Compositions and methods for using engineered deubiquitinases for probing ubiquitin-dependent cellular processes
View Patent ↗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.
1 . A method of treating or ameliorating the effects of an inherited ion channelopathy in a human subject, the method comprising administering to the subject a recombinant engineered deubiquitinase (DUB) comprising:
a) a catalytic unit comprising the catalytic domain of a deubiquitinase;
b) a protein binder comprising an antibody, or antigen binding fragment thereof, that specifically binds a target substrate protein for deubiquitination by the engineered DUB; and
c) a variable linker between the catalytic unit and the protein binder.
2 . The method of claim 1 , wherein the inherited ion channelopathy comprises epilepsy, migraine, neuropathic pain, cardiac arrhythmias, long QT syndrome, Brugada syndrome, cystic fibrosis, hyperinsulinemic hypoglycemia, or Bartter syndrome.
3 . The method of claim 1 , wherein the inherited ion channelopathy comprises Brugada syndrome.
4 . The method of claim 1 , wherein the inherited ion channelopathy comprises long QT syndrome.
5 . The method of claim 1 , wherein the inherited ion channelopathy comprises cystic fibrosis.
6 . The method of claim 1 , wherein the inherited ion channelopathy comprises cardiac arrhythmias.
7 . The method of claim 1 , wherein the inherited ion channelopathy comprises epilepsy.
8 . The method of claim 1 , wherein the antibody is a nanobody, scFv, (scFv)2, Fab, Fab′, F(ab′)2, Fv, diabody, or a DARPin.
9 . The method of claim 1 , wherein the antibody is a scFv, (scFv)2, Fab, Fab′, F(ab′)2, Fv, antibody mimetic, or diabody.
10 . The method of claim 1 , wherein the antibody is a single domain antibody (dAb).
11 . The method of claim 1 , wherein the antibody is a nanobody.
12 . The method of claim 3 , wherein the antibody is a single domain antibody (dAb).
13 . The method of claim 3 , wherein the antibody is a nanobody.
14 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne and the antibody is a single domain antibody (dAb).
15 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne and the antibody is a nanobody.
16 . The method of claim 3 , wherein the catalytic unit comprises the catalytic domain of Cezanne and the antibody is a single domain antibody (dAb).
17 . The method of claim 3 , wherein the catalytic unit comprises the catalytic domain of Cezanne and the antibody is a nanobody.
18 . The method of claim 1 , wherein the catalytic unit is selective for a particular ubiquitin linkage type.
19 . The method of claim 1 , wherein the catalytic unit is not selective for a particular ubiquitin linkage type.
20 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase, wherein the deubiquitinase is from the ubiquitin specific proteases (USP) family, the ovarian tumor proteases (OTU) family, the ubiquitin C-terminal hydrolases (UCH) family, the Josephin domain (Josephin) family, the motif interacting with ubiquitin-containing novel DUB (MINDY) family, or the JAB1/MPN/Mov34 metalloenzyme domain (JAMM) family.
21 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase from the UCH family.
22 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase from the USP family.
23 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of USP21.
24 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase from the OTU family.
25 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne, OTUD1, OTUD4, TRABID, or OTULIN.
26 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne.
27 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of OTUD1.
28 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of OTUD4.
29 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of TRABID.
30 . The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of OTULIN.