MegaTALS: hyperspecific reagents for targeted gene modification and correction

MegaTALS：用于靶向基因修饰和校正的超特异性试剂

• 批准号: 8629497
• 负责人: BARRY L. STODDARD
• 金额: $ 33.44万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8629497
• 关键词: Address; Animal Model; Biological Models; Cell Line; Cellular Assay; Cleaved cell; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Cystic Fibrosis; DNA; DNA Repair; DNA Sequence; Deoxyribonucleases; Development; Disease; Engineering; Epithelium; Gene Conversion; Gene Delivery; Gene Targeting; Gene-Modified; Genes; Genetic Recombination; Genome engineering; Genomics; Globin; Hematopoietic; Hemoglobinopathies; Hereditary Disease; Homing; Human; Human Cell Line; Human Genetics; Human Genome; In Vitro; Individual; Laboratories; Letters; Lung; Medical; Methods; Modeling; Modification; Molecular; Mutagenesis; Outcome; Pathology; Patients; Preparation; Property; Proteins; Protocols documentation; Reagent; Recruitment Activity; Repetitive Sequence; Research; Science; Site; Solid; Specificity; Structure; Subfamily lentivirinae; System; Technology; Testing; Therapeutic; Tissues; Toxic effect; Transfection; Work; Zinc Fingers; arm; biological systems; endonuclease; falls; gene correction; gene therapy; genome-wide; human disease; in vivo; meetings; nuclease; programs; public health relevance; repaired; scaffold; therapeutic target

Project summary LAGLIDADG homing endonucleases ('LHEs', also termed 'meganucleases'), zinc finger nucleases ('ZFNs') TAL effector nucleases '(TALENs') and CRISPR nucleases are DNA cleavage systems that are used for genome engineering and corrective gene therapy. These systems display specificity profiles that correspond to varying amounts of off-target activity in the human genome. To address this issue, we have (1) developed methods for LHE engineering that is appropriate for academic laboratories (PNAS 2011); (2) determined the structure and recognition mechanism of a TAL effector (Science 2012); and (3) created and characterized a hyperspecific gene targeting scaffold termed a 'MegaTAL' endonuclease, that exploits the combined properties and mechanisms of TAL effectors and meganucleases. Aim 1: Create engineered MegaTALs to target two individual human disease-associated loci, then correlate their in vitro properties to their activities in transfected human cell lines. We have generated meganucleases that nick or cleave DNA target sites associated with two significant human genetic disorders (hemoglobinopathies and cystic fibrosis). The first of these therapeutic targets requires ex vivo disruption of a silencing region in the -globin locus in patient-derived hematopoietic cell lines, whereas the second application requires in vivo targeted gene correction in the lung epithelium. We hypothesize that the MegaTALs will display both increased cleavage activity (by localizing the endonuclease to the target) and exceptional specificity for that same DNA sequence. We will test the hypothesis by characterizing: (1) the effect of the TAL anchor on gene conversion levels; and (2) the effect of the TAL anchor on genome-wide cleavage profiles and off-target activities. Aim 2: Augment the MegaTAL scaffold with tailored nucleolytic activities and molecular recruitment domains that can reduce undesired repair outcomes and/or enhance gene conversion activity. We have developed strategies both to diversify the nucleolytic activity of the LHE (to generate site specific nickases and cleavases for the same targets) and to recruit corrective DNA templates and/or recombination machinery to the target by the meganuclease. We hypothesize that these constructs can be exploited to further reduce or eliminate undesirable off-target activity, mutagenesis and toxicity, and will test that hypothesis using a panel of cellular assays for DNA repair, fidelity and viability as described in the proposal.

项目摘要 LAGLIDADG归巢核酸内切酶（“LHE”，也称为“大范围核酸酶”），锌指核酸酶（“ZFN”） TAL效应物核酸酶（TALEN）和CRISPR核酸酶是用于DNA切割的DNA切割系统， 基因工程和矫正基因疗法。这些系统显示特异性概况， 人类基因组中不同量的脱靶活性。为了解决这个问题，我们（1）开发了 适用于学术实验室的LHE工程方法（PNAS 2011）;（2）确定 TAL效应器的结构和识别机制（Science 2012）;以及（3）创建并表征了TAL效应器的结构和识别机制。 超特异性基因靶向支架称为“MegaTAL”内切核酸酶，其利用了组合的 TAL效应子和大范围核酸酶的性质和机制。 目标1：创建工程MegaTAL以靶向两个单独的人类疾病相关基因座，然后 将它们的体外性质与它们在转染的人细胞系中的活性相关联。我们已经生成 与两种重要的人类遗传疾病相关的切口或切割DNA靶位点的大范围核酸酶 （血红蛋白病和囊性纤维化）。这些治疗靶点中的第一个需要离体破坏细胞内的免疫原性。 在患者来源的造血细胞系中，第二个基因沉默区位于-珠蛋白基因座中，而第二个基因沉默区位于-珠蛋白基因座中。 应用需要肺上皮中的体内靶向基因校正。我们假设 MegaTAL将显示增加的切割活性（通过将核酸内切酶定位于靶标）和 对相同的DNA序列有特殊的特异性。我们将通过描述以下特征来测试假设：（1） TAL锚对基因转化水平的影响;和（2）TAL锚对全基因组的影响。 切割谱和脱靶活性。 目的2：通过定制的溶核活性和分子募集增强MegaTAL支架 这些结构域可以减少不期望的修复结果和/或增强基因转化活性。我们有 开发了使LHE的溶核活性多样化的策略（产生位点特异性切口酶， 用于相同靶标的切割酶）和募集校正DNA模板和/或重组机制以用于相同靶标的重组。 大范围核酸酶的靶点。我们假设这些结构可以被利用来进一步减少或 消除不需要的脱靶活性、诱变和毒性，并将使用一组 如提案中所述的DNA修复、保真度和活力的细胞测定。