It is concerning that patients developed leukemia from insertional oncogenesis both early and late, 15 years after transplantation of retroviral-based engineered cells 7. While most patients treated with first generation of gene therapy survived and benefited from the therapy, a substantial fraction (>25%) of patients developed leukemia from insertional oncogenesis 4, 5, 6. This strategy has resulted in both successes and setbacks. Using integrating viral vectors, such as gamma-retroviral and lentiviral vectors, extra copies of a functional IL2RG gene are semi-randomly integrated into the genome of SCID-X1 patient-derived CD34 + HSPCs. Gene therapy is an alternative therapy to allo-HSCT. The importance of achieving gene correction in long-term hematopoietic stem cells (LT-HSCs) to achieve sustained clinical benefit is demonstrated by the waning of a functional immune system in patients who do not derive their immune system from LT-HSCs with a wild-type IL2RG gene. Because of the selective advantage of lymphoid progenitors expressing normal IL2RG, however, only a small number of genetically corrected hematopoietic stem and progenitor cells (HSPCs) are needed to reconstitute T-cell immunity 2, 3. Although allogeneic hematopoietic cell transplant (allo-HCT) is considered the standard of care for SCID-X1, it holds significant risks due to potential incomplete immune reconstitution, graft versus host disease (GvHD) and a decreased survival rate in the absence of an human leukocyte antigen (HLA)-matched sibling donor 1. Without early treatment, affected male infants die in the first year of life from infections. The gene encodes a shared subunit of the receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21. X-linked sSevere cCombined iImmunodeficiency (SCID-X1) is a primary immune deficiency disorder (PID) caused by mutations in the IL2RG gene on the X chromosome. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl. We achieve high levels of targeting frequencies (median 45%) in CD34 + HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system.
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