Nucleobase editors comprising nucleic acid programmable DNA binding proteins

Patent metadata

Field	Value
Publication number	US-11268082-B2
Application number	US-201815934945-A
Country	US
Kind code	B2
Filing date	Mar 23, 2018
Priority date	Mar 23, 2017
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of nucleic acid programmable DNA binding proteins (napDNAbp), e.g., Cpf1 or variants thereof, and nucleic acid editing proteins or protein domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of a napDNAbp (e.g., CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute) and nucleic acid editing proteins or domains, are provided.

First claim

What is claimed is:

1. A fusion protein comprising: (i) a nucleic acid programmable DNA binding protein (napDNAbp); (ii) a cytidine deaminase domain; and (iii) two uracil glycosylase inhibitor (UGI) domains, wherein the napDNAbp is a CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein.

2. A complex comprising the fusion protein of claim 1 , and a guide RNA bound to the napDNAbp of the fusion protein.

3. A method for editing a target nucleic acid molecule, the method comprising contacting the target nucleic acid molecule with the complex of claim 2 , wherein the guide RNA comprises at least 10 contiguous nucleotides complementary to the target nucleic acid molecule, thereby editing the target nucleic acid molecule.

4. A method comprising contacting a nucleic acid molecule with the complex of claim 2 .

5. An isolated cell comprising the complex of claim 2 .

6. A pharmaceutical composition comprising the complex of claim 2 .

7. An isolated cell comprising the fusion protein of claim 1 .

8. A pharmaceutical composition comprising the fusion protein of claim 1 .

9. A method comprising delivering the fusion protein of claim 1 to the inner ear of a subject.

10. A method comprising delivering the fusion protein of claim 1 to a zebrafish embryo.

11. The fusion protein of claim 1 , wherein the cytidine deaminase domain is a deaminase from the apolipoprotein B mRNA-editing complex (APOBEC) family.

12. The fusion protein of claim 1 , wherein the cytidine deaminase domain comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 49-84, wherein said at least 85% amino acid sequence identity is based on an alignment against any one of SEQ ID NOs: 49-84 by NCBI Constraint-based Multiple Alignment Tool (COBALT).

13. A kit comprising a nucleic acid construct comprising:
(a) a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises (i) a nucleic acid programmable DNA binding protein (napDNAbp), wherein the napDNAbp is a CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein, (ii) a cytidine deaminase domain, and (iii) two uracil glycosylase inhibitor (UGI) domains; and
(b) a heterologous promoter that drives expression of the sequence of (a).

14. A polynucleotide encoding a fusion protein, wherein the fusion protein comprises (i) a nucleic acid programmable DNA binding protein (napDNAbp), wherein the napDNAbp is a CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein: (ii) a cytidine deaminase domain; and (iii) two uracil glycosylase inhibitor (UGI) domains.

15. A vector comprising the polynucleotide of claim 14 .

16. The vector of claim 15 , wherein the vector comprises a heterologous promoter driving expression of the polynucleotide.

17. An isolated cell comprising a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises (i) a nucleic acid programmable DNA binding protein (napDNAbp), wherein the napDNAbp is a CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein: (ii) a cytidine deaminase domain; and (iii) two uracil glycosylase inhibitor (UGI) domains.

18. A fusion protein comprising: (i) a nucleic acid programmable DNA binding protein (napDNAbp); (ii) a cytidine deaminase domain; (iii) a first uracil glycosylase inhibitor (UGI) domain; and (iv) a second uracil glycosylase inhibitor (UGI) domain.

19. The fusion protein of claim 18 , wherein the nucleic acid programmable DNA binding protein (napDNAbp) is a CasX, CasY, Cpf1, Cpf1 nickase, dCpf1, C2c1, C2c2, C2c3, Cas9, dCas9, Cas9 nickase or Argonaute protein.

20. The fusion protein of claim 19 , wherein the napDNAbp is a dCas9 or Cas9 nickase.

21. The fusion protein of claim 19 , wherein the napDNAbp comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 34 or 38.

22. The fusion protein of claim 18 , wherein the cytidine deaminase domain comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 49-84, wherein said at least 85% amino acid sequence identity is based on an alignment against any one of SEQ ID NOs: 49-84 by NCBI Constraint-based Multiple Alignment Tool (COBALT).

23. The fusion protein of claim 18 , wherein the fusion protein comprises the structure:
NH 2 -[cytidine deaminase domain]-[napDNAbp]-[first UGI domain]-[second UGI domain]-COOH;
NH 2 -[first UGI domain]-[second UGI domain]-[cytidine deaminase domain]-[napDNAbp]-COOH;
NH 2 -[napDNAbp]-[cytidine deaminase domain]-[first UGI domain]-[second UGI domain]-COOH; or
NH 2 -[first UGI domain]-[second UGI domain]-[napDNAbp]-[cytidine deaminase domain]-COOH;
wherein each instance of “]-[” comprises an optional linker.

24. The fusion protein of claim 18 , wherein the cytidine deaminase domain and the napDNAbp are linked via a linker comprising the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605).

25. A complex comprising the fusion protein of claim 18 and a guide RNA bound to the napDNAbp of the fusion protein.

26. A method for editing a target nucleic acid molecule, the method comprising contacting the target nucleic acid molecule with the fusion protein of claim 18 and a guide RNA, wherein the guide RNA comprises at least 10 contiguous nucleotides complementary to the target nucleic acid molecule, thereby editing the target nucleic acid molecule.

Assignees

• Harvard College

Inventors

• Liu David R
• Komor Alexis Christine
• Chen Liwei
• Rees Holly A

Classifications

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title