van der Loo, J. C. M.Swaney, W. P.Grassman, E.Terwilliger, A.Higashimoto, T.Schambach, A.Hacein-Bey-Abina, S.Nordling, D. L.Cavazzana-Calvo, M.Thrasher, A. J.Williams, D. A.Reeves, L.Malik, P.2025-07-072025-07-072012van der Loo JC, Swaney WP, Grassman E, et al. Critical variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency. Gene Ther. 2012;19(8):872-876. doi:10.1038/gt.2012.37https://hdl.handle.net/1805/49184Patients 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.en-USPublisher PolicySCID-X1Self-inactivatingGamma-retrovirusProcess developmentClinical-gradeArticle