Engineered meganucleases specific for recognition sequences in the PCSK9 gene

• US 11,680,254 B2
• Filed: 04/20/2018
• Issued: 06/20/2023
• 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
• Primary Examiner(s): Dunston, Jennifer
• Application Number:
  US16/606,856
• Publication Number:
  US 20200131489A1
• Time in Patent Office: 1,887 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 binds and cleaves a recognition sequence consisting of SEQ ID NO:

4 within a proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO;

6.

2. A polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease that binds and cleaves a recognition sequence consisting of SEQ ID NO:

4 within a PCSK9 gene, wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO;

6.

3. The polynucleotide of claim 2, wherein said polynucleotide is an mRNA.

4. A recombinant DNA construct comprising a nucleic acid sequence encoding an engineered meganuclease that binds and cleaves a recognition sequence consisting of SEQ ID NO:

4 within a PCSK9 gene, wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO;

6.

5. A viral vector comprising a nucleic acid sequence encoding an engineered meganuclease that binds and cleaves a recognition sequence consisting of SEQ ID NO:

4 within a PCSK9 gene, wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO;

6.

6. The viral vector of claim 5, wherein said viral vector is a recombinant AAV vector.

7. The viral vector of claim 6, wherein said recombinant AAV vector is serotype 8.

8. The viral vector of claim 6, wherein said nucleic acid sequence encoding said engineered meganuclease is operably linked to a liver-specific promoter.