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


ABSTRACT: The ?-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.

SUBMITTER: Ghani K 

PROVIDER: S-EPMC6610700 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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

Ghani Karim K   Boivin-Welch Michael M   Roy Sylvie S   Dakiw-Piaceski Angela A   Barbier Martin M   Pope Elena E   Germain Lucie L   Caruso Manuel M  

Molecular therapy. Methods & clinical development 20190607


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.  ...[more]

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