MegaTALS: hyperspecific reagents for targeted gene modification and correction

项目总结 LAGLIDADG归巢核酸内切酶(LHEs)，又称巨核酸酶(MeganDNA Ase)，锌指核酸酶(ZFN) TAL效应核酸酶和CRISPR核酸酶是DNA裂解系统，用于 基因组工程和矫正基因疗法。这些系统显示与以下各项对应的特异性配置文件 人类基因组中不同数量的脱靶活动。为了解决这个问题，我们开发了(1) 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)创建并表征了一种 超特异性基因靶向支架被称为‘MegaTAL’内切酶，它利用结合的 TAL效应子和巨核酸酶的性质和机制。 目标1：创建针对两个单独的人类疾病相关基因的工程MegaTAL，然后 将它们的体外特性与其在转基因人类细胞系中的活性相关联。我们已经产生了 切割或切割与两种重大人类遗传性疾病相关的DNA靶点的巨核酸酶 (血红蛋白病和囊性纤维化)。这些治疗靶点中的第一个需要体外干扰 在患者来源的造血细胞系中-珠蛋白基因座的沉默区域，而第二 应用需要在肺上皮中进行体内靶向基因校正。我们假设 MegaTALs将同时表现出更高的切割活性(通过将核酸内切酶定位到目标)和 同样的DNA序列具有特殊的特异性。我们将通过以下特征来检验该假设：(1) TAL锚对基因转化水平的影响；以及(2)TAL锚对全基因组的影响 卵裂图谱和偏离目标的活动。 目标2：通过量身定制的核溶解活性和分子招募来增强MegaTAL支架 可以减少不希望的修复结果和/或增强基因转化活性的结构域。我们有 开发了使LHE的核溶解活性多样化的策略(产生位点特异的镍酶和 相同靶点的裂解酶)，并招募矫正的DNA模板和/或重组机制到 以巨核酸酶为靶标。我们假设这些构造可以被利用来进一步减少或 消除不受欢迎的脱靶活动、诱变和毒性，并将使用一组 建议中描述的DNA修复、保真度和生存能力的细胞分析。

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.