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

外周血库和脑库中的广谱 HIV 基因编辑策略

• 批准号: 10551252
• 负责人: Ilker Kudret Sariyer
• 金额: $ 68.54万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10551252
• 关键词: Animals; BCAR1 gene; Back; Bar Codes; Base Sequence; Binding; Biodistribution; Bioinformatics; Biological; Biological Assay; Biology; Blood; Brain; CD4 Positive T Lymphocytes; Cell physiology; Cells; Central Nervous System; Cicatrix; Cloning; Collection; Complex; Computational Biology; Coupling; DNA; Data; Development; Disease; Double Strand Break Repair; Effectiveness; Enzymatic Biochemistry; Enzymes; Event; Excision; Explosion; Funding; Gene Expression; Generations; Genes; Genetic Transcription; Genetic Variation; Genome; Guide RNA; HIV; HIV Genome; HIV Infections; HIV-1; Human immunodeficiency virus test; In Vitro; Individual; Infection; Knowledge; Legal patent; Lentivirus Vector; Link; Macrophage; Mediating; Microglia; Modeling; Molecular; Mus; Mutation; Nature; Nested PCR; Outcome; Patients; Peripheral; Peripheral Blood Mononuclear Cell; Plasmids; Process; Provirus Integration; Reproducibility; Research; Resources; Ribonucleoproteins; Sampling; Shuttle Vectors; System; T-Lymphocyte; Technology; Testing; Tissue Sample; Tissues; Training; Variant; Viral; Viral Load result; Viral Physiology; Viral Proteins; Viral reservoir; Virus; antiretroviral therapy; computational pipelines; deep learning; delivery vehicle; design; effective therapy; humanized mouse; improved; in vivo; in vivo evaluation; innovation; insertion/deletion mutation; monocyte; mouse model; nonhuman primate; novel; prediction algorithm; promoter; repaired; tat Protein; tool; treatment optimization; treatment strategy; vector

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.

项目摘要/摘要 联合抗逆转录病毒疗法(CART)在降低病毒载量和抑制HIV-1感染方面有效， 然而，仍然没有治愈HIV-1疾病的方法。这在一定程度上是由于潜伏感染细胞的形成。 在组织和细胞储存库中含有整合的前病毒。规则间隔成簇的回文重复 (CRISPR)基因编辑已经显示出作为艾滋病毒治愈策略的前景。今天使用的所有CaS酶都会启动结合 通过识别一个Protspacer邻近基序(PAM)，随后是引导RNA(GRNA)之间的互补 并以DNA为靶标诱导DNA裂解。内源性细胞继发的双链断裂修复 已经证明，过程导致了由Protspacer和侧翼决定的非随机突变分布 序列上下文。此外，整合前病毒序列中的自然遗传变异已被显示 降低CRISPR介导的编辑效率，这对gRNA的有效性至关重要 选拔过程。在过去的资助期间，我们设计了一种获得专利的计算管道，以选择广泛的- 光谱spCas9 gRNAs，可解释HIV序列在大量 使用预测算法或功能分析的个体和没有偏离目标的影响。初步数据 这里展示了gRNA在其他组织隔间(脑)和跨亚型中的有效性。在……里面 除了这些gRNA设计方面的进步，该团队还展示了在交付和 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检测技术结合 用来扩展已知的新型CaS酶如何编辑DNA靶标的计算生物学 体外和体内检测HIV-1感染患者样本以优化治疗策略(Cas：gRNA 组合)选择以考虑组织隔间(外围)内和跨组织间的HIV序列变异 VS脑)和亚型。新资源，如多重慢病毒表达系统(MILE)和 整合生物事件的哺乳动物合成细胞记录仪(MSCRIBE)将被用于研究CAS 在单细胞水平上的酶学和HIV-1的重新激活。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且可重现的编辑结果 核苷酸序列。为了验证这一假设，将使用三个具体目标：(I)开发一个通用模型 Cas：gRNA组合修复结果，(Ii)使用分子识别Cas：gRNA对的功能影响 以及(Iii)关于HIV的联合CA：gRNA对的体外和体内测试。这些目标将延长 了解使用艾滋病毒平台的所有生物学领域的CRIPSR编辑技术。它将在细胞中做到这一点 对外周和中枢神经系统(T、单核细胞、小胶质细胞)以及在不同激活状态下的HIV研究很重要 各州。特别是针对HIV，它将把Cas：gRNA设计扩展到其他组织和亚型之间的开发 针对艾滋病毒-1准物种的一种非常重要和创新的方法。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愈方法的治疗策略。