Broad-spectrum HIV gene editing strategies in peripheral and brain reservoirs

项目概要/摘要 联合抗逆转录病毒疗法（cART）可有效降低病毒载量和抑制HIV-1感染， 然而，仍然没有治愈HIV-1疾病方法。这部分是由于潜伏感染细胞的形成 在组织和细胞库中携带整合的前病毒。规则间隔的重复序列 （CRISPR）基因编辑已显示出作为HIV治疗策略的前景。目前使用的所有Cas酶都启动结合 通过识别前间区序列邻近基序（PAM），随后是引导RNA（gRNA）之间的互补性， 和靶DNA以诱导DNA切割。内源性细胞因子介导的双链断裂修复 已经表明，这些过程导致由原型间隔区和侧翼决定的非随机突变分布 序列上下文。此外，已显示整合前病毒序列内的天然遗传变异 降低CRISPR介导的编辑效率，这对gRNA的功效至关重要 选择过程。在过去的资助期间，我们设计了一个专利的计算管道，以选择广泛的- spCas 9 gRNA谱，其解释了大量的 使用预测算法或功能性测定法，对个体进行分析，并且没有脱靶效应。初步数据 这里呈现的显示了gRNA在其他组织区室（脑）和跨亚型中具有功效。在 除了这些gRNA设计方面的进展外，该团队还展示了在递送方面的重大进展， 在小动物研究和非人类灵长类动物研究中的有效性。为了更好地利用 CRISPR/Cas基因编辑，该项目将利用新的高通量生物测定结合国家的， 本领域的计算生物学扩展了关于新的Cas酶如何编辑DNA靶标的已知内容， 体外和体外测试HIV-1感染的患者样品，以优化治疗策略（Cas：gRNA 组合）选择以解释组织区室（外周）内和跨组织区室的HIV序列变异 与脑）和亚型。新的资源，如多重慢病毒表达系统（MuLE）和 哺乳动物合成细胞记录仪整合生物事件（mSCRIBE）将用于研究Cas 酶学和HIV-1在单细胞水平的再活化。我们假设，Cas：gRNA靶向将诱导 基于酶、靶标和周围环境可预测的安全和可重现的编辑结果 核苷酸序列。为了验证这一假设，将使用三个具体目标：（i）开发通用模型 （ii）使用分子生物学方法鉴定Cas：gRNA对的功能影响， 记录器，和（iii）组合的Cas：gRNA对对HIV的离体和体内测试。这些目标将扩大 利用HIV平台，了解CRIPSR编辑技术在生物学所有领域的应用。它会在细胞中完成 重要的是HIV研究在外周和中枢神经系统（T，单核细胞，小胶质细胞）和不同的激活 states.特别是对于HIV，它将把Cas：gRNA设计扩展到其他组织和亚型之间， 这是一个非常重要和创新的方法，以针对HIV-1准种。这将导致一个非常有效的 使用CRISPR基因编辑作为艾滋病毒感染和疾病的潜在治疗方法的治疗策略。

Project Summary/Abstract Combination antiretroviral therapy (cART) is effective at reducing viral load and suppressing HIV-1 infection, however, there still is no cure for HIV-1 disease. This is due in part to the formation of latently infected cells harboring integrated proviruses in tissue and cell reservoirs. Clustered regularly interspaced palindromic repeats (CRISPR) gene editing has shown promise as an HIV cure strategy. All Cas enzymes in use today initiate binding by recognizing a protospacer adjacent motif (PAM) followed by the complementarity between guide RNA (gRNA) and target DNA to induce DNA cleavage. Subsequent double-strand break repair by endogenous cellular processes has been shown to result in a non-random mutational distribution dictated by protospacer and flanking sequence context. Furthermore, natural genetic variation within integrated proviral sequences has been shown to decrease the CRISPR-mediated editing efficiency which is critically dependent for efficacy of the gRNA selection process. The past funding period, we have designed a patented computational pipeline to select broad- spectrum spCas9 gRNAs that account for HIV sequence variation within and between large numbers of individuals and that have no off-target effect using predictive algorithms or functional assays. Preliminary data presented here shows gRNAs have efficacy in other tissue compartments (brain) and across subtypes. In addition to these gRNA design advances, the team also showed major advancements in delivery and effectiveness in small animal studies and non-human primate studies. In order to better harness the utility of CRISPR/Cas gene editing, this project will utilize novel high-throughput biologic assays combined with state-of- the art computational biology to expand what is known about how novel Cas enzymes edit the DNA target in vitro and test HIV-1-infected patient samples ex vivo and in vivo to optimize treatment strategy (Cas:gRNA combination) selection to account for HIV sequence variation within and across tissue compartments (periphery vs brain) and subtypes. Novel resources like the Multiple Lentiviral Expression System (MuLE) and the Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE) will be leveraged to study Cas enzymology and HIV-1 reactivation at the single-cell level. We hypothesize that, Cas:gRNA targeting will induce safe and reproducible editing outcomes that are predictablly based on the enzyme, target, and surrounding nucleotide sequence. To interrogate this hypothesis, three Specific Aims will be used: (i) develop a generic model of Cas:gRNA combination repair outcomes, (ii) identify functional impact of Cas:gRNA pairs using molecular recorders, and (iii) ex vivo and in vivo testing of combined Cas:gRNA pairs on HIV. These Aims will extend the knowledge of CRIPSR editing technologies for all fields of biology using the HIV platform. It will do this in cells important for HIV research in the periphery and the CNS (T, monocyte, microglia) and under different activation states. For HIV specifically, it will extend Cas:gRNA design into other tissues and between subtypes to develop a highly significant and innovative approach to target the HIV-1 quasispecies. This will result in a highly effective treatment strategy for using CRISPR gene-editing as a potential cure for HIV infection and disease.