METHODS AND COMPOSITIONS FOR TREATMENT OF A GENETIC CONDITION

• US 20160090607A1
• Filed: 12/10/2015
• Published: 03/31/2016
• Est. Priority Date: 05/15/2013
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Conway, Anthony, Cost, Gregory J., DeKelver, Russell, Rebar, Edward J., Reik, Andreas, Urnov, Fyodor, Wang, Jianbin, Zhang, H. Steve
• Granted Patent: US 9,902,974 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C07K 2/00 Peptides of undefined number of amino acids; Derivatives thereof
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21004 Type II site-specific deoxyribonuclease (3.1.21.4)

Abstract

Methods and compositions comprising single guide RNAs targeted to endogenous gene for genetic alteration of cells are provided.

Claims

1. A fusion protein comprising a Cas protein and a functional domain.

2. The fusion protein of claim 1, wherein the functional domain is a transcriptional activation domain, a transcriptional repression domain or a nuclease domain.

3. The fusion protein of claim 2, wherein the nuclease domain is a FokI endonuclease domain.

4. The fusion protein of claim 3, wherein the FokI endonuclease domain comprises one or more mutations in the dimerization domain.

5. A dimer comprising first and second fusion proteins according to claim 3.

6. The dimer of claim 5, wherein the first fusion protein comprises a FokI endonuclease domain comprising one or more mutations in the cleavage (catalytic?) domain.

7. A pharmaceutical composition comprising a fusion protein according to claim 1.

8. A fusion protein according to claim 1 in association with a single guide RNA (sgRNA).

9. A polynucleotide encoding a fusion protein according to claim 1.

10. A method of modifying the expression of an endogenous gene in a cell, the method comprising the steps of:

administering to the cell a nucleic acid molecule comprising a single guide RNA that recognizes a target site in the endogenous gene and a polynucleotide according to claim 8, wherein the fusion protein associates with the single guide RNA on the target site, thereby modifying the expression of the endogenous gene.

11. The method of claim 10, wherein the fusion protein comprises a transcriptional activation domain and expression of the endogenous gene is increased.

12. The method of claim 10, wherein the fusion protein comprises a transcriptional repression domain and expression of the endogenous gene is inhibited.

13. The method of claim 10, wherein the fusion protein comprises a nuclease domain and the endogenous gene is cleaved.

14. The method of claim 10 wherein the fusion protein comprises a nuclease domain and the endogenous gene is nicked.

15. The method of claim 10, wherein the cell is a mammalian or plant cell.

16. The method of claim 15, wherein the mammalian cell is a stem cell.

17. The method of claim 10, wherein the endogenous gene is selected from the group consisting of a mammalian β

globin gene (HBB), a gamma globin gene (HBG1), a B-cell lymphoma/leukemia 11A (BCL11A) gene, a Kruppel-like factor 1 (KLF1) gene, a CCR5 gene, a CXCR4 gene, a PPP1R12C (AAVS1) gene, an hypoxanthine phosphoribosyltransferase (HPRT) gene, an albumin gene, a Factor VIII gene, a Factor IX gene, a Leucine-rich repeat kinase 2 (LRRK2) gene, a Hungtingin (Htt) gene, a rhodopsin (RHO) gene, a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, a surfactant protein B gene (SFTPB), a T-cell receptor alpha (TRAC) gene, a T-cell receptor beta (TRBC) gene, a programmed cell death 1 (PD1) gene, a Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) gene, an human leukocyte antigen (HLA) A gene, an HLA B gene, an HLA C gene, an HLA-DPA gene, an HLA-DQ gene, an HLA-DRA gene, a LMP7 gene, a Transporter associated with Antigen Processing (TAP) 1 gene, a TAP2 gene, a tapasin gene (TAPBP), a class II major histocompatibility complex transactivator (CIITA) gene, a dystrophin gene (DMD), a glucocorticoid receptor gene (GR), an IL2RG gene and an RFX5 gene.

18. The method of claim 10, wherein the endogenous gene is selected from the group consisting of a plant FAD2 gene, a plant FAD3 gene, a plant ZP15 gene, a plant KASII gene, a plant MDH gene, and a plant EPSPS gene.