RESUMEN
BACKGROUND: Sickle cell disease is caused by a defect in the ß-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease. OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product, has a targeted disruption of the HBG1 and HBG2 (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny. METHODS: We performed a tiling CRISPR-Cas9 screen of the HBG1 and HBG2 promoters by electroporating CD34+ cells obtained from healthy donors with Cas9 complexed with one of 72 guide RNAs, and we assessed the fraction of fetal hemoglobin-immunostaining erythroblasts (F cells) in erythroid-differentiated progeny. The gRNA resulting in the highest level of F cells (gRNA-68) was selected for clinical development. We enrolled participants with severe sickle cell disease in a multicenter, phase 1-2 clinical study to assess the safety and adverse-effect profile of OTQ923. RESULTS: In preclinical experiments, CD34+ HSPCs (obtained from healthy donors and persons with sickle cell disease) edited with CRISPR-Cas9 and gRNA-68 had sustained on-target editing with no off-target mutations and produced high levels of fetal hemoglobin after in vitro differentiation or xenotransplantation into immunodeficient mice. In the study, three participants received autologous OTQ923 after myeloablative conditioning and were followed for 6 to 18 months. At the end of the follow-up period, all the participants had engraftment and stable induction of fetal hemoglobin (fetal hemoglobin as a percentage of total hemoglobin, 19.0 to 26.8%), with fetal hemoglobin broadly distributed in red cells (F cells as a percentage of red cells, 69.7 to 87.8%). Manifestations of sickle cell disease decreased during the follow-up period. CONCLUSIONS: CRISPR-Cas9 disruption of the HBG1 and HBG2 gene promoters was an effective strategy for induction of fetal hemoglobin. Infusion of autologous OTQ923 into three participants with severe sickle cell disease resulted in sustained induction of red-cell fetal hemoglobin and clinical improvement in disease severity. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT04443907.).