Destroying the HIV-1 provirus by utilizing components of the CRISPR/Cas system

利用 CRISPR/Cas 系统的组件破坏 HIV-1 原病毒

• 批准号: 8659862
• 负责人: MICHAEL W FANGER
• 金额: $ 45.16万
• 依托单位: CELDARA MEDICAL, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-07 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8659862
• 关键词: Address; Anti-Retroviral Agents; Binding; Cell Line; Cell Nucleus; Cells; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Combination Drug Therapy; Complex; DNA; DNA Damage; DNA Repair; DNA Sequence; Defense Mechanisms; Development; Enzymes; Eukaryotic Cell; Foundations; Frequencies; Gene Targeting; Genes; Genetic Transcription; Genome; Goals; Guide RNA; HIV; HIV-1; Histidine; Human; Human Genome; Hybrids; Immune; Invaded; Length; Life; Life Cycle Stages; Liposomes; Lymphoid Tissue; Modeling; Modification; Molecular; Monitor; Mus; Nuclear; Nucleotides; Patients; Pharmaceutical Preparations; Phase; Plasmids; Population; Production; Program Development; Prokaryotic Cells; Proteins; Proviruses; RNA; RNA Sequences; Reagent; SIV; Series; Small Business Innovation Research Grant; Specificity; Study models; System; T-Lymphocyte; Therapeutic; Toxic effect; Transfection; Translating; Vaccines; Viral; Viral Genes; Viral Genome; Virion; Virus Diseases; Work; Zebrafish; base; cell type; cleft therapy; design; design and construction; effective therapy; endonuclease; in vivo; innovation; macrophage; meetings; nanoparticle; novel; novel therapeutics; nuclease; pre-clinical; prevent; public health relevance; research study; therapy development

DESCRIPTION (provided by applicant): The goal of this project is to develop a plasmid-based therapy that will destroy the integrated HIV-1 DNA (HIV- 1 provirus) in infected cells. Current HIV-1 anti-retroviral therapy (ARV) consists of a cocktail of drugs that blocks viral binding and replication, but does not destroy the HIV-1 provirus. In addition, ARV does not impact the large reservoir of latent HIV-infected cells present in lymphoid tissues and in the gut of infected patients. Moreover, patients must remain on ARV for life, or viral replication will resume once treatment is interrupted. We postulate that cleaving one or more regions within the HIV-1 provirus will impair the ability of an infected cell to transcribe viral genes and to make new vira RNA genomes, and subsequently prevent the production of infectious virions. In addition, this approach would also target and destroy the HIV-1 provirus in latent HIV-infected cells, a population of cells for which anti-retroviral therapy is ineffective. Our approach involves adaptin an immune defense mechanism used by the majority of prokaryotes to block infection by viruses. This defense mechanism is called CRISPR/Cas, in reference to the two major components of this system. The first component is an RNA molecule termed CRISPR RNA, or crRNA, transcribed from the prokaryotic CRISPR locus, and which is complementary to a region in the invading viral genome. The second component is a nuclease enzyme termed Cas (for CRISPR-associated) that is encoded from the cas locus and which binds to the crRNA to form a hybrid molecular complex. Key to this approach is the design of specific guide RNAs that not only are complementary to a unique region in the target gene, but also do not bind to any other region in the genome. We have identified and cloned 9 different guide RNAs that meet all of the requisite criteria for binding to the HIV-1 proviral DNA sequence: they bind to a unique 20 nucleotide region in the HIV provirus, they recognize a region that meets the requirement for the Cas endonuclease activity, and they do not recognize any other region in the human genome, including other endogenous retroviral sequences. In Phase I we will: (1) modify the sequence of the plasmids that produce guide RNAs complementary to unique regions within the HIV-1 proviral DNA in order to increase expression and hybridization with the humanized Cas9 protein, and (2) determine the extent to which the guide RNA:hCas9 hybrid molecular complex binds to and degrades the HIV-1 proviral DNA in HIV-infected macrophages, a primary cell type that represents a pool of latent HIV-infected cells [6]. Our long-term objective is to develop a therapy that can be used in conjunction with existing ARVs to not only prevent the production of infectious virions from infected cells, but that would target the large reservoir of HIV-1 infected cells, and obviate the need for life-long ARV treatment.

描述（由申请人提供）：该项目的目标是开发一种基于质粒的疗法，该疗法将破坏受感染细胞中整合的 HIV-1 DNA（HIV-1 原病毒）。目前的 HIV-1 抗逆转录病毒疗法 (ARV) 由多种药物组成，可阻止病毒结合和复制，但不会破坏 HIV-1 原病毒。此外，ARV 不会影响感染患者淋巴组织和肠道中存在的大量潜伏 HIV 感染细胞。此外，患者必须终生继续服用抗逆转录病毒药物，否则一旦治疗中断，病毒复制就会恢复。我们假设，切割 HIV-1 原病毒内的一个或多个区域将损害受感染细胞转录病毒基因和制造新病毒 RNA 基因组的能力，从而阻止感染性病毒粒子的产生。此外，这种方法还将靶向并破坏潜伏的HIV感染细胞中的HIV-1原病毒，这是抗逆转录病毒治疗无效的细胞群。我们的方法涉及适应大多数原核生物用来阻止病毒感染的免疫防御机制。这种防御机制被称为 CRISPR/Cas，参考该系统的两个主要组成部分。第一个成分是一种称为 CRISPR RNA 或 crRNA 的 RNA 分子，从原核 CRISPR 基因座转录而来，与入侵病毒基因组中的一个区域互补。第二个成分是一种称为 Cas（CRISPR 相关）的核酸酶，由 cas 位点编码，与 crRNA 结合形成杂合分子复合物。这种方法的关键是设计特定的引导RNA，它不仅与目标基因中的独特区域互补，而且不与基因组中的任何其他区域结合。我们已经鉴定并克隆了 9 种不同的向导 RNA，它们满足与 HIV-1 前病毒 DNA 序列结合的所有必要标准：它们与 HIV 前病毒中独特的 20 个核苷酸区域结合，它们识别满足结合要求的区域。 Cas核酸内切酶活性，并且它们不识别人类基因组中的任何其他区域，包括其他内源逆转录病毒序列。在第一阶段，我们将：(1) 修改产生与 HIV-1 前病毒 DNA 内独特区域互补的向导 RNA 的质粒序列，以增加表达以及与人源化 Cas9 蛋白的杂交，以及 (2) 确定其程度指导 RNA:hCas9 杂合分子复合物与 HIV 感染的巨噬细胞中的 HIV-1 前病毒 DNA 结合并降解，巨噬细胞是一种代表潜伏 HIV 感染细胞库的主要细胞类型 [6]。我们的长期目标是开发一种可与现有抗逆转录病毒药物联合使用的疗法，不仅可以防止受感染细胞产生传染性病毒颗粒，而且可以针对大量 HIV-1 感染者 细胞，并消除了终生 ARV 治疗的需要。