Improved lentiviral vector titers from a multi-gene knockout packaging line.
Mol Ther Oncolytics
; 23: 582-592, 2021 Dec 17.
Article
em En
| MEDLINE
| ID: mdl-34938858
ABSTRACT
Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2'-5'-oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to â¼2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded â¼7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by â¼2-fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to â¼11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.
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MEDLINE
Tipo de estudo:
Prognostic_studies
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article