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 基因组编辑切除原病毒以治疗艾滋病毒的令人兴奋的前景。 然而，其临床应用的一个主要障碍是如何将其有效地递送至潜伏感染细胞并 特别是在体内。该提案的总体目标是通过开发一种新的类慢病毒来缩小这一差距 颗粒融合 Resicle (LVLP-R) 将特异性递送多重 Cas12a 核糖核蛋白 (RNP) 和 mRNA 通过 MS2 外壳蛋白 (MCP) 到达体内表达 CD4 的细胞，以切除 HIV 原病毒和辅助受体 CCR5。这种 CD4 靶向 LVLP-R (LVLP-R-CD4) 将提供 Cas12a 蛋白/mRNA 和多重引导 由于缺乏慢病毒，RNA (gRNA) 具有更高的切除效率和更低的脱靶可能性 逆转录和整合。我们假设 LVLP-R-CD4 与 Cas12a RNP/mRNA 可以 同时在体内切除 HIV 前病毒 DNA 和 CCR5。我们的初步数据支持这一假设 以及关于成功 RNP/mRNA LVLP 传递和体内多重基因组编辑的现有报告。瞄准 I，我们将优化我们已建立的 VSVG 假型 LVLP-R (LVLP-R-V) Cas12a RNP/mRNA 递送 体外和体内 HIV 原病毒和 CCR5 切除。在目标 II 中，我们将开发和优化 DARPin 或 HIV Env 介导的 LVLP-R-CD4 可有效将 Cas12a RNP/mRNA 传递至人类 CD4 T 细胞进行基因组分析 体外和体内编辑。在目标 III 中，我们将使用以下方法评估 HIV 前病毒和 CCR5 切除对于 HIV 治愈的效果： LVLP-R-CD4 在 HIV 感染的 ART 抑制的人源化 BLT 小鼠中传递多重 Cas12a RNP/mRNA。 我们专注于将 CD4 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申请的 CRISPR/Cas基因组编辑切除T细胞中的HIV原病毒DNA； (2) CD4 T细胞是主要的潜在储存库 HIV感染细胞； (3) 本提案的研究团队在慢病毒研究方面拥有丰富的专业知识 使用各种方法（包括人源化小鼠模型）递送 CD4 T 细胞并进行 HIV 感染。 这项高回报提案将探索一种新的 LVLP-R-CD4 递送优势 LbCas12a RNP/mRNA CD4 细胞并切除 HIV 原病毒和 CCR5 作为一种新的治疗策略。完成拟议的研究将 提供了一种将基因组编辑器传递到体内 CD4 细胞的新工具，并可能提供一种新的基因治疗方法 HIV 和其他 T 细胞相关疾病。