Department of Laboratory Medicine, University of Washington, Seattle, USA.
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, USA.
BMC Biol. 2018 Jul 11;16(1):75. doi: 10.1186/s12915-018-0544-1.
RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have therefore been proposed as a curative approach for HIV. However, most studies to date have focused on molecular clones with ideal target site recognition and do not account for target site variability observed within and between patients. For clinical success and broad applicability, guide RNA (gRNA) selection must account for circulating strain diversity and incorporate the within-host diversity of HIV.
We identified a set of gRNAs targeting HIV LTR, gag, and pol using publicly available sequences for these genes and ranked gRNAs according to global conservation across HIV-1 group M and within subtypes A-C. By considering paired and triplet combinations of gRNAs, we found triplet sets of target sites such that at least one of the gRNAs in the set was present in over 98% of all globally available sequences. We then selected 59 gRNAs from our list of highly conserved LTR target sites and evaluated in vitro activity using a loss-of-function LTR-GFP fusion reporter. We achieved efficient GFP knockdown with multiple gRNAs and found clustering of highly active gRNA target sites near the middle of the LTR. Using published deep-sequence data from HIV-infected patients, we found that globally conserved sites also had greater within-host target conservation. Lastly, we developed a mathematical model based on varying distributions of within-host HIV sequence diversity and enzyme efficacy. We used the model to estimate the number of doses required to deplete the latent reservoir and achieve functional cure thresholds. Our modeling results highlight the importance of within-host target site conservation. While increased doses may overcome low target cleavage efficiency, inadequate targeting of rare strains is predicted to lead to rebound upon cART cessation even with many doses.
Target site selection must account for global and within host viral genetic diversity. Globally conserved target sites are good starting points for design, but multiplexing is essential for depleting quasispecies and preventing viral load rebound upon therapy cessation.
RNA 引导的 CRISPR/Cas9 系统可设计用于突变或切除潜伏感染细胞中的整合 HIV 基因组，因此被提议作为 HIV 的治疗方法。然而，迄今为止的大多数研究都集中在具有理想靶位点识别的分子克隆上，并且没有考虑到患者内和患者间观察到的靶位点变异性。为了临床成功和广泛适用性，向导 RNA (gRNA) 的选择必须考虑到循环株的多样性，并纳入 HIV 体内的多样性。
我们使用这些基因的公开序列鉴定了一组靶向 HIV LTR、gag 和 pol 的 gRNA，并根据 HIV-1 组 M 中的全局保守性和亚型 A-C 内的保守性对 gRNA 进行了排序。通过考虑 gRNA 的配对和三联体组合，我们发现了一组靶位点的三联体，使得该组中的至少一个 gRNA 存在于所有全球可用序列的 98%以上。然后，我们从高度保守的 LTR 靶位点列表中选择了 59 个 gRNA，并使用功能丧失 LTR-GFP 融合报告器在体外评估其活性。我们使用多个 gRNA 实现了 GFP 的有效敲低，并发现高度活性的 gRNA 靶位点聚类在 LTR 的中间附近。使用来自 HIV 感染患者的已发表深度测序数据，我们发现全球保守位点在体内也具有更大的靶标保守性。最后，我们基于体内 HIV 序列多样性和酶效率的变化分布开发了一个数学模型。我们使用该模型估计了耗尽潜伏储库并达到功能治愈阈值所需的剂量数。我们的建模结果强调了体内靶位点保守性的重要性。虽然增加剂量可以克服低靶标切割效率，但如果靶向稀有株的效果不佳，则预计在停止 cART 后即使剂量很多也会导致病毒载量反弹。
靶标选择必须考虑全球和体内病毒遗传多样性。全球保守的靶标是设计的良好起点，但多重化对于耗尽准种和防止治疗停止后病毒载量反弹至关重要。
