Klatt Denise, Sereni Lucia, Liu Boya, Genovese Pietro, Schambach Axel, Verhoeyen Els, Williams David A, Brendel Christian
Gene Therapy Program, Dana Farber/Boston Children's Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA 02115, USA.
Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Mol Ther Nucleic Acids. 2024 Nov 13;35(4):102389. doi: 10.1016/j.omtn.2024.102389. eCollection 2024 Dec 10.
The baboon endogenous retrovirus (BaEV) glycoprotein is superior to the commonly used vesicular stomatitis virus glycoprotein (VSVg) for retroviral gene transfer into resting hematopoietic stem cells and lymphocyte populations. The derivative BaEVRLess (lacking the R domain) produces higher viral titers compared with wild-type BaEV, but vector production is impaired by syncytia formation and cell death of the HEK293T cells due to the high fusogenic activity of the glycoprotein. This lowers viral titers, leads to increased batch-to-batch variability, and impedes the establishment of stable packaging cell lines essential for the economical production of viral supernatants. Here, we show that knockout of the entry receptor ASCT2 in HEK293T producer cells eliminates syncytia formation, resulting in a 2-fold increase in viral titers, reduced toxicity of viral supernatants, and enables the generation of stable packaging cell lines. In successive steps, we stably integrated BaEVRLess and α-retroviral a.Gag/Pol expression cassettes and isolated clones supporting titers up to 10 to 10 infectious particles/mL, a 10-fold increase in concentrated viral titers. The additional overexpression of CD47 and knockout of β2-microglobulin in the packaging cell line are tailored for future use in gene therapy applications by reducing non-specific uptake by macrophages and the immunogenicity of viral particles.
与常用的水泡性口炎病毒糖蛋白（VSVg）相比，狒狒内源性逆转录病毒（BaEV）糖蛋白在将逆转录病毒基因转移到静止造血干细胞和淋巴细胞群体方面表现更优。与野生型BaEV相比，衍生的BaEVRLess（缺乏R结构域）产生的病毒滴度更高，但由于糖蛋白的高融合活性，HEK293T细胞形成多核体并死亡，导致载体生产受损。这降低了病毒滴度，导致批次间变异性增加，并阻碍了建立稳定的包装细胞系，而稳定的包装细胞系对于经济地生产病毒上清液至关重要。在这里，我们表明，在HEK293T生产细胞中敲除进入受体ASCT2可消除多核体形成，使病毒滴度提高2倍，降低病毒上清液的毒性，并能够生成稳定的包装细胞系。在后续步骤中，我们稳定整合了BaEVRLess和α-逆转录病毒a.Gag/Pol表达盒，并分离出支持滴度高达10至10个感染性颗粒/毫升的克隆，浓缩病毒滴度提高了10倍。通过减少巨噬细胞的非特异性摄取和病毒颗粒的免疫原性，包装细胞系中CD47的额外过表达和β2-微球蛋白的敲除是为未来基因治疗应用量身定制的。
