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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.

Author information

1
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.
2
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.
3
Section of Dermatology, The Hospital for Sick Children and University of Toronto, Toronto, ON, Canada.

Abstract

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.

KEYWORDS:

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

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