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

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, and provides methods comprising administering to a subject a nucleic acid encoding the recombinant engineered DUB to treat or ameliorate an inherited ion channelopathy. The disclosure further provides a method of screening mutations causing an inherited ion channelopathy 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 based on ubiquitination status, and identifying treatable mutations when surface density and/or total expression is recovered. The present disclosure also provides methods of treating or ameliorating the effects of acute/chronic viral infections in a subject using a nucleic acid encoding the recombinant engineered DUB.

Integral surface membrane proteins including ion channels, transporters, and receptors are vital to the survival and function of all cells, and impaired surface trafficking of membrane proteins underlies diverse diseases ranging from cystic fibrosis to cardiac arrhythmias. A vast number of inherited channel mutations result in channels with defects in trafficking to the surface membrane, and understanding the underlying cause of loss-of-function is critical for employing a personalized strategy to treat each disease. The disclosure relates to a platform for high-throughput screening of disease-causing mutations to diagnose underlying pathological mechanisms and to inform on the best treatment options for particular mutations, thereby advancing personalized medicine for these rare diseases.

Ubiquitination is a mechanism that regulates membrane protein trafficking, stability, and function, and deubiquitinases (DUBs) provide salience to ubiquitin signaling through the revision and removal of ubiquitin chains. The disclosure describes engineered DUBs, including linkage-selective and non-selective catalytic domains fused to protein binders such as nanobodies, to sculpt specific ubiquitin chain types on target proteins. The disclosure further takes advantage of conserved mechanisms by which pathogens exploit host ubiquitin machinery to provide a generalizable, modular, and transferrable approach to selectively target diverse infectious processes and combat viral reliance on ubiquitination necessary for sustaining infection.

Claims Coverage

The independent claim recites six main inventive features related to a method of treatment using a nucleic acid encoding a recombinant engineered deubiquitinase for specified diseases in a human subject.

The independent claim covers a method of treatment in a human subject by administering a nucleic acid encoding a recombinant engineered DUB characterized by (1) a catalytic unit comprising a deubiquitinase catalytic domain, (2) a protein binder that specifically binds a target substrate protein, (3) a variable linker between catalytic unit and binder, and (4) application to the recited diseases including cystic fibrosis, long QT syndrome, Brugada syndrome, Bartter Syndrome, and epilepsy.

Stated Advantages

Documented Applications