Critical variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency.
Critical variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency.
复制标题
影响用于治疗 X 连锁严重联合免疫缺陷的自失活 γ-逆转录病毒载体的临床级生产的关键变量。
作者：
vanderLoo,JCM;Swaney,WP;Grassman,E;Terwilliger,A;Higashimoto,T;Schambach,A;Hacein-Bey-Abina,S;Nordling,DL;Cavazzana-Calvo,M;Thrasher,AJ;Williams,DA;Reeves,L;Malik,P
X 连锁严重联合免疫缺陷 (SCID-X1) 患者在使用表达白细胞介素 2 受体 (IL2RG) 共同伽马链的伽马逆转录病毒载体 (gRV) 进行基因治疗后成功治愈。然而，20 名患者中有 5 名因整合载体长末端重复序列 (LTR) 中的病毒增强子激活细胞原癌基因而患上白血病。这些事件促使人们设计了具有自失活 (SIN) LTR 的 gRV 载体，以增强载体的安全性。在此，我们报告了使用长臂猿白血病病毒包膜假型化的临床级 SIN IL2RG gRV 的生产，用于 SCID-X1 的新基因治疗试验，并重点介绍了被发现对基于转染的大规模 SIN gRV 生产至关重要的变量。成功的临床生产需要仔细选择不预先添加谷氨酰胺的培养基、减少包装细胞与细胞解离酶的接触以及洗涤缓冲液中存在阳离子。临床载体滴度高；根据集落形成单位测定和免疫缺陷型 NOD 中的异种移植测定，转导了 68-70% 的正常人 CD34+ 细胞。 CB17-Prkdc scid/J（非肥胖糖尿病/严重联合免疫缺陷 (NOD/SCID)）和 NOD。 Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NOD/SCID gamma (NSG)))小鼠；并导致人 SCID-X1 CD34+ 细胞在体外产生 T 细胞。该载体在多中心国际 I/II 期试验中获得认证并发布用于治疗 SCID-X1。
Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68–70% normal human CD34+ cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD. CB17-Prkdc scid/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD. Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34+ cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial.