Genome Editing by Homologous Recombination to Create HIV Resistant Immune System

通过同源重组进行基因组编辑以创建抗 HIV 免疫系统

• 批准号: 8993696
• 负责人: Matthew H Porteus
• 金额: $ 20万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8993696
• 关键词: Acquired Immunodeficiency Syndrome; Adverse effects; Allogenic; Anti-Retroviral Agents; Autologous; Berlin; Binding; CCR5 gene; CD34 gene; CXCR4 gene; Cell Line; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Development; Drug resistance; Effectiveness; Engineering; Evaluation; Gene-Modified; Generations; Genes; Genetic; Genetic Engineering; Goals; Guide RNA; HIV; Health; Hematopoietic; Hematopoietic System; Human; Hybrids; Immune; Immune system; Infection; Infusion procedures; Knock-in Mouse; Knock-out; Lead; Left; Life; Macaca mulatta; Mediating; Membrane; Methods; Modern Medicine; Mutate; Mutation; Nonhomologous DNA End Joining; Operon; Patients; Pharmacotherapy; Population; Property; Receptor Gene; Resistance; Safety; Stem cell transplant; Stem cells; T-Lymphocyte; Testing; Time; Toxic effect; Virus; Virus Diseases; Zinc Fingers; base; cellular engineering; combinatorial; cost; endonuclease; gene therapy; genetic approach; genetically modified cells; genome editing; genotoxicity; homologous recombination; innovation; interest; killings; macrophage; neutralizing antibody; nonhuman primate; nuclease; public health relevance; receptor; small molecule; stem; success; synthetic biology; viral resistance; virus genetics

 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.

 描述(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，艾滋病是一种最终致命的疾病。该病毒目前感染了全球3500万人，仅在2013年，艾滋病就导致了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，病毒感染本身不是致命的，相反，病毒通过逐渐破坏导致艾滋病的免疫系统间接杀死患者。因此，一个有效的治疗方法将需要创建一个抵抗艾滋病毒破坏的免疫系统。用具有艾滋病毒抵抗力的基因工程细胞替换造血系统，也可能导致无菌治愈(彻底消灭患者体内的病毒)，方法是消除病毒的敏感庇护所细胞，并随着时间的推移用基因工程抗性细胞取代它们。对此，其他人则专注于利用工程核酸酶介导的基因组编辑，通过非同源末端连接来突变和干扰CCR5共同受体基因的表达，作为一种遗传方法来阻止HIV最初进入细胞。相反，我们的重点是利用同源重组介导的基因组编辑在CCR5基因座上敲入抗HIV基因的鸡尾酒，从而同时表达多种抗HIV基因保护因子，并扰乱CCR5基因-一种形式的遗传车。通过使细胞具有一种新的特性，我们将基因组编辑原理与合成生物学结合起来。在这项建议中，我们专注于建立一个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CD34+造血干细胞和祖细胞(HSPC)的CCR5基因。我们的假设是，我们将有效和安全地产生一种抗艾滋病毒的免疫系统(包括T细胞和巨噬细胞)，这将为艾滋病毒提供稳定的长期基因治疗。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基因的组合最有效，并且不会扰乱修饰细胞的正常功能。目的2确定在CD34+HSPC中使用CCR5-TALEN介导的基因组编辑的有效性和安全性，并将其与已开发的CCR5-ZFN进行比较。目的3确定在CD34+HSPC中使用CCR5-CRISPR介导的基因组编辑的有效性和安全性，并将其与已开发的CCR5-ZFN进行比较。总而言之，这些目标将确定最有效的抗艾滋病毒操纵子和最佳核酸酶传递平台，以产生稳定工程的艾滋病毒耐药细胞。