Methods and compositions for treatment of a genetic condition

• US 9,873,894 B2
• Filed: 05/15/2014
• Issued: 01/23/2018
• Est. Priority Date: 05/15/2013
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Conway, Anthony, Cost, Gregory J., Dekelver, Russell, Rebar, Edward J., Reik, Andreas, Urnov, Fyodor, Wang, Jianbin, Zhang, H. Steve
• Primary Examiner(s): Galisteo Gonzalez, Antonio
• Application Number:
  US14/278,903
• Publication Number:
  US 20150056705A1
• Time in Patent Office: 1,349 Days
• Field of Search: None
• 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 an endogenous mammalian β globin gene (HBB), gamma globin gene (HBG1), B-cell lymphoma/leukemia 11A (BCL11A) gene, Kruppel-like factor 1 (KLF1) gene, CCR5 gene, CXCR4 gene, PPP1R12C (AAVS1) gene, hypoxanthine phosphoribosyltransferase (HPRT) gene, albumin gene, Factor VIII gene, Factor IX gene, Leucine-rich repeat kinase 2 (LRRK2) gene, Hungtingin (Htt) gene, rhodopsin (RHO) gene, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, surfactant protein B gene (SFTPB), T-cell receptor alpha (TRAC) gene, T-cell receptor beta (TRBC) gene, programmed cell death 1 (PD1) gene, Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) gene, human leukocyte antigen (HLA) A gene, a LMP7 gene, a Transporter associated with Antigen Processing (TAP) gene, tapasin gene (TAPBP), a class II major histocompatibility complex transactivator (CIITA) gene, a dystrophin gene (DMD), a glucocorticoid receptor gene (GR), IL2RG gene or RFX5 for genetic alteration of cells are provided.

Claims

1. A single-guide RNA that binds to an endogenous gene selected from the group consisting of 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, an hypoxanthine phosphoribosyltransferase (HPRT) gene, an albumin 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 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 DRA 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, and an RFX5 gene, wherein the single-guide RNA is selected from the group consisting of SEQ ID NOs;

149-158 and 167-215.

2. A system comprising the single-guide RNA of claim 1 and a functional domain that associates with the single-guide RNA.

3. The system of claim 2, wherein the functional domain is selected from the group consisting of a transcriptional activation domain, a transcriptional repression domain and a nuclease domain.

4. The system of claim 3, wherein the functional domain is a Type IIS restriction enzyme nuclease domain or a Cas protein.

5. The system of claim 3, wherein the functional domain is a transcriptional activation domain.

6. The system of claim 3, wherein the functional domain is a transcriptional repression domain.

7. The system of claim 4, wherein the Cas protein comprises one more nuclease cleavage domains.

8. A mammalian cell comprising the system of claim 2.

9. The mammalian cell of claim 8, wherein the mammalian cell is a stem cell.

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 first nucleic acid molecule comprising a single guide RNA according to claim 1 that recognizes a target site in the endogenous gene and a second nucleic acid molecule that encodes a functional domain, wherein the functional domain 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 functional domain is selected from the group consisting of a transcriptional activation domain, a transcriptional repression domain and a nuclease domain.

12. The method of claim 11, wherein the functional domain is a TypeIIS restriction enzyme nuclease domain or a Cas protein.

13. The method of claim 11, wherein the functional domain is a transcriptional activation domain and expression of the endogenous gene is increased.

14. The method of claim 11, wherein the functional domain is a transcriptional repression domain and expression of the endogenous gene is inhibited.

15. The method of claim 11, wherein the functional domain is a nuclease and the endogenous gene is cleaved.

16. The method of claim 15, where the nuclease domain is comprised by the Cas protein.

17. The method of claim 16, where the Cas protein comprises one more nuclease cleavage domains.

18. The method of claim 10, wherein the cell is a mammalian cell.

19. The method of claim 18, wherein the mammalian cell is a stem cell.