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-12351845-B2
Publication Date
2025-07-08
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, inter alia, 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 present disclosure further provides a method of treating or ameliorating the effects of an inherited ion channelopathy in a subject, comprising administering to the subject a nucleic acid encoding the recombinant engineered DUB aforementioned, and a method of treating or ameliorating the effects of acute/chronic viral infections in a subject, comprising administering to the subject a nucleic acid encoding the recombinant engineered DUB aforementioned.
The background explains that impaired surface trafficking of membrane proteins underlies diverse diseases ranging from cystic fibrosis to cardiac arrhythmias and that ubiquitination regulates membrane protein trafficking, stability and function. The disclosure addresses a need for a platform for high-throughput screening of disease-causing mutations to diagnose underlying pathological mechanisms and provides a novel therapeutic opportunity for gene therapy and targeted correction of ubiquitin-dependent trafficking defects.
The detailed description discloses embodiments in which the catalytic unit can be selective for all ubiquitin linkage types or selective for particular ubiquitin linkage types, and states that the catalytic unit can comprise catalytic domains from DUB families including USP, OTU, UCH, Josephin, MINDY, and JAMM. The disclosure further specifies that the protein binder can be selected from intracellular antibody fragments, scFvs, nanobodies, antibody mimetics, monobodies, DARPins, lipocalins, and targeting sequences, and gives embodiments such as nanoUSP21, nanoOTUD1, nanoOTUD4, nanoCezanne, nanoTRABID, and nanoOTULIN with stated linkage selectivities.
Claims Coverage
The independent claim presents a method with five main inventive features related to a recombinant engineered DUB and its therapeutic administration.
Recombinant engineered deubiquitinase
A recombinant engineered deubiquitinase (DUB) comprising a catalytic unit, a protein binder that specifically binds a target substrate protein for deubiquitination, and a variable linker between the catalytic unit and the protein binder.
Catalytic unit comprising catalytic domain
A catalytic unit comprising the catalytic domain of a deubiquitinase.
Protein binder that specifically binds target substrate
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 catalytic unit and protein binder
A variable linker positioned between the catalytic unit and the protein binder.
Administering nucleic acid encoding engineered DUB
A method of treating or ameliorating the effects of an inherited ion channelopathy in a human subject comprising administering to the subject a nucleic acid molecule encoding the recombinant engineered DUB as defined above.
The independent claim covers a therapeutic method that administers a nucleic acid encoding a modular engineered DUB composed of a catalytic domain, a substrate-specific protein binder (antibody or antigen binding fragment), and a variable linker, with the DUB module itself and the administration method as the inventive elements.
Stated Advantages
Provides a novel therapeutic opportunity for gene therapy and targeted correction of ubiquitin-dependent trafficking defects.
Enables a platform for high-throughput screening of disease-causing mutations to diagnose underlying pathological mechanisms and inform personalized treatment options.
Offers a generalizable approach to selectively target diverse infectious processes by combating viral reliance on ubiquitination and providing a modular and transferrable approach for battling emerging or chronic infections.
Documented Applications
Treating or ameliorating inherited ion channelopathies in a subject by administering a nucleic acid encoding the recombinant engineered DUB, including but not limited to: epilepsy, migraine, neuropathic pain, cardiac arrhythmias, long QT syndrome, Brugada syndrome, cystic fibrosis, hyperinsulinemic hypoglycemia, and Bartter syndrome.
Screening mutations causing inherited ion channelopathies for a trafficking-deficient mutation that is treatable by the recombinant engineered DUB, by measuring surface density and/or total expression, determining ubiquitination status, selecting and co-expressing a recombinant engineered DUB, and identifying treatable mutations when expression is recovered.
Treating or ameliorating acute or chronic viral infections in a subject by administering a nucleic acid encoding the recombinant engineered DUB to target ubiquitin-dependent processes exploited by pathogens.
Providing a recombinant expression vector comprising a nucleic acid that encodes the recombinant engineered DUB, and cells transformed with such vector.
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