Intramural Research Program, National Institute on Drug Abuse, Biomedical Research Center, Suite 200, 251 Bayview Boulevard, Baltimore, MD 21224, USA.
Intramural Research Program, National Institute on Drug Abuse, Biomedical Research Center, Suite 200, 251 Bayview Boulevard, Baltimore, MD 21224, USA.
Mol Ther. 2019 Jan 2;27(1):151-163. doi: 10.1016/j.ymthe.2018.10.002. Epub 2018 Oct 11.
Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.
研究人员利用 CRISPR/Cas9 基因编辑系统特异性靶向 HIV 高度保守区域，导致体外和体内感染性和发病机制降低。我们利用一种特殊的细胞外囊泡，称为“gesicle”，以核糖核蛋白形式高效、短暂地递送电针对 HIV 长末端重复序列（LTR）的 Cas9。Gesicles 通过表达水疱性口炎病毒糖蛋白和包装蛋白作为其货物而产生，从而绕过了转基因递送的需求，并允许更精细地控制 Cas9 表达。通过纳米粒子和 Western blot 分析，我们验证了 HEK293FT 细胞产生含有 Cas9 的 gesicles。将 gesicles 应用于 CHME-5 小胶质细胞，通过 Western blot 可快速但短暂地转移 Cas9，Western blot 可在 1 小时检测到，但在治疗后 24 小时不可检测。预先加载靶向 HIV LTR 的 guide RNA 的 Cas9 蛋白的 gesicle 递送至 HIV-NanoLuc CHME-5 细胞，导致 LTR 区域发生突变和拷贝数丢失。最后，我们证明这种治疗导致在基础条件下和在用促炎因子脂多糖（LPS）或肿瘤坏死因子 alpha（TNF-α）刺激后，前病毒活性降低。这些数据表明，gesicles 是递送 CRISPR/Cas9 技术的可行替代方法。
