The impact of lentiviral vector genome size and producer cell genomic to gag-pol mRNA ratios on packaging efficiency and titre

Lentiviral vectors are showing success in the clinic, but producing enough vector to meet the growing demand is a major challenge. Furthermore, next-generation gene therapy vectors encode multiple genes resulting in larger genome sizes, which is reported to reduce titers. A packaging limit has not b...

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Published inMolecular therapy. Methods & clinical development Vol. 21; pp. 574 - 584
Main Authors Sweeney, Nathan P., Vink, Conrad A.
Format Journal Article
LanguageEnglish
Published Elsevier Inc 11.06.2021
American Society of Gene & Cell Therapy
Elsevier
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Summary:Lentiviral vectors are showing success in the clinic, but producing enough vector to meet the growing demand is a major challenge. Furthermore, next-generation gene therapy vectors encode multiple genes resulting in larger genome sizes, which is reported to reduce titers. A packaging limit has not been defined. The aim of this work was to assess the impact of genome size on the production of lentiviral vectors with an emphasis on producer cell mRNA levels, packaging efficiency, and infectivity measures. Consistent with work by others, vector titers reduced as genome size increased. While genomic infectivity accounted for much of this effect, genome sizes exceeding that of clinical HIV-1 isolates result in low titers due to a combination of both low genomic infectivity and decreased packaging efficiency. Manipulating the relative level of genomic RNA to gag-pol mRNA in the producer cells revealed a direct relationship between producer cell mRNA levels and packaging efficiency yet could not rescue packaging of oversized genomes, implying a de facto packaging defect. However, independent of genome size, an equimolar ratio between wild-type gag-pol mRNA and vector genomic RNA in producer cells was optimal for titer. [Display omitted] Lentiviral vector titers reduce as genome size increases. By studying producer cell mRNA, we find that, independent of genome size, an equimolar ratio of vector genomic RNA to gag-pol mRNA is optimal for packaging efficiency and titer but also reveals a de facto packaging defect for oversized vector genomes.
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ISSN:2329-0501
2329-0501
DOI:10.1016/j.omtm.2021.04.007