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

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）由一种药物混合物组成，这种药物可以阻止病毒的结合和复制，但不能破坏HIV-1原病毒。此外，ARV不会影响存在于淋巴组织和感染患者肠道中的大量潜伏hiv感染细胞。此外，患者必须终生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，在HIV-1原病毒内切割一个或多个区域将损害被感染细胞转录病毒基因和制造新病毒RNA基因组的能力，并随后阻止传染性病毒粒子的产生。此外，这种方法还将瞄准并摧毁潜伏的hiv感染细胞中的HIV-1前病毒，这是抗逆转录病毒治疗无效的细胞群。我们的方法包括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，参考了该系统的两个主要组成部分。第一个成分是一种RNA分子，称为CRISPR RNA，或crRNA，从原核CRISPR位点转录而来，与入侵病毒基因组中的一个区域互补。第二个成分是一种被称为Cas （CRISPR-associated）的核酸酶，它由Cas位点编码，并与crRNA结合形成杂交分子复合物。这种方法的关键是设计特定的引导rna，不仅与靶基因的独特区域互补，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，它们符合与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Cas内切酶活性要求的区域，并且它们不识别人类基因组中的任何其他区域，包括其他内源性逆转录病毒序列。在第一阶段，我们将：(1)修改产生与HIV-1前病毒DNA内独特区域互补的引导RNA的质粒序列，以增加与人源化Cas9蛋白的表达和杂交；(2)确定在hiv感染的巨噬细胞（一种代表潜伏hiv感染细胞池的原代细胞类型[6]）中，引导RNA:hCas9杂交分子复合物与HIV-1前病毒DNA结合并降解的程度。我们的长期目标是开发一种可以与现有抗逆转录病毒药物联合使用的治疗方法，不仅可以防止受感染细胞产生感染性病毒粒子，而且可以针对HIV-1感染的大量储存库

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.