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.

摘要 研究揭示了CRIPSR/Cas基因组编辑切除前病毒治愈HIV的令人兴奋的前景。 然而，它们临床应用的一个主要障碍是如何有效地将其输送到潜伏感染的细胞和 特别是在活体内。这项提议的总体目标是通过开发一种新的类慢病毒来弥合这一差距 粒子融合蛋白(LVLP-R)，将专门递送多路传输的Cas12a核糖核蛋白(RNP)和 MS2外壳蛋白(MCP)转导体内表达CD4细胞的mRNA对HIV前病毒和辅受体的清除作用 CCR5.这种针对CD4的LVLP-R(LVLP-R-CD4)将提供Cas12a蛋白/mRNA和多路复用指南 由于缺乏慢病毒，具有更高的切除效率和降低了脱靶潜力的RNA(GRNAs) 反转录和整合。我们假设带有Cas12a RNP/mRNA的LVLP-R-CD4可以 体内同时摘除HIV前病毒DNA和CCR5。这一假设得到了我们初步数据的支持 以及已有的关于RNP/mRNA LVLP传递和体内多重基因组编辑的成功报道。在AIM I，我们将优化我们建立的VSVG伪型LVLP-R(LVLP-R-V)对Cas12a RNP/mRNA的交付 用于HIV前病毒和CCR5切除的体外和体内实验。在AIM II中，我们将开发和优化DARPin或HIV Env介导的LVLP-R-CD4高效携带Cas12a RNP/mRNA到人CD4T细胞的基因组研究 在体外和体内编辑。在AIM III中，我们将评估HIV前病毒和CCR5切除对HIV治愈的影响 在HIV感染的ART抑制的人源化BLT小鼠中，LVLP-R-CD4递送多路Cas12a RNP/mRNA。 我们专注于将CD4T细胞作为我们的主要测试平台，因为(1)我们小组是第一个应用 CRISPR/Cas基因组编辑以切除T细胞中的HIV前病毒DNA；(2)CD4T细胞是主要的潜在储存库 以及(3)这项建议中的研究小组在研究慢病毒方面拥有广泛的专业知识。 使用各种方法，包括人源化小鼠模型，对CD4T细胞的传递和艾滋病毒感染进行研究。 这一高额奖励提案将探索一种新的LVLP-R-CD4递送优势LbCas12a RNP/mRNA以 CD4细胞和切除HIV前病毒和CCR5作为一种新的治疗策略。建议的研究完成后， 提供了一种新的工具，将基因组编辑程序运送到体内的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。