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Mol Ther Methods Clin Dev. 2019 Jun 7;14:90-99. doi: 10.1016/j.omtm.2019.05.013. eCollection 2019 Sep 13.

Generation of High-Titer Self-Inactivated γ-Retroviral Vector Producer Cells.

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CHU de Québec-Université Laval Research Center (Oncology Division), Université Laval Cancer Research Center, and Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Université Laval, Québec, QC G1R 2J6, Canada.
CHU de Québec-Université Laval Research Center (Regenerative Medicine Division) and Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX, and Department of Surgery, Faculty of Medicine, Université Laval, Québec, QC, G1J 1Z4, Canada.
Section of Dermatology, The Hospital for Sick Children and University of Toronto, Toronto, ON, Canada.


The γ-retroviral vector is a gene delivery vehicle that is commonly used in gene therapy. Despite its efficacy, its strong enhancers contributed to malignant transformations in some hematopoietic stem cell (HSC) gene therapy trials. A safer version without viral enhancers (SIN) is available, but its production is cumbersome, as high titers can only be obtained in transient transfection. Our aim was to develop a system that could easily generate high-titer SIN vectors from stable producer cells. The use of the cytomegalovirus enhancer-promoter sequence to generate the full-length genomic RNA combined to sequences that decrease transcriptional readthrough (WPRE and strong polyadenylation sequences) led to 6 × 106 infectious units (IU)/mL of a SIN GFP vector in transient transfection. The incorporation of a blasticidin selection cassette to the retroviral plasmid allowed the generation of stable clones in the 293Vec packaging cells that release 2 × 107 IU/mL and 1.4 × 107 IU/mL of a SIN GFP and a SIN PIGA vector, respectively. A titer of 1.8 × 106 IU/mL was obtained with a SIN vector containing the long 8.9-kb COL7A1 cDNA. Thus, an efficient process was established for the generation of stable 293Vec-derived retrovirus producer cells that release high-titer SIN vectors.


SIN γ-retroviral vector; epidermolysis bullosa; gene transfer; packaging cell; type VII collagen

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