mutation that is treatable by the recombinant engineered DUB, comprising measuring surface density and/or total expression, determining ubiquitination status, selecting and co-expressing a recombinant engineered DUB based on ubiquitination status, and identifying mutations as treatable if surface density and/or total expression is recovered. The disclosure also provides a method of treating or ameliorating the effects of acute/chronic viral infections in a subject by administering to the subject a nucleic acid encoding the recombinant engineered DUB.
Claims Coverage
The claims include one independent claim. Main inventive features include an RNA molecule encoding a recombinant engineered DUB, and three core structural elements of the engineered DUB.
RNA molecule encoding a recombinant engineered deubiquitinase
An RNA molecule encoding a recombinant engineered deubiquitinase (DUB).
Catalytic unit comprising the catalytic domain of a deubiquitinase
A catalytic unit comprising the catalytic domain of a deubiquitinase.
Protein binder comprising an antibody or antigen binding fragment that specifically binds a target substrate protein
A protein binder comprising an antibody, or antigen binding fragment thereof, that specifically binds a target substrate protein for deubiquitination by the engineered DUB.
Variable linker between the catalytic unit and the protein binder
A variable linker between the catalytic unit and the protein binder.
The independent claim covers an RNA molecule encoding an engineered DUB defined by a catalytic domain, a substrate-specific protein binder (antibody or antigen-binding fragment), and a variable linker connecting these elements.
Stated Advantages
Provides methods for treating or ameliorating inherited ion channelopathies by administering a nucleic acid encoding the engineered DUB.
Provides a platform for high-throughput screening of disease-causing channel mutations to diagnose trafficking-deficient mutations and inform personalized treatment options.
Offers a novel therapeutic opportunity for gene therapy and targeted correction of ubiquitin-dependent trafficking defects.
Provides a modular and transferable approach to selectively target ubiquitin-dependent processes exploited by diverse infectious pathogens and to treat acute/chronic viral infections.
Documented Applications
Treating or ameliorating inherited ion channelopathies, including long QT syndrome, Brugada syndrome, and cystic fibrosis, by administering a nucleic acid encoding the recombinant engineered DUB.
Screening mutations causing inherited ion channelopathies for trafficking-deficient mutations treatable by the recombinant engineered DUB, using measurements of surface density/total expression and determination of ubiquitination status followed by co-expression of selected engineered DUBs.
Treating or ameliorating acute or chronic viral infections by administering a nucleic acid encoding the recombinant engineered DUB to a subject.
Provision of a recombinant expression vector comprising a nucleic acid that encodes the recombinant engineered DUB and cells transformed with said vector.
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