Browsing by Author "Srifa, Waracharee"

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    Gene correction for SCID-X1 in long-term hematopoietic stem cells
    (Springer Nature, 2019) Pavel-Dinu, Mara; Wiebking, Volker; Dejene, Beruh T.; Srifa, Waracharee; Mantri, Sruthi; Nicolas, Carmencita E.; Lee, Ciaran; Bao, Gang; Kildebeck, Eric J.; Punjya, Niraj; Sindhu, Camille; Inlay, Matthew A.; Saxena, Nivedita; DeRavin, Suk See; Malech, Harry; Roncarolo, Maria Grazia; Weinberg, Kenneth I.; Porteus, Matthew H.; Bioengineering
    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. 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. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. 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. 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. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.
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    Highly efficient editing of the β-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease
    (Oxford University Press, 2019) Park, So Hyun; Lee, Ciaran M.; Dever, Daniel P.; Davis, Timothy H.; Camarena, Joab; Srifa, Waracharee; Zhang, Yankai; Paikari, Alireza; Chang, Alicia K.; Porteus, Matthew H.; Sheehan, Vivien A.; Bao, Gang; Bioengineering
    Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the β-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16–19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.