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

摘要/摘要 尽管有抗逆转录病毒疗法(ART)的抑制，但HIV患者中的病毒并未被消除，而且可以 在抗逆转录病毒治疗中断后反弹，导致全面感染。因此，消除病毒库的策略是 急需之物。最近发现的名为CRISPR的基因编辑技术具有巨大的潜力 根除HIV-1。CRISPR由Cas9核酸酶和嵌合引导RNA(GRNA)组成。当Cas9 和针对HIV序列的gRNA存在于潜伏感染的细胞中，它可以导致干扰 整合的前病毒基因组，永久灭活病毒。使用CRISPR面临的最大挑战 消除HIV的方法是缺乏体内人类T细胞的递送系统，T细胞是主要的细胞 HIV-1病毒。 这是响应RFA-AI-18-016的R01应用程序 艾滋病毒治疗学“。为了解决在体内向人T细胞运送Cas9/gRNA的挑战，我们 建议使用一种T细胞靶向慢病毒，其趋向性由抗人CD7的抗体引导 在所有人类T细胞上高水平表达的分子，包括作为主要储存库的静息T细胞 潜伏的艾滋病毒。为了解决载体整合和结构性Cas9表达的问题，我们生成了 慢病毒与Cas9核糖核蛋白预先包装在一起，没有整合DNA组件。证明- 在病毒学抑制的HIV感染的人源化小鼠中进行的概念外研究表明，干扰 CCR5，HIV-1的共同受体，与这种系统性方法一起导致了不含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 在临床环境中产生了令人振奋的结果的传统“休克和杀戮”方法的局限性。 该建议有三个具体目标： 在具体目标1中，我们将设计和测试针对HIV dna的广谱gRNA。 预计将突变整合的艾滋病毒前病毒的一段或整个长度的方法。这个 该方法将在ART抑制的人源化小鼠身上进行测试，以确定其对病毒库和反弹的影响。一个 还将对毒性、偏离目标的影响和病毒逃逸进行全面调查。具体而言 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 HIV患者潜伏病毒准种的广谱gRNA。这些研究将使用HIV-1RNA Sort-Seq，一种量化可诱导复制能力HIV储备库的新方法。以特定的目标 3，我们在慢病毒载体中加入了战略变化，以降低载体相关的免疫原性和 允许单一或多次但快速给药方案，以增强效力。 这些原则证明研究的结果可望为今后的工作奠定坚实的平台 研究一种可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.