Generation of High-Titer Self-Inactivated γ-Retroviral Vector Producer Cells
| Autor: | Michael Boivin-Welch, Elena Pope, Sylvie Roy, Lucie Germain, Karim Ghani, Manuel Caruso, Angela Dakiw-Piaceski, Martin A Barbier |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Polyadenylation viruses Genetic enhancement packaging cell SIN γ-retroviral vector Article Viral vector 03 medical and health sciences type VII collagen 0302 clinical medicine Retrovirus Plasmid Genetics epidermolysis bullosa Vector (molecular biology) gene transfer Molecular Biology Gene biology Chemistry fungi biology.organism_classification Virology Titer 030104 developmental biology 030220 oncology & carcinogenesis Molecular Medicine |
| Zdroj: | Molecular Therapy. Methods & Clinical Development |
| ISSN: | 2329-0501 |
| DOI: | 10.1016/j.omtm.2019.05.013 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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