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

摘要/总结 尽管抗逆转录病毒疗法（ART）可以抑制病毒，但艾滋病毒患者中的病毒并未被消除，并且可能 抗逆转录病毒治疗中断后引起全面感染的反弹。因此，消除病毒储库的策略是 迫切需要。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核酸酶和嵌合引导RNA（gRNA）组成。当Cas9 设计用于靶向HIV序列的gRNA存在于潜伏感染的细胞中， 整合的前病毒基因组，永久灭活病毒。使用CRISPR的最大挑战是 消除HIV的方法是缺乏人T细胞的体内递送系统， HIV-1 这是响应RFA-AI-18-016“基因的体内靶向递送”的R 01应用程序 治疗艾滋病的药物”。为了解决Cas9/gRNA在体内递送至人T细胞的挑战，我们 提出使用靶向T细胞的慢病毒，其嗜性由针对人CD 7的抗体指导， 分子在所有人类T细胞上高水平表达，包括静息T细胞，其是免疫调节的主要储存库。 潜伏的艾滋病病毒。为了解决载体整合和组成型Cas9表达的问题，我们已经产生了 用Cas9核糖核蛋白预包装的慢病毒，没有整合的DNA组分。证据- 在病毒学抑制的HIV感染的人源化小鼠中进行的概念研究表明， CCR 5，HIV-1的辅助受体，采用这种系统性方法导致无ART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，因此它解决了受感染细胞的感染。 传统的“休克和杀死”方法的局限性，这些方法在临床环境中产生了有希望的结果。 该建议有三个具体目标： 在具体目标1中，我们将在两个独立的实验中设计和测试靶向HIV DNA的广谱gRNA。 预期突变整合的HIV前病毒的片段或切除整合的HIV前病毒的整个长度的方法。的 将在ART抑制的人源化小鼠中测试方法对病毒储库和反弹的影响。一 还将对毒性、脱靶效应和病毒逃逸进行全面调查。在特定 目的2，我们将在患者细胞来源的人源化小鼠中进行功能研究，以确定 来自HIV+患者的潜伏病毒准种上的广谱gRNA。这些研究将使用HIV-1 RNA Sort-Seq，一种定量可诱导复制能力HIV储库的新方法。具体目标 3，我们在慢病毒载体中引入策略性改变以降低载体相关的免疫原性， 允许具有增强效力的单次或多次但快速的给药方案。 这些原理验证研究的结果有望为未来的研究建立一个坚实的平台。 研究一种可能大大有助于治愈艾滋病毒/艾滋病的方法。

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.