From St. Jude Children's Research Hospital, Memphis, TN (A.S., J.S.H., Y.Z.); Memorial Sloan Kettering Cancer Center, New York (J.-J.B., M.C.); the Novartis Institutes for BioMedical Research, Cambridge, MA (P.B., M.A., A.L., X.Z., S.C., T.M., Y.Y., Y.L., J.Y., V.W.C.Y., S.C.S., S.D.V.); University of Chicago Medicine, Chicago (R.P., J.L.L.); Children's Hospital Los Angeles, Los Angeles (N.K.); IRCCS San Raffaele Hospital, Milan (F.C.); and the Novartis Institutes for BioMedical Research, Basel, Switzerland (U.N.).
N Engl J Med. 2023 Aug 31;389(9):820-832. doi: 10.1056/NEJMoa2215643.
Sickle cell disease is caused by a defect in the β-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 and (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny.
We performed a tiling CRISPR-Cas9 screen of the and 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.
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.
CRISPR-Cas9 disruption of the and 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.).
镰状细胞病是由成人血红蛋白的β-珠蛋白亚单位缺陷引起的。镰状血红蛋白在缺氧条件下聚合，产生变形的红细胞，导致血管阻塞，导致进行性器官损伤和早逝。红细胞中胎儿血红蛋白水平升高可预防镰状细胞病的并发症。OTQ923 是一种经过聚类规则间隔短回文重复序列 (CRISPR)-Cas9 编辑的 CD34+造血干细胞和祖细胞 (HSPC) 产品，其靶向破坏 和 (γ-珠蛋白) 基因启动子，增加红细胞祖细胞中胎儿血红蛋白的表达。
我们通过用 Cas9 复合物与 72 个向导 RNA 之一电穿孔来自健康供体的 CD34+细胞，对 和 启动子进行了平铺 CRISPR-Cas9 筛选，并评估了红细胞分化产物中胎儿血红蛋白免疫染色红细胞 (F 细胞) 的分数。导致最高水平 F 细胞的 gRNA（gRNA-68）被选中进行临床开发。我们在一项多中心、1-2 期临床研究中招募了患有严重镰状细胞病的参与者，以评估 OTQ923 的安全性和不良影响概况。
在临床前实验中，用 CRISPR-Cas9 和 gRNA-68 编辑的 CD34+HSPC（来自健康供体和镰状细胞病患者）具有持续的靶标编辑，没有脱靶突变，并在体外分化或异种移植到免疫缺陷小鼠后产生高水平的胎儿血红蛋白。在该研究中，三名参与者在骨髓清除性预处理后接受了自体 OTQ923，并随访了 6 至 18 个月。在随访结束时，所有参与者均有植入和稳定的胎儿血红蛋白诱导（总血红蛋白中胎儿血红蛋白的百分比，19.0 至 26.8%），胎儿血红蛋白广泛分布在红细胞中（红细胞中 F 细胞的百分比，69.7%至 87.8%）。镰状细胞病的表现在随访期间下降。
CRISPR-Cas9 破坏 和 基因启动子是诱导胎儿血红蛋白的有效策略。将自体 OTQ923 输注到 3 名严重镰状细胞病患者中，导致红细胞胎儿血红蛋白持续诱导和疾病严重程度的临床改善。（由诺华制药公司资助；ClinicalTrials.gov 编号，NCT04443907。）
