Methods and compositions for gene correction

• US 9,631,187 B2
• Filed: 10/29/2015
• Issued: 04/25/2017
• Est. Priority Date: 01/05/2011
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc., Whitehead Institute for Biomedical Research
• Original Assignee: Sangamo Biosciences, Inc., Whitehead Institute for Biomedical Research
• Inventors: Jaenisch, Rudolf, Laganiere, Josee, Soldner, Frank, Zhang, Lei
• Primary Examiner(s): Saidha, Tekchand
• Assistant Examiner(s): EKSTROM, RICHARD C
• Application Number:
  US14/926,847
• Publication Number:
  US 20160046683A1
• Time in Patent Office: 544 Days
• Field of Search: None
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• 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 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for correction and/or mutation of genes associated with Parkinson'"'"'s Disease as well as clones and animals derived therefrom.

Claims

1. A polynucleotide encoding the fusion protein comprising a cleavage domain and a zinc-finger protein (ZFP) that binds to a target site in a leucine rich repeat kinase 2 (LRRK2) or α

-synuclein (SCNA) gene in a genome, wherein the ZFP comprises 5 or 6 engineered zinc-finger binding domains, each zinc finger domain comprising a recognition helix region designated F1 to F5 or F1 to F6, wherein the zinc finger protein comprises the recognition helix regions as shown in a single row of Table 2 or Table 4 and further wherein the target site is selected from the group consisting of SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

46, SEQ ID NO;

36 and SEQ ID NO;

37.

2. A method for cleaving a LRRK2 gene or a SCNA gene in a cell, the method comprising:

(a) introducing a first polynucleotide according to claim 1 into the cell under conditions such that the fusion protein is expressed and the gene is cleaved.

3. The method of claim 2, wherein the first polynucleotide encodes first and second fusion proteins that are expressed in the cell.

4. The method of claim 2, further comprising introducing a second polynucleotide encoding an additional fusion protein comprising a zinc finger nuclease (ZFN), wherein the additional ZFN is expressed in the cell.

5. A method of modifying a LRRK2 gene or a SCNA gene in a genome of a cell, the method comprising,

cleaving the LRRK2 or SCNA gene according to the method of claim 2, wherein cleavage results in modification by non-homologous end joining or homology directed repair.

6. The method of claim 5, further comprising introducing an exogenous sequence into the cell, wherein cleavage results in integration of the exogenous sequence into the genome.

7. The method of claim 6, wherein the modification comprises correction of a mutation in the gene.

8. The method of claim 6, wherein the modification comprises introduction of a mutation in the gene.

9. The method of claim 8, wherein the mutation is in the SCNA gene and is selected from the group consisting of A53T, A30P, and E46K mutations.

10. The method of claim 8, wherein the mutation is in the LRRK2 gene and is selected from the group consisting of G2019S, G2385R, R1441C and R1628P.

11. The method of claim 5, wherein one allele is modified.

12. The method of claim 5, wherein both alleles are modified.

13. The method of claim 5, where-in the modification comprises inactivation of the gene.

14. A kit comprising a polynucleotide according to claim 1 and instructions for cleaving a LRRK2 gene or a SCNA gene.