Engineered transcription activator-like effector (tale) domains and uses thereof

Patent metadata

Field	Value
Publication number	US-2016201040-A1
Application number	US-201414913458-A
Country	US
Kind code	A1
Filing date	Aug 22, 2014
Priority date	Aug 22, 2013
Publication date	Jul 14, 2016
Grant date	—

Abstract

Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides engineered TALE domains and TALEs comprising such engineered domains, e.g., TALE nucleases (TALENs), TALE transcriptional activators, TALE transcriptional repressors, and TALE epigenetic modification enzymes, with improved specificity and methods for generating and using such TALEs.

First claim

1 . An isolated Transcription Activator-Like Effector (TALE) domain, wherein
(i) the isolated TALE domain is an N-terminal TALE domain and the net charge of the isolated N-terminal domain is less than the net charge of the canonical N-terminal domain (SEQ ID NO: 1) at physiological pH; or (ii) wherein the isolated TALE domain is a C-terminal TALE domain and the net charge of the C-terminal domain is less than the net charge of the canonical C-terminal domain (SEQ ID NO: 22) at physiological pH.

2 . An isolated N-terminal or C-terminal TALE domain, wherein
(i) the isolated TALE domain is an N-terminal TALE domain and the binding energy of the N-terminal domain to a target nucleic acid molecule is smaller than the binding energy of the canonical N-terminal domain (SEQ ID NO: 1); or (ii) the isolated TALE domain is a C-terminal TALE domain and the binding energy of the C-terminal domain to a target nucleic acid molecule is smaller than the binding energy of the canonical C-terminal domain (SEQ ID NO: 22).

3 . The isolated TALE domain of claim 1 , wherein the net charge of the C-terminal domain is less than or equal to +6, less than or equal to +5, less than or equal to +4, less than or equal to +3, less than or equal to +2, less than or equal to +1, less than or equal to 0, less than or equal to −1, less than or equal to −2, less than or equal to −3, less than or equal to −4, or less than or equal to −5.

4 . The isolated TALE domain of claim 1 , wherein the C-terminal domain comprises an amino acid sequence that differs from the canonical C-terminal domain sequence in that at least one cationic amino acid residue of the canonical C-terminal domain sequence is replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH.

5 . The isolated TALE domain of claim 1 , wherein the N-terminal domain comprises an amino acid sequence that differs from the canonical N-terminal domain sequence in that at least one cationic amino acid residue of the canonical N-terminal domain sequence is replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH.

6 . The isolated TALE domain of claim 1 , wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 cationic amino acid(s) in the isolated TALE domain is/are replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH.

7 . The isolated TALE domain of claim 4 , wherein the at least one cationic amino acid residue is arginine (R) or lysine (K).

8 . The isolated TALE domain of claim 4 , wherein the amino acid residue that exhibits no charge or a negative charge at physiological pH is glutamine (Q) or glycine (G).

9 . (canceled)

10 . The isolated TALE domain of claim 1 , wherein the C-terminal domain comprises one or more of the following amino acid replacements:K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q .

11 . The isolated TALE domain of claim 1 , wherein the C-terminal domain comprises a Q3 variant sequence (K788Q, R792Q, K801Q).

12 . The isolated TALE domain of claim 1 , wherein the C-terminal domain comprises a Q7 variant sequence (K777Q, K778Q, K788Q, R789Q, R792Q, R793Q, R801Q).

13 . The isolated TALE domain of claim 1 , wherein the N-terminal domain is a truncated version of the canonical N-terminal domain.

14 . The isolated TALE domain of claim 1 , wherein the C-terminal domain is a truncated version of the canonical C-terminal domain.

15 . The isolated TALE domain of claim 14 , wherein the truncated domain comprises less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 25% of the residues of the canonical domain.

16 . The isolated TALE domain of claim 14 , wherein the truncated C-terminal domain comprises less than 60, less than 50, less than 40, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, or less than 20 amino acid residues.

17 . The isolated TALE domain of claim 14 , wherein the truncated C-terminal domain comprises 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 residues.

18 . The isolated TALE domain of claim 1 , wherein the isolated TALE domain is comprised in a TALE molecule comprising the structure
[N-terminal domain]-[TALE repeat array]-[C-terminal domain]-[effector domain]
or
[effector domain]-[N-terminal domain]-[TALE repeat array]-[C-terminal domain].

19 . The isolated TALE domain of claim 18 , wherein the effector domain comprises a nuclease domain, a transcriptional activator or repressor domain, a recombinase domain, or an epigenetic modification enzyme domain.

20 . The isolated TALE domain of claim 18 , wherein the TALE molecule binds a target sequence within a gene known to be associated with a disease or disorder.

21 . A Transcription Activator-Like Effector Nuclease (TALEN), comprising
(a) a nuclease cleavage domain; (b) a C-terminal domain conjugated to the nuclease cleavage domain; (c) a TALE repeat array conjugated to the C-terminal domain; and (d) an N-terminal domain conjugated to the TALE repeat array, wherein
(i) the net charge of the N-terminal domain is less than the net charge of the canonical N-terminal domain (SEQ ID NO: 1) at physiological pH; and/or
(ii) the net charge of the C-terminal domain is less than the net charge of the canonical C-terminal domain (SEQ ID NO: 22) at physiological pH.

22 . A Transcription Activator-Like Effector Nuclease (TALEN), comprising
(a) a nuclease cleavage domain; (b) a C-terminal domain conjugated to the nuclease cleavage domain; (c) a TALE repeat array conjugated to the C-terminal domain; and (d) an N-terminal domain conjugated to the TALE repeat array, wherein
(i) the binding energy of the N-terminal domain to a target nucleic acid molecule is smaller than the binding energy of the canonical N-terminal domain (SEQ ID NO: 1); and/or
(ii) binding energy of the C-terminal domain to a target nucleic acid molecule is smaller than the binding energy of the canonical C-terminal domain (SEQ ID NO: 22).

23 - 57 . (canceled)

58 . A method of preparing an engineered TALEN, the method comprising:
replacing at least one amino acid in the canonical N-terminal TALEN domain and/or the canonical C-terminal TALEN domain with an amino acid having a no charge or a negative charge at physiological pH; and/or truncating the N-terminal TALEN domain and/or the C-terminal TALEN domain to remove a positively charged fragment;
thus generating an engineered TALEN having an N-terminal domain and/or a C-terminal domain of a decreased net charge.

59 - 70 . (canceled)

Assignees

• Harvard College

Inventors

• Liu David R
• Guilinger John Paul
• Pattanayak Vikram

Classifications

• A61P31/18

  for HIV · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C12N15/01 Primary

  Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor · CPC title

• C12Q1/68

  involving nucleic acids · CPC title