ENGINEERED MEGANUCLEASES SPECIFIC FOR RECOGNITION SEQUENCES IN THE PCSK9 GENE

• US 20200131489A1
• Filed: 04/20/2018
• Published: 04/30/2020
• Est. Priority Date: 04/21/2017
• Status: Active Grant

• Current Assignee: Precision BioSciences Incorporated
• Original Assignee: Precision BioSciences Incorporated
• Inventors: Bartsevich, Victor, Jantz, Derek, Smith, James Jefferson, Lape, Janel
• Granted Patent: US 11,680,254 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 38/00 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 Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/6424 Serine endopeptidases (3.4.21)

Abstract

The present invention encompasses engineered meganucleases which recognize and cleave a recognition sequence within the human PCSK9 gene. The present invention also encompasses methods for using such engineered meganucleases in a pharmaceutical composition and in methods for treating or reducing the symptoms of cholesterol-related disorders, such as hypercholesterolemia. Further, the invention encompasses pharmaceutical compositions comprising engineered meganuclease proteins, nucleic acids encoding engineered meganucleases, and the use of such compositions for treating cholesterol-related disorders, such as hypercholesterolemia.

Claims

1. An engineered meganuclease that recognizes and cleaves a recognition sequence consisting of SEQ ID NO:

4, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and wherein said first subunit comprises;

(a) an amino acid sequence having at least 95% sequence identity to residues 7-153 of any one of SEQ ID NOs;

6-14; and

(b) a first hypervariable (HVR1) region having at least 95% sequence identity to an amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs;

6-14;

and wherein said second subunit binds to a second recognition half-site of said recognition sequence and wherein said second subunit comprises;

(i) an amino acid sequence having at least 95% sequence identity to residues 198-344 of any one of SEQ ID NOs;

6-14; and

(ii) a second hypervariable (HVR2) region having at least 95% sequence identity to an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs;

6-14.

3. The engineered meganuclease of claim 1, wherein said HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs:

6-14.

4. The engineered meganuclease of claim 1, wherein said HVR1 region further comprises residues corresponding to residues 48, 50, 71, and 73 of SEQ ID NO:

8.

5. The engineered meganuclease of claim 1, wherein said HVR1 region comprises residues 24-79 of any one of SEQ ID NOs:

6-14.

7. The engineered meganuclease of claim 1, wherein said HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs:

6-14.

8. The engineered meganuclease of claim 7, wherein said HVR2 region further comprises a residue corresponding to residue 258 of SEQ ID NO:

12.

9. The engineered meganuclease of claim 1, wherein said HVR2 region comprises residues 215-270 of any one of SEQ ID NOs:

6-14.

10. The engineered meganuclease of claim 1, wherein said first subunit comprises a residue corresponding to residue 80 of any one of SEQ ID NOs:

6-14.

11. The engineered meganuclease of claim 1, wherein said second subunit comprises a residue corresponding to residue 271 of any one of SEQ ID NOs:

6-14.

12. The engineered meganuclease of claim 1, wherein said first subunit comprises residues 7-153 of any one of SEQ ID NOs:

6-14.

13. The engineered meganuclease of claim 1, wherein said second subunit comprises residues 198-344 of any one of SEQ ID NOs:

6-14.

14. The engineered meganuclease of claim 1, wherein said engineered meganuclease comprises a linker, wherein said linker covalently joins said first subunit and said second subunit.

15. The engineered meganuclease of claim 1, wherein said engineered meganuclease comprises the amino acid sequence of any one of SEQ ID NOs:

6-14.

16. A polynucleotide comprising a nucleic acid sequence encoding said engineered meganuclease of claim 1.

17. The polynucleotide of claim 16, wherein said polynucleotide is an mRNA.

19. A recombinant DNA construct comprising a nucleic acid sequence encoding said engineered meganuclease of claim 1.

21. The recombinant DNA construct of claim 19 further comprising a nucleic acid sequence that encodes a viral vector comprising said nucleic acid sequence encoding said engineered meganuclease.

23. The recombinant DNA construct of claim 21, wherein said viral vector is a recombinant AAV vector.

24. A viral vector comprising a nucleic acid sequence encoding said engineered meganuclease of claim 1.

26. The viral vector of claim 24, wherein said viral vector is a recombinant AAV vector.

28. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and:

(a) a nucleic acid encoding said engineered meganuclease of claim 1;

or

(b) said engineered meganuclease of claim 1.

29. The pharmaceutical composition of claim 28, wherein said nucleic acid encoding said engineered meganuclease is an mRNA.

31. The pharmaceutical composition of claim 28, wherein said pharmaceutical composition comprises a viral vector comprising said nucleic acid.

33. The pharmaceutical composition of claim 29, wherein said mRNA is encapsulated within lipid nanoparticles.

34. A method for reducing expression of PCSK9 in a subject, said method comprising delivering to a target cell in said subject:

(a) an effective amount of a nucleic acid encoding said engineered meganuclease of claim 1, wherein said engineered meganuclease is expressed in said target cell;

or

(b) an effective amount of said engineered meganuclease of claim 1;

wherein said engineered meganuclease recognizes and cleaves a recognition sequence comprising SEQ ID NO;

4 in said target cell and reduces expression of PCSK9 when compared to a control cell.

50. The engineered meganuclease of claim 1, wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO:

6.

2. (canceled)

6. (canceled)

18. (canceled)

20. (canceled)

22. (canceled)

25. (canceled)

27. (canceled)

30. (canceled)

32. (canceled)

35-49. -49. (canceled)