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 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 β-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications.

通常用于治疗血红蛋白病的慢病毒载体 (LVs) 通常对人类造血干细胞和祖细胞 (HSPCs) 具有低滴度和次优的基因转移效率，阻碍了体外基因治疗的临床转化和商业化。我们观察到，在包装细胞和释放的载体颗粒中，高百分比的 β-珠蛋白 LV 病毒基因组 RNA 在 3' 端是不完整的。不完整的载体基因组阻碍了靶细胞的逆转录，限制了稳定的基因转移到 HSPCs。通过结合对载体设计和生产的三种修改（将载体长度缩短至 5.3 kb；在包装过程中表达 HIV-1 Tat 蛋白；以及以PKR-/-包装细胞）在 HSPC 中载体滴度增加 30 倍，载体感染性增加 3 倍。这些方法可以改善 β-珠蛋白和其他复杂 LV 的制造，以增强基因传递，并可能促进临床应用。