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