Gene correction of SCID-related genes in hematopoietic stem and progenitor cells

• US 9,616,090 B2
• Filed: 07/30/2015
• Issued: 04/11/2017
• Est. Priority Date: 07/30/2014
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Conway, Anthony, Cost, Gregory J., Holmes, Michael C., Urnov, Fyodor
• Primary Examiner(s): BURKHART, MICHAEL D
• Application Number:
  US14/813,924
• Publication Number:
  US 20160030477A1
• Time in Patent Office: 621 Days
• Field of Search: None
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61K 38/1793 for cytokines; for lymphokines; for interferons
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: C07K 14/4705 stimulating, promoting or activating activity
• CPC Class Codes: C07K 14/7155 for interleukins [IL]
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present disclosure is in the field of genome engineering, particularly targeted integration of a functional SCID-related genes (e.g., IL2RG, RAG1 and/or RAG2 gene) into the genome of a cell for provision of proteins lacking or deficient in SCID.

Claims

1. A nuclease comprising a DNA-binding domain and cleavage domain, wherein the DNA-binding domain is a zinc finger protein (ZFP), a TAL-effector domain, or a single RNA (sgRNA) and further wherein the DNA-binding domain binds to a target site in intron 1 of an endogenous IL2RG gene or intron 1 or 2 of an endogenous RAG gene such that the nuclease cleaves the IL2RG or RAG gene.

2. The nuclease of claim 1, wherein the DNA-binding domain comprises a zinc finger protein that binds to a sequence comprising a target site as shown in any of SEQ ID NOs:

47-58 or 81-83.

3. The nuclease of claim 2, wherein the zinc finger protein comprises recognition helix regions as shown in a single row of Table 1.

4. The nuclease of claim 1, wherein the sgRNA comprises a DNA-binding guide RNA as shown in Table 5.

5. The nuclease of claim 1, wherein the TALE-effector domain comprises hypervariable diresidues (RVDs) as shown in in a single row of Table 3.

6. A polynucleotide encoding a nuclease according to claim 1.

7. A host cell comprising an exogenous sequence selected from the group consisting of:

a sequence encoding an IL2RG polypeptide integrated into intron 1 of an endogenous IL2RG gene;

a sequence encoding a RAG polypeptide integrated into intron 1 or 2 of an endogenous RAG gene; and

combinations thereof, wherein the exogenous sequence is integrated into the host cell using a nuclease according to claim 1.

8. The host cell of claim 7, wherein the exogenous sequence comprises a cDNA selected from the group consisting of a sequence comprising exons 2 through 8 of a wild type IL2RG gene;

a sequence comprising a full-length IL2RG gene;

a sequence comprising exon 3 of a wild type RAG gene and a sequence comprising a full-length RAG gene.

9. The host cell of claim 7, wherein the cell is a hematopoietic stem cell or an induced pluripotent stem cell (iPSC).

10. A method for cleaving an endogenous SCID-related gene in a cell, the method comprising:

introducing, into the cell, one or more polynucleotides according to claim 6 to the cell such that the nuclease(s) is(are) expressed and the one or more SCID-related genes are cleaved.

11. A method for targeted integration of a gene encoding a protein lacking or deficient in a subject with SCID, the method comprising:

cleaving an endogenous SCID-related gene in a cell according to the method of claim 10 in the presence of a donor sequence encoding at least a functional fragment of the protein lacking or deficient in the subject with SCID such that the donor sequence is integrated into the endogenous SCID-related gene.

12. The method of claim 11, wherein the donor comprises a cDNA selected from the group consisting of a sequence comprising exons 2 through 8 of a wild type IL2RG gene;

a sequence comprising a full-length IL2RG gene;

a sequence comprising exon 3 of a wild type RAG gene and a sequence comprising a full-length RAG gene.

13. The method of claim 11, wherein the donor is carried on a viral or non-viral vector.

14. The method of claim 13, wherein the viral vector is an adeno-associated vector (AAV).

15. The method of claim 11, wherein the gene lacking or deficient in the subject with SCID is expressed under control of endogenous genetic control elements.

16. The method of claim 11, wherein the donor sequence comprises an exogenous promoter that drives expression of the gene lacking or deficient in the subject with a SCID.

17. A method of treating or preventing SCID in a subject, the method comprising administering a host cell according to claim 7 to the subject.

18. A kit comprising a polynucleotide according to claim 6.

19. A kit comprising a host cell according to claim 7.