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

Lentiviral vectors (LV) commonly used for the treatment of hemoglobinopathies often have low titers and sub-optimal gene transfer efficiency for human hematopoietic stem and progenitor cells (HSPC), 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 towards the 3’ end in packaging cells and in released vector particles. The incomplete vector genomes impeded reverse transcription in target cells, limiting stable gene transfer to HSPC. By combining three modifications to vector design and production (shortening the vector length to 5.3-kb; expressing 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 HSPC. These approaches may improve the manufacturing of β-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications. Examination of viral RNA truncation in 3 lentiviral vectors of different lengths