Lentivirus-like particle specific delivery of Cas12 ribonucleoprotein (RNP) to HIV reservoir cells in vivo for an HIV cure

总结 研究揭示了CRIPSR/Cas基因组编辑切除前病毒用于HIV治疗的令人兴奋的前景。 然而，其临床应用的主要障碍是如何将其有效地递送至潜伏感染的细胞， 特别是在体内。这项提案的总体目标是通过开发一种新的慢病毒样蛋白来缩小这一差距。 颗粒融合树脂（LVLP-R），将特异性递送多重Cas 12 a核糖核蛋白（RNP）和 通过MS 2外壳蛋白（MCP）将mRNA体内输送至表达CD 4的细胞，以切除HIV前病毒和辅助受体 CCR 5。这种靶向CD 4的LVLP-R（LVLP-R-CD 4）将递送Cas 12 a蛋白/mRNA和多重引导物。 由于缺乏慢病毒载体，具有增加的切除效率和降低的脱靶潜力的RNA（gRNA） 逆转录和整合。我们假设LVLP-R-CD 4与Cas 12 a RNP/mRNA可以通过调节LVLP-R-CD 4的表达来调节LVLP-R-CD 4的表达。 在体内同时切除HIV前病毒DNA和CCR 5。我们的初步数据支持这一假设 以及关于成功的RNP/mRNA LVLP递送和体内多重基因组编辑的已有报道。在Aim中 I，我们将优化我们建立的VSVG-假型LVLP-R（LVLP-R-V）递送Cas 12 a RNP/mRNA， 用于HIV前病毒和CCR 5切除。在Aim II中，我们将开发和优化DARPin或HIV Env介导的LVLP-R-CD 4，有效地将Cas 12 a RNP/mRNA递送至人CD 4 T细胞用于基因组 体外和体内编辑。在目标III中，我们将评估HIV前病毒和CCR 5切除对HIV治愈的作用， 多重Cas 12 a RNP/mRNA在HIV感染的ART抑制的人源化BLT小鼠中的LVLP-R-CD 4递送。 我们专注于靶向CD 4 T细胞作为我们的主要测试平台，因为（1）我们的团队是第一个应用 CRISPR/Cas基因组编辑以切除T细胞中的HIV前病毒DNA;（2）CD 4 T细胞是主要的潜在储存库 艾滋病毒感染的细胞;（3）这项提案中的研究小组在研究慢病毒方面具有广泛的专业知识 使用各种方法，包括人源化小鼠模型，对CD 4 T细胞的递送和HIV感染进行研究。 这项高回报的提案将探索一种新的LVLP-R-CD 4递送ILbCas 12 a RNP/mRNA， CD 4细胞和切除HIV前病毒和CCR 5作为一种新的治疗策略。完成拟议的研究将 提供了一种新的工具，在体内将基因组编辑器递送到CD 4细胞，并可能提供一种新的基因治疗方法。 艾滋病和其他T细胞相关疾病。

Summary Studies have revealed the exciting promise of CRIPSR/Cas genome editing to excise provirus for HIV cure. However, a major barrier to their clinical application is how to deliver it to latently infected cells effectively and specifically in vivo. The overall objective of this proposal is to close this gap by developing a new lentivirus-like particle fusogenic resicle (LVLP-R) that will specifically deliver multiplexed Cas12a ribonucleoprotein (RNP) and mRNA via MS2 coat protein (MCP) to CD4-expressing cells in vivo for excision of HIV proviruses and coreceptor CCR5. This CD4-targeted LVLP-R (LVLP-R-CD4) will deliver Cas12a protein/mRNA and multiplexed guide RNAs (gRNAs) with an increased excision efficiency and reduced off-target potential due to lack of lentiviral reverse transcription and integration. We hypothesize that LVLP-R-CD4 with Cas12a RNP/mRNA can simultaneously excise HIV proviral DNA and CCR5 in vivo. This hypothesis is supported by our preliminary data and pre-existing reports on successful RNP/mRNA LVLP delivery and multiplex genome editing in vivo. In Aim I, we will optimize our established VSVG-pseudotyped LVLP-R (LVLP-R-V) delivery of Cas12a RNP/mRNA both in vitro and in vivo for HIV proviral and CCR5 excision. In Aim II, we will develop and optimize DARPin or HIV Env-mediated LVLP-R-CD4 that efficiently delivers Cas12a RNP/mRNA to human CD4 T cells for genome editing in vitro and in vivo. In Aim III, we will evaluate HIV proviral and CCR5 excision for an HIV cure using LVLP-R-CD4 delivery of multiplexed Cas12a RNP/mRNA in HIV-infected ART-suppressed humanized-BLT mice. We focus on targeting CD4 T cells as our primary test platform because (1) our group was the first to apply CRISPR/Cas genome editing to excise HIV proviral DNA in T cell; (2) CD4 T cells are the major latent reservoir cells of HIV infection; and (3) the research team in this proposal has extensive expertise in studying lentiviral delivery and HIV infection of CD4 T cells using various approaches, including the humanized-mouse model. This high-reward proposal will explore a new LVLP-R-CD4 delivery of advantaged LbCas12a RNP/mRNA to CD4 cells and excise HIV provirus and CCR5 as a novel cure strategy. Completion of the proposed studies will offer a novel tool to deliver genome editors to CD4 cells in vivo and may provide a new gene therapy approach to HIV and other T cell-related diseases.