Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
Transplant Diagnostics, Thermo Fisher Scientific, Canoga Park, CA, 91303, USA.
Gene Ther. 2018 Oct;25(7):454-472. doi: 10.1038/s41434-018-0039-2. Epub 2018 Sep 6.
Lentiviral vector mobilization following HIV-1 infection of vector-transduced cells poses biosafety risks to vector-treated patients and their communities. The self-inactivating (SIN) vector design has reduced, however, not abolished mobilization of integrated vector genomes. Furthermore, an earlier study demonstrated the ability of the major product of reverse transcription, a circular SIN HIV-1 vector comprising a single- long terminal repeat (LTR) to support production of high vector titers. Here, we demonstrate that configuring the internal vector expression cassette in opposite orientation to the LTRs abolishes mobilization of SIN vectors. This additional SIN mechanism is in part premised on induction of host PKR response to double-stranded RNAs comprised of mRNAs transcribed from cryptic transcription initiation sites around 3'SIN-LTR's and the vector internal promoter. As anticipated, PKR response following transfection of opposite orientation vectors, negatively affects their titers. Importantly, shRNA-mediated knockdown of PKR rendered titers of SIN HIV-1 vectors comprising opposite orientation expression cassettes comparable to titers of conventional SIN vectors. High-titer vectors carrying an expression cassette in opposite orientation to the LTRs efficiently delivered and maintained high levels of transgene expression in mouse livers. This study establishes opposite orientation expression cassettes as an additional PKR-dependent SIN mechanism that abolishes vector mobilization from integrated and episomal SIN lentiviral vectors.
HIV-1 感染转导细胞后的慢病毒载体动员给接受载体治疗的患者及其社区带来了生物安全风险。然而，自我失活（SIN）载体设计减少了，但并未消除整合载体基因组的动员。此外，先前的一项研究表明，逆转录的主要产物，即包含单个长末端重复序列（LTR）的圆形 SIN HIV-1 载体，能够支持高载体滴度的产生。在这里，我们证明了将内部载体表达盒配置为与 LTRs 相反的方向会消除 SIN 载体的动员。这种额外的 SIN 机制部分基于诱导宿主 PKR 对来自 3'SIN-LTR 和载体内部启动子周围隐秘转录起始位点的 mRNA 转录产生的双链 RNA 的反应。正如预期的那样，反方向载体转染后 PKR 反应会对其滴度产生负面影响。重要的是，PKR 的 shRNA 介导敲低使包含反方向表达盒的 SIN HIV-1 载体的滴度与传统 SIN 载体的滴度相当。携带与 LTR 相反方向的表达盒的高滴度载体能够有效地递送至小鼠肝脏，并维持高水平的转基因表达。这项研究确立了反方向表达盒作为一种额外的 PKR 依赖性 SIN 机制，可消除整合和附加型 SIN 慢病毒载体的载体动员。
