Disruption of latent HIV-1 proviruses using CRISPR/Cas endonucleases

 描述(由申请人提供)： 而体内HIV-1感染活化的CD4+细胞通常会导致产生 感染导致细胞死亡，少数被感染的T细胞重新进入静息状态，这可能导致潜伏的HIV-1感染。潜伏的HIV-1感染是由整合的HIV-1前病毒引起的，这些前病毒在转录上完全沉默，但可以通过适当的抗原或试剂刺激从潜伏期重新激活。虽然高效抗逆转录病毒疗法(HAART)可以防止HIV-1传播并显著降低病毒载量，但由于HIV-1感染细胞的逐渐死亡，一小部分潜伏感染HIV-1的T细胞保持完好。因此，当停止HAART时，HIV-1可能会迅速重新出现，并重新构成高水平感染。由于潜伏感染的T细胞池寿命极长，事实证明，仅使用HAART是不可能治愈HIV-1感染患者的，因为这些患者完全没有病毒，能够停止HAART而不会出现病毒反弹。因为根据定义，潜伏感染HIV-1的T细胞几乎不可能与其他静止的T细胞区分开来，因此耗尽潜伏池的努力以前主要集中在尝试在HAART存在的情况下使用例如HDAC抑制剂激活潜伏感染细胞中存在的前病毒。然而，越来越明显的是，这种方法无法有效地激活大多数潜在的前病毒。这里提出的另一种策略是使用载体递送的RNA引导的内切酶来靶向并摧毁潜伏的HIV-1前病毒，该内切酶属于细菌抗病毒RNA引导的DNA内切酶家族。在这一应用中，我们提出了几种有效和特异性地破坏潜伏的HIV-1前病毒储存库的方法，使用慢病毒载体递送Ca9蛋白和针对HIV-1基因组的一个或多个高度保守的区域的小引导RNA。我们将首先优化前病毒靶向和Cas9和单引导RNA(SgRNAs)的表达，并最大化慢病毒载体滴度。其次，我们将测试Cas9/sgRNA组合的各种瞬时表达模式，并分析它们在培养中摧毁潜伏的HIV-1前病毒的能力，最后，我们将使用HIV-1感染的人源化小鼠模型，确定慢病毒载体传递的Cas9/sgRNA复合体在体内清除潜在的HVI-1前病毒储库的能力。

 DESCRIPTION (provided by applicant): While the infection of activated CD4+ cells by HIV-1 in vivo normally results in a productive viral infection leading to cell death, a small number of infected T cells re-enter the resting state and this can lead to a latent HIV-1 infection. Latent HIV-1 infections result from integrated HIV-1 proviruses that are entirely transcriptionally silent yet can be reactivated from latency by stimulation by an appropriate antigen or reagent. While highly active antiretroviral therapy (HAART) can prevent HIV-1 spread and dramatically reduce viral loads, due to the progressive death of productively HIV-1-infected cells, a small population of latently HIV-1-infected T cells remains intact. As a result, when HAART is discontinued, HIV-1 can rapidly reappear and reconstitute a high level infection. Because the latently infected T-cell pool is extremely long lived, it has proven impossible to "cure" HIV-1 infected patients using HAART alone in the sense that these patients become entirely virus free and are able to discontinue HAART without virus rebound. Because latently HIV-1 infected T cells are, almost by definition, impossible to distinguish from other resting T cells, efforts to deplete the latent pool have previously focused on attempts to activate the proviruses present in latently infected cells using, for example, HDAC inhibitors, in the presence of HAART. However, it has become increasingly clear that this approach is unable to effectively activate the majority of latent proviruses. An alternative strategy, proposed here, is to target and destroy latent HIV-1 proviruses using vector-delivered RNA-guided endonucleases belonging to the CRISPR/Cas family of bacterial antiviral RNA- guided DNA endonucleases. In this application, we propose several approaches to effectively and specifically destroy the latent HIV-1 proviral reservoir using lentiviral vector delivery of Ca9 proteins and small guide RNAs that target one or more highly conserved regions of the HIV-1 genome. We will first optimize proviral targeting and expression of Cas9 and single guide RNAs (sgRNAs) and maximize lentiviral vector titer. Secondly, we will test various transient expression modalities for Cas9/sgRNA combinations and analyze their ability to destroy latent HIV-1 proviruses in culture, and finally, we will determine the ability of lentiviral vector- delivered Cas9/sgRNA complexes to ablate the latent HVI-1 proviral reservoir in vivo, using a humanized mouse model of HIV-1 infection.