beta-Globin Lentiviral Vectors Have Reduced Titers due to Incomplete Vector RNA Genomes and Lowered Virion Production.
Publication Year:
2021
PubMed ID:
33186538
Funding Grants:
Public Summary:
Lentiviral vectors (LVs) commonly used for the treatment of hemoglobin abnormalities often have low efficiency in human hematopoietic stem and progenitor cells (HSPCs), hindering clinical translation and commercialization for gene therapy. We observed that incomplete vector genomes impeded viral reverse transcription in target cells, limiting stable gene transfer to HSPCs. By combining three modifications to vector design and production there was a 30-fold increase in vector amount and a 3-fold increase in vector ability to infect HSPCs. These approaches may improve the manufacturing of beta-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications.
Scientific Abstract:
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 beta-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 target 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 beta-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications.