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 host cell comprising an exogenous sequence integrated into intron 1 of an endogenous IL2RG gene or intron 1 or 2 of an endogenous RAG gene using a nuclease comprising a zinc finger protein, a TAL-effector domain or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence comprising a target site as shown in any of SEQ ID NOs:47-60 or 81-83 or a target site bound by any SEQ ID NO:85-95.
2. The host cell of claim 1 , wherein the nuclease comprises the zinc finger protein that comprises recognition helix regions as shown in a single row of Table 1.
3. The host cell of claim 1 , wherein the sgRNA comprises a DNA-binding guide RNA as shown in Table 5.
4. The host cell of claim 1 , wherein the TALE-effector domain comprises hypervariable diresidues (RVDs) as shown in in a single row of Table 3.
5. The host cell of claim 1 , wherein the exogenous sequence is 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.
6. The host cell of claim 5 , 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.
7. The host cell of claim 1 , wherein the cell is a hematopoietic stem cell or an induced pluripotent stem cell (iPSC).
8. A method of treating or preventing SCID or Omenn Syndrome in a subject, the method comprising administering a host cell according to claim 1 to the subject.
9. The method of claim 8 , wherein the cell is a hematopoietic stem cell.