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

慢病毒样颗粒将 Cas12 核糖核蛋白 (RNP) 特异性递送至体内 HIV 储存细胞以治疗 HIV

• 批准号: 10598912
• 负责人: Wenhui Hu
• 金额: $ 91.51万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10598912
• 关键词: BLT mice; Binding; Biodistribution; CCR5 gene; CD4 Positive T Lymphocytes; Cell Line; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; Data; Disease; Dose; Engineering; Excision; GAG Gene; Genome; Glycoproteins; Guide RNA; HIV; HIV Genome; HIV Infections; HIV-1; HIV/AIDS; Human; Immunity; In Vitro; Infection; Knock-out; Lentivirus; MS2 coat protein; Mediating; Messenger RNA; Mission; Modeling; Monkeys; Mus; Peptides; Phase I Clinical Trials; Positioning Attribute; Proteins; Proviruses; Public Health; RNA; RNA Binding; Reporter; Reporting; Research; Resistance; Rest; Reverse Transcription; Ribonucleoproteins; SIV; Specificity; T-Lymphocyte; Testing; Transcript; Transgenic Mice; United States National Institutes of Health; Vesicle; Viral; Virus; Work; adeno-associated viral vector; antiretroviral therapy; clinical application; gene therapy; genome editing; high reward; humanized mouse; immune activation; immunogenicity; improved; in vivo; innovation; mRNA delivery; mouse model; novel; novel therapeutics; particle; pharmacokinetics and pharmacodynamics; prevent; response; tool; vector

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.

概括 研究揭示了CRIPSR/CAS基因组编辑对HIV治疗的消费病毒的令人兴奋的希望。 但是，其临床应用的主要障碍是如何有效地将其传递给潜在受感染的细胞，以及 特别是体内。该提案的总体目的是通过开发一种新的慢病毒来缩小这一差距 颗粒融合式雷斯（LVLP-R），该雷斯将特异性提供多重的CAS12A核糖核蛋白（RNP）和 通过MS2涂层蛋白（MCP）在体内表达CD4的细胞的mRNA，以切除HIV病毒和crocector CCR5。该针对CD4的LVLP-R（LVLP-R-CD4）将提供CAS12A蛋白/mRNA和多路复用指南 RNA（GRNA）的切除效率提高，并且由于缺乏慢病毒而降低了脱靶潜力 逆转录和集成。我们假设使用CAS12A RNP/mRNA的LVLP-R-CD4可以 同时在体内消除艾滋病毒前病毒DNA和CCR5。我们的初步数据支持了这一假设 以及有关成功的RNP/mRNA LVLP递送和多重基因组编辑的预先报道。目标 i，我们将优化我们已建立的VSVG-PESEUDYTYPERED LVLP-R（LVLP-R-V）CAS12A RNP/mRNA的交付 体外和体内用于HIV病毒和CCR5切除。在AIM II中，我们将开发和优化DARPIN或HIV Env介导的LVLP-R-CD4有效地将CAS12A RNP/mRNA传递到人CD4 T细胞中的基因组 在体外和体内编辑。在AIM III中，我们将评估HIV病毒和CCR5切除术，以使用HIV治疗 在HIV感染的ART抑制的人源化BLT小鼠中，多路复用CAS12A RNP/mRNA的LVLP-R-CD4递送。 我们专注于将CD4 T细胞作为主要测试平台，因为（1）我们的组是第一个应用的组 T细胞中的CRISPR/CAS基因组编辑为消费税HIV病毒DNA； （2）CD4 T细胞是主要的潜在储层 HIV感染细胞； （3）该提案中的研究团队在研究慢病毒方面具有广泛的专业知识 CD4 T细胞的递送和HIV感染使用各种方法，包括人源化鼠标模型。 这个高回报的提案将探索有利的LBCAS12A RNP/mRNA的新的LVLP-R-CD4提供 CD4细胞和消除HIV病毒和CCR5作为一种新型的治愈策略。拟议研究的完成将 提供一种新颖的工具将基因组编辑器在体内运送给CD4细胞，并可能提供一种新的基因治疗方法 艾滋病毒和其他与T细胞相关的疾病。