title: Critical variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency.
abstract: 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.
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229-3039, USA. Han.vanderloo@cchmc.org
authors.forename: J C M
authors.initials: JC
authors.lastname: van der Loo
authors.forename: W P
authors.initials: WP
authors.lastname: Swaney
authors.forename: E
authors.initials: E
authors.lastname: Grassman
authors.forename: A
authors.initials: A
authors.lastname: Terwilliger
authors.forename: T
authors.initials: T
authors.lastname: Higashimoto
authors.forename: A
authors.initials: A
authors.lastname: Schambach
authors.forename: S
authors.initials: S
authors.lastname: Hacein-Bey-Abina
authors.forename: D L
authors.initials: DL
authors.lastname: Nordling
authors.forename: M
authors.initials: M
authors.lastname: Cavazzana-Calvo
authors.forename: A J
authors.initials: AJ
authors.lastname: Thrasher
authors.forename: D A
authors.initials: DA
authors.lastname: Williams
authors.forename: L
authors.initials: L
authors.lastname: Reeves
authors.forename: P
authors.initials: P
authors.lastname: Malik
mesh_terms: D000818:Animals; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D053631:Interleukin Receptor Common gamma Subunit / Q000235:genetics*; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D012190:Retroviridae / Q000235:genetics*; D020079:Terminal Repeat Sequences; D014161:Transduction, Genetic; D053632:X-Linked Combined Immunodeficiency Diseases / Q000628:therapy*
chemical_list: D053631:Interleukin Receptor Common gamma Subunit