Gene correction for rag2 deficiency in human stem cells
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsMara Pavel-Dinu, Matthew H. Porteus
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Abstract
The present disclosure provides methods and compositions for treating RAG2 deficiencies in subjects, comprising genetically modifying cells from the subjects ex vivo by integrating a functional, codon-optimized RAG 2 cDNA at the endogenous RAG2 locus.
Claims35
A method of genetically modifying a cell from a subject with a Recombination-Activating Gene 2 (RAG2) deficiency, the method comprising:
- introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the RAG2 gene, an RNA-guided nuclease, and a homologous donor template comprising a RAG2 cDNA comprising a nucleotide sequence having at least 80% identity to SEQ ID NO:5 or SEQ ID NO:6, flanked by a first and a second RAG2 homology region;
- wherein:
- the sgRNA binds to the nuclease and directs it to a target sequence within the RAG2 gene, whereupon the nuclease cleaves the gene at the target sequence, and wherein:
- the cDNA is integrated by homology directed recombination (HDR) at the site of the cleaved RAG2 gene, such that the cDNA is expressed under the control of the endogenous RAG2 promoter, thereby providing functional RAG2 protein product in the cell.
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A homologous donor template comprising:
- (i) a RAG2 cDNA comprising a nucleotide sequence comprising at least 80% identity to SEQ ID NO:5 or SEQ ID NO:6;
- (ii) a first RAG2 homology region located to one side of the cDNA within the donor template;
- and
- (iii) a second RAG2 homology region located to the other side of the cDNA within the donor template.
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An isolated, genetically modified iPSC or HSPC comprising an exogenous, codon-optimized RAG2 cDNA integrated at the endogenous RAG2 locus, wherein the integrated cDNA comprises a nucleotide sequence having at least 80% identity to SEQ ID NO:5 or 6.
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Dependencies are inferred from the claim text.