IP Library › Granted Patent US 12,655,182
Granted Patent B2
US 12,655,182 · App. 17/627,035 · Granted Jun 16, 2026

Evolved botulinum neurotoxins and uses thereof

Inventors: David R. Liu (Cambridge, MA); Travis R. Blum (Cambridge, MA)
Assignees: President and Fellows of Harvard College; The Broad Institute, Inc.
C07K14/33C12N9/52A61K38/00C12Y304/24069
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Quick Facts
Patent No.
US 12,655,182
App. No.
17/627,035
Granted
Jun 16, 2026
Kind
B2
Abstract

The disclosure provides amino acid sequence variants of Botulinum neurotoxin F (BoNT F) proteases that cleave SNARE proteins (e.g., VAMP1, VAMP7, etc.) and methods of evolving the same. In some embodiments, proteases described by the disclosure are useful for cleaving proteins found in a cell, that is in an intracellular environment. In some embodiments, proteases described by the disclosure are useful for treating diseases associated with increased or aberrant VAMP7 expression or activity, for example, cancer, transplantation rejection, graft-versus-host disease, and neurological disorders. Some aspects of this disclosure provide methods for generating BoNT protease variants by continuous directed evolution.

Claims (20)

1 . A protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO.: 6, wherein the protein comprises at least three amino acid mutations selected from the group consisting of R303H, T335S, S350G, I354V, S166Y, S167I, M174T, D175G, E200K, K31N, V106A, Y113D, E200G, R240L, V131G, S141T, N99S, R240C, F360L, G177D, N184H, R240F, Y113S, V131F, N184K, S69L, Y72H, K96N, N184T, S148N, I150V, A158P, L381M, N396H, P410L, Y372H, I412N, D414G, D418Y, Y372N, E423K, Y244C, V193M, Y372P, N165S, S224I, A281V, E66D, R41H, A232S, and V106A.

2 . The protein of claim 1 , wherein the protein comprises at least four amino acid mutations selected from the group consisting of R303H, T335S, S350G, 1354V, S166Y, S167I, M174T, D175G, E200K, K31N, V106A, Y113D, E200G, R240L, V131G, S141T, N99S, R240C, F360L, G177D, N184H, R240F, Y113S, V131F, N184K, S69L, Y72H, K96N, N184T, S148N, I150V, A158P, L381M, N396H, P410L, Y372H, I412N, D414G, D418Y, Y372N, E423K, Y244C, V193M, Y372P, N165S, S224I, A281V, E66D, R41H, A232S, and V106A.

3 . The protein of claim 1 , wherein the protein comprises the amino acid sequence as set forth in any one of SEQ ID NOs.: 13-31.

4 . The protein of claim 1 , wherein the protein further comprises a neurotoxin HCC domain.

5 . The protein of claim 4 , wherein the HCC domain comprises SEQ ID NO.: 11.

6 . The protein of claim 1 , wherein the protein comprises at least three amino acid mutations selected from the group consisting of S69L, Y72H, V106A, S148N, I150V, A158P, S166Y, S167I, G177D, N184H, E200G, S224I, A232S, R240L, S350G, F360L, Y372N, L381M, N396H, and P410L.

7 . A pharmaceutical composition comprising the protein of claim 1 and a pharmaceutically acceptable excipient.

8 . The protein of claim 1 , wherein the protein further comprises a neurotoxin translocation domain (HCN).

9 . The protein of claim 8 , wherein the HCN domain comprises SEQ ID NO.: 10.

10 . The protein of claim 1 , wherein the protein comprises the amino acid mutation S166Y.

11 . The protein of claim 1 , wherein the protein comprises at least three amino acid mutations selected from the group consisting of R41H, K96N, S166Y, R240L, R240C, Y372H, and D414G.

12 . The protein of claim 1 , wherein the protein comprises at least three amino acid mutations selected from the group consisting of S166Y, N184H, N184K, R240L, S350G, F360L, Y372H, P410L, and D414G.

13 . The protein of claim 1 , wherein the protein comprises at least three amino acid mutations selected from the group consisting of S166Y, N184K, R240L, S350G, F360L, Y372H, N396H, P410L, and E423K.

14 . The protein of claim 1 , wherein the protein comprises at least three amino acid mutations selected from the group consisting of V106A, N165S, S166Y, S167I, N184K, R240L, S350G, F360L, Y372H, N396H, P410L, and E423K.

15 . The protein of claim 1 , wherein the protein comprises at least three amino acid mutations selected from the group consisting of V106A, Y113D, Y113S, S166Y, S167I, N184H, N184K, N184S, E200K, R240L, Y244C, S350G, F360L, Y372H, N396H, P410L, and E423K.

16 . The protein of claim 1 , wherein the protein comprises at least three amino acid mutations selected from the group consisting of E66D, V106A, S166Y, S167I, D175G, N184K, E200G, S224I, R240L, R240F, T335S, S350G, F360L, Y372H, N396H, P410L, and D418Y.

17 . The protein of claim 1 , wherein the protein comprises at least three amino acid mutations selected from the group consisting of Y72H, N99S, V106A, V131G, S141T, S166Y, S167I, M174T, N184T, V193M, E200G, S224I, R240L, R240F, S350G, F360L, Y372H, N396H, and P410L.

18 . The protein of claim 1 , wherein the protein comprises at least three amino acid mutations selected from the group consisting of Y72H, V106A, V131G, S141T, S166Y, S167I, M174T, E200G, S224I, R240L, S350G, F360L, Y372H, N396H, and P410L.

19 . A host cell comprising the protein of claim 1 .

20 . The host cell of claim 19 , wherein the host cell is a bacterial cell, yeast cell, or mammalian cell.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 2, 2022
From: BLUM, TRAVIS R.
To: THE BROAD INSTITUTE, INC.
Reel/Frame 061628/0366 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 2, 2022
From: LIU, DAVID R.
To: HOWARD HUGHES MEDICAL INSTITUTE
Reel/Frame 061628/0469 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 2, 2022
From: HOWARD HUGHES MEDICAL INSTITUTE
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 061628/0485 →
Continuity (2)
Provisional Application 62874443 · Jul 15, 2019
Related Publication 20220259269A1 · Aug 18, 2022
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