Zucchelli Eleonora, Pema Monika, Stornaiuolo Anna, Piovan Claudia, Scavullo Cinzia, Giuliani Erica, Bossi Sergio, Corna Stefano, Asperti Claudia, Bordignon Claudio, Rizzardi Gian-Paolo, Bovolenta Chiara
MolMed S.p.A., 20132 Milano, Italy; Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK.
MolMed S.p.A., 20132 Milano, Italy.
Mol Ther Methods Clin Dev. 2017 Jan 11;4:102-114. doi: 10.1016/j.omtm.2017.01.002. eCollection 2017 Mar 17.
Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies. Their optimization is therefore very important for the field of vectorology and gene therapy. A key molecule for LV function is the envelope because it guides cell entry. The most commonly used in transiently produced LVs is the vesicular stomatitis virus glycoprotein (VSV-G) envelope, whose continuous expression is, however, toxic for stable LV producer cells. In contrast, the feline endogenous retroviral RD114-TR envelope is suitable for stable LV manufacturing, being well tolerated by producer cells under constitutive expression. We have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34 cells. To further improve RD114-TR-pseudotyped LV cell entry by increasing envelope expression, we codon-optimized the RD114-TR open reading frame (ORF). Here we show that, despite the RD114-TRco precursor being produced at a higher level than the wild-type counterpart, it is unexpectedly not duly glycosylated, exported to the cytosol, and processed. Correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in vivo, and, consequently, the unprocessed precursor is incorporated into LVs, making them inactive.
慢病毒载体（LVs）是一种极具价值的基因转移工具，目前已应用于基础、应用和临床研究。因此，其优化对于载体学和基因治疗领域非常重要。LV功能的关键分子是包膜，因为它引导细胞进入。瞬时产生的LVs中最常用的是水泡性口炎病毒糖蛋白（VSV-G）包膜，然而，其持续表达对稳定的LV生产细胞有毒性。相比之下，猫内源性逆转录病毒RD114-TR包膜适用于稳定的LV生产，在组成型表达下能被生产细胞良好耐受。我们之前报道了用RD114-TR假型化的LVs成功进行瞬时和稳定生产，可有效转导T淋巴细胞和CD34细胞。为了通过增加包膜表达进一步改善RD114-TR假型化LV的细胞进入，我们对RD114-TR开放阅读框（ORF）进行了密码子优化。在此我们表明，尽管RD114-TRco前体的产生水平高于野生型对应物，但出乎意料的是，它没有被正确糖基化、输出到细胞质并进行加工。体内功能表面和跨膜亚基中前体的正确切割受到阻碍，因此，未加工前体被整合到LVs中，使其失去活性。
