Genome Editing by Homologous Recombination to Create HIV Resistant Immune System

 描述（由申请人提供）：人类免疫缺陷病毒（HIV）是获得性免疫缺陷综合征（AIDS）的病原体，这是一种最终致命的疾病。该病毒目前感染了全世界3500万人，仅在2013年，艾滋病就造成150万人死亡。艾滋病毒和艾滋病之间关系的一个关键特征是，病毒感染本身并不致命，而是病毒通过逐步破坏免疫系统间接杀死患者，导致艾滋病。因此，功能性治疗需要建立一个抵抗艾滋病毒破坏的免疫系统。用经过改造的细胞来替代造血系统，赋予艾滋病病毒抗性，也可能通过消除病毒的易感庇护细胞，并随着时间的推移用经过改造的抗性细胞来取代它们，从而实现灭菌治疗（从患者身上完全消除病毒）。为此，其他人已经专注于使用工程化核酸酶介导的基因组编辑，通过非同源末端连接来突变和破坏CCR 5共受体基因的表达，作为阻断HIV初始进入细胞的遗传方法。相反，我们的重点是使用同源重组介导的基因组编辑在CCR 5位点敲入抗HIV基因的混合物，从而同时表达多种抗HIV遗传保护因子并破坏CCR 5基因-一种遗传cART形式。通过改造细胞使其具有新的特性，我们将基因组编辑的原理与合成生物学相结合。在该提案中，我们专注于建立一种高效且无细胞毒性的抗HIV基因鸡尾酒（“抗HIV操纵子”），并确定最佳和最安全的核酸酶平台，以靶向将该抗HIV操纵子插入CD 34+造血干细胞和祖细胞（HSPC）中的CCR 5基因。我们的假设是，我们将有效和安全地产生一个抗艾滋病毒的免疫系统（包括T细胞和巨噬细胞），这将提供一个稳定的长期遗传治愈艾滋病毒。为了实现这一目标，我们有三个具体目标。目标1将确定哪种抗HIV基因的组合是最有效的，并且不会干扰修饰细胞的正常功能。目的2将确定在CD 34 + HSPCS中使用CCR 5-TALEN介导的基因组编辑的有效性和安全性，并将其与开发的CCR 5-ZFN进行比较。目的3将确定在CD 34 + HSPCS中使用CCR 5-CRISPR介导的基因组编辑的有效性和安全性，并将其与开发的CCR 5-ZFN进行比较。总的来说，这些目标将确定最有效的抗HIV操纵子和最佳的核酸酶递送平台，用于产生稳定工程化的HIV抗性细胞。

 DESCRIPTION (provided by applicant): The human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS), an ultimately lethal condition. The virus currently infects 35 million people worldwide and in 2013 alone, AIDS caused 1.5 million deaths. A key feature of the relationship between HIV and AIDS is that the viral infection itself is not lethal, but instead the virus indirectly kills the patient through the progressive destruction of the immune system leading to AIDS. Thus, a functional cure would entail creating an immune system that is resistant to destruction by HIV. Replacement of the hematopoietic system with cells engineered to confer HIV resistance might also lead to a sterilizing cure (the complete abolition of the virus from the patient) through the virus eliminating its susceptible sanctuary cells and having them replaced by engineered resistant cells over time. Toward this, others have focused on using engineered nuclease mediated genome editing by non-homologous end-joining to mutate and disrupt expression of the CCR5 co-receptor gene as a genetic approach to blocking initial HIV entry into cells. Our focus instead is to use homologous recombination mediated genome editing to knock-in a cocktail of anti-HIV genes at the CCR5 locus thereby simultaneously expressing multiple anti-HIV genetic protective factors and disrupting the CCR5 gene-a form of genetic cART. By engineering cells to have a new property we are combining the principles of genome editing with synthetic biology. In this proposal we focus on establishing a highly effective and non-cytotoxic cocktail of anti-HIV genes (an "anti-HIV operon") and determining the best and safest nuclease platform to target the insertion of this anti-HIV operon into the CCR5 gene in CD34+ hematopoietic stem and progenitor cells (HSPCs). Our hypothesis is that we will effectively and safely generate an HIV resistant immune system (including T-cells and macrophages) that will provide a stable long-term genetic cure to HIV. To achieve this goal we have three specific aims. Aim 1 will determine which combination of anti-HIV genes are the most effective and do not perturb the normal functioning of the modified cells. Aim 2 will determine the effectiveness and safety of using CCR5-TALEN mediated genome editing in CD34+ HSPCS and compare this to the developed CCR5-ZFNs. Aim 3 will determine the effectiveness and safety of using CCR5-CRISPR mediated genome editing in CD34+ HSPCS and compare this to the developed CCR5-ZFNs. Collectively these aims will identify the most effective anti-HIV operon and the optimal nuclease delivery platform for generation of stably engineered HIV resistant cells.