T cell-targeted lentiviral vectors with Cas9/RNP for the in vivo gene therapy of HIV-AIDS

带有 Cas9/RNP 的 T 细胞靶向慢病毒载体用于 HIV-AIDS 体内基因治疗

• 批准号: 10322128
• 负责人: Priti Kumar
• 金额: $ 76.22万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-23 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10322128
• 关键词: Acquired Immunodeficiency Syndrome; Address; Affect; Affinity; Antibodies; Automobile Driving; Binding; CCR5 gene; CD7 gene; CRISPR/Cas technology; CXCR4 gene; Cell Line; Cell model; Cell physiology; Cells; Clinical; Clonal Expansion; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Integration; Detection; Dimensions; Disease remission; Dose; Future; Gene Delivery; Genetic Transcription; Genome; Goals; Guide RNA; HIV; HIV-1; HIV/AIDS; Human; Immune system; Individual; Infection; Interruption; Intervention; Investigation; Length; Lentivirus; Lentivirus Vector; Measurement; Methodology; Mutate; Outcome; Patients; Provirus Integration; Proviruses; RNA; RNA delivery; Regimen; Rest; Ribonucleoproteins; Shock; Solid; Sorting; System; T memory cell; T-Lymphocyte; Techniques; Technology; Testing; Therapeutic; Toxic effect; Translating; Tropism; Viral; Viral reservoir; Viremia; Virus; Virus Activation; Virus Latency; Virus Replication; Withdrawal; antiretroviral therapy; combinatorial; design; gene discovery; gene therapy; hematopoietic tissue; humanized antibody; humanized mouse; immunogenicity; improved; in vivo; innovation; latent HIV reservoir; latent virus activation; nanobodies; next generation sequencing; nonhuman primate; novel; nuclease; pre-clinical; reactivation from latency; receptor; response; siRNA delivery; targeted delivery; therapeutic gene; vector; viral rebound; virtual

ABSTRACT/SUMMARY Despite suppression by antiretroviral therapy (ART), virus is not eliminated in HIV patients and can rebound causing full-blown infection upon ART interruption. Thus, a strategy to eliminate the virus reservoir is urgently needed. The recently discovered gene editing technique called CRISPR has tremendous potential for eradicating HIV-1. CRISPR is comprised of a Cas9 nuclease and chimeric guide RNA (gRNA). When Cas9 and gRNA designed to target HIV sequences are present in latently-infected cells, it can result in disruption of the integrated proviral genome, permanently inactivating the virus. The biggest challenge to using the CRISPR approach for HIV elimination is the absence of an in vivo delivery system for human T cells, the major cellular of HIV-1. This is a R01 application in response to RFA-AI-18-016, “Targeted In Vivo Delivery of Gene Therapeutics for HIV Cure”. To address the challenge of Cas9/gRNA delivery to human T cells in vivo, we propose the use of a T cell-targeting lentivirus whose tropism is guided by antibodies to human CD7, a molecule expressed at high levels on all human T cells, including resting T cells which are a major reservoir for latent HIV. To address concerns of vector integration and constitutive Cas9 expression, we have generated lentiviruses that are pre-packaged with Cas9 ribonucleoproteins with no integrating DNA components. Proof- of-concept studies in virologically-suppressed HIV-infected humanized mice demonstrate that disrupting CCR5, the coreceptor for HIV-1, with this systemic approach results in ART-free virologic remission. Importantly, as the approach does not require activation or elimination of the infected cell, it addresses the limitations of conventional `Shock and Kill' approaches that have yielded promising results in clinical settings. The proposal has three specific aims- In Specific Aim 1, we will design and test broad-spectrum gRNAs targeting HIV DNA in two independent approaches expected to mutate a segment or excise the entire length of the integrated HIV provirus. The approach will be tested in ART-suppressed humanized mice for impact on virus reservoir and rebound. A comprehensive investigation of toxicity, off-target effects and virus escape will also be undertaken. In Specific Aim 2, we will perform functional studies in patient-cell derived humanized mice to determine the effects of broad-spectrum gRNAs on latent virus quasispecies from HIV+ patients. The studies will employ HIV-1 RNA Sort-Seq, a novel methodology to quantitate the inducible replication-competent HIV reservoir. In Specific Aim 3, we incorporate strategic changes in the lentiviral vector to reduce vector-associated immunogenicity and permit a single or multiple but rapid-dosing regimen with enhanced potency. The outcome of these proof-of-principle studies is expected to establish a solid platform for future studies on an approach that could significantly contribute towards a cure for HIV-AIDS.

摘要/总结 尽管通过抗逆转录病毒疗法 (ART) 进行抑制，但 HIV 患者体内的病毒并未被消除，并且可以 ART 中断后反弹导致全面感染。因此，消除病毒库的策略是 急需。最近发现的基因编辑技术 CRISPR 具有巨大的潜力 根除 HIV-1。 CRISPR 由 Cas9 核酸酶和嵌合向导 RNA (gRNA) 组成。当Cas9 并且针对 HIV 序列设计的 gRNA 存在于潜伏感染的细胞中，它可能会导致 整合的原病毒基因组，永久灭活病毒。使用 CRISPR 的最大挑战 消除 HIV 的方法是缺乏人类 T 细胞的体内递送系统，T 细胞是主要的细胞 HIV-1。 这是响应 RFA-AI-18-016“基因定向体内递送”的 R01 应用程序 HIV 治愈疗法”。为了解决将 Cas9/gRNA 递送到体内人类 T 细胞的挑战，我们 建议使用一种靶向 T 细胞的慢病毒，其趋向性由人类 CD7 抗体引导， 分子在所有人类 T 细胞上高水平表达，包括静息 T 细胞，静息 T 细胞是 潜伏的艾滋病毒。为了解决载体整合和组成型 Cas9 表达的问题，我们生成了 预包装有 Cas9 核糖核蛋白、无整合 DNA 成分的慢病毒。证明- 对病毒学抑制的 HIV 感染人源化小鼠进行的概念研究表明，破坏 CCR5 是 HIV-1 的辅助受体，采用这种系统方法可实现无需 ART 的病毒学缓解。 重要的是，由于该方法不需要激活或消除受感染的细胞，因此它解决了 传统的“电击杀伤”方法的局限性，但在临床环境中取得了有希望的结果。 该提案有三个具体目标—— 在具体目标 1 中，我们将在两个独立的环境中设计和测试针对 HIV DNA 的广谱 gRNA 预期突变整合的HIV原病毒的一个片段或切除整个长度的方法。这 该方法将在 ART 抑制的人源化小鼠中进行测试，以了解对病毒储存和反弹的影响。一个 还将对毒性、脱靶效应和病毒逃逸进行全面调查。具体来说 目标 2，我们将在源自患者细胞的人源化小鼠中进行功能研究，以确定 HIV+患者潜伏病毒准种的广谱gRNA。该研究将使用 HIV-1 RNA Sort-Seq，一种定量可诱导复制的 HIV 病毒库的新方法。特定目标 3、我们对慢病毒载体进行了策略性改变，以降低载体相关的免疫原性和 允许单次或多次但快速的给药方案，且效力增强。 这些原理验证研究的结果预计将为未来建立一个坚实的平台 研究一种可以显着促进艾滋病毒/艾滋病治愈的方法。