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 nucleic acid molecule encoding 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 nucleic acid molecule is an RNA molecule.
7. The method of claim 1 , wherein the nucleic acid molecule is an mRNA molecule.
8. The method of claim 1 , wherein the nucleic acid molecule is a DNA molecule.
9. The method of claim 1 , wherein the nucleic acid molecule is a cDNA molecule.
10. The method of claim 1 , wherein the nucleic acid molecule is contained within a vector.
11. The method of claim 6 , wherein the vector is a viral vector.
12. The method of claim 7 , wherein the viral vector is an adenoviral vector.
13. The method of claim 6 , wherein the vector is a plasmid vector.
14. The method of claim 1 , wherein the nucleic acid molecule is a DNA molecule contained within a viral vector.
15. The method of claim 1 , wherein the antibody is a nanobody, scFv, (scFv)2, Fab, Fab′, F(ab)2, Fv, diabody, or a DARPin.
16. The method of claim 1 , wherein the antibody is a scFv, (scFv)2, Fab, Fab′, F(ab)2, Fv, antibody mimetic, or diabody.
17. The method of claim 1 , wherein the antibody is a single domain antibody (dAb).
18. The method of claim 1 , wherein the antibody is a nanobody.
19. The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne and the antibody is a single domain antibody (dAb).
20. The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne and the antibody is a nanobody.
21. The method of claim 1 , wherein the protein binder specifically binds an ion channel.
22. The method of claim 21 , wherein the ion channel is KCNQ1, HERG, or CFTR.
23. The method of claim 1 , wherein the catalytic unit is selective for a particular ubiquitin linkage type.
24. 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.
25. The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase from the USP family.
26. The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase from the OTU family.
27. The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne, OTUD1, OTUD4, TRABID, or OTULIN.
28. The method of claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne.
29. The method of claim 1 , wherein the catalytic unit comprises
(a) the catalytic domain of USP21 and is capable of non-selectively eliminating all ubiquitin linkage types;
(b) the catalytic domain of OTUD1 and is capable of selectively eliminating K63 ubiquitin linkages;
(c) the catalytic domain of OTUD4 and is capable of selectively eliminating K48 ubiquitin linkages;
(d) the catalytic domain of Cezanne and is capable of selectively eliminating K11 ubiquitin linkages;
(e) the catalytic domain of TRABID and is capable of selectively eliminating K29 ubiquitin linkages, K33 ubiquitin linkages, or K29 and K33 ubiquitin linkages; or
(f) the catalytic domain of OTULIN and is capable of selectively eliminating Met1 ubiquitin linkages.
30. The method of claim 1 , wherein the catalytic unit is non-selective for a particular ubiquitin linkage type.