β-Globin Lentiviral Vectors Have Reduced Titers due to Incomplete Vector RNA Genomes and Lowered Virion Production

Jiaying Han, Kevin Tam, Feiyang Ma, Curtis Tam, Bamidele Aleshe, Xiaoyan Wang, Jason P. Quintos, Marco Morselli, Matteo Pellegrini, Roger P. Hollis, Donald B. Kohn
2020 Stem Cell Reports  
Lentiviral vectors (LVs) commonly used for the treatment of hemoglobinopathies often have low titers and sub-optimal gene transfer efficiency for human hematopoietic stem and progenitor cells (HSPCs), hindering clinical translation and commercialization for ex vivo gene therapy. We observed that a high percentage of β-globin LV viral genomic RNAs were incomplete toward the 3' end in packaging cells and in released vector particles. The incomplete vector genomes impeded reverse transcription in
more » ... arget cells, limiting stable gene transfer to HSPCs. By combining three modifications to vector design and production (shortening the vector length to 5.3 kb; expressing HIV-1 Tat protein during packaging; and packaging in PKR-/- cells) there was a 30-fold increase in vector titer and a 3-fold increase in vector infectivity in HSPCs. These approaches may improve the manufacturing of β-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications.
doi:10.1016/j.stemcr.2020.10.007 pmid:33186538 pmcid:PMC7897704 fatcat:l4bajrqljjaiddmh7utaboee5u