MegaTALS: hyperspecific reagents for targeted gene modification and correction

项目摘要 锌指核酸酶、TAL效应核酸酶、CRISPR-Cas9核酸酶和 巨核酸酶/TAL效应器融合(MegaTALs，这是本项目的重点)都是高度特异的 核酸酶可以在单个基因组位置产生单链或双链断裂。这其中的每一个 核酸酶平台正在被开发用于广泛的应用，包括基础研究、工业 农业基因组工程、细胞疗法(例如CAR T细胞)和直接基因疗法。 尽管CRISPR核酸酶现在是几乎所有基因组工程的首选系统，但它们的实用性和 治疗应用的性能并不是一个解决的问题。对于临床使用，核酸酶性能是 由其包装和传递的简易性、其在活细胞中的活性和专一性以及 相互竞争的DNA修复结果。对于这样的目的，MegaTAL核酸酶表现出几种有利的性质， 包括单体结构、小尺寸、高活性和特异性以及独特的切割机制 产生3‘DNA悬垂。我们已经产生了大量的工程MegaTAL核酸酶，并已经 描述了它们在原代人类细胞和转基因生物中的体外和体内表现，如 在全文中总结了本项目的说明。 虽然所有这四个平台都在研究和用于基因治疗，但它们的优化 属性和行为(特别是通过同源性驱动的校正来驱动基因修改，而不是 通过突变末端连接破坏基因)是一项重要的持续优先事项。对于任何核酸酶，其动力学 DNA结合、切割和解离(以及每一步相应的亲和力和半衰期)可以改变 DSB损伤的组成、结构和动态行为可能影响每条途径 不同的。这可能会导致修复结果的显著差异，正如我们的初步数据所表明的那样。 在这一续订申请中，我们建议利用我们设计的核酸酶结构和最近发表的 两个特定目的的结果：(1)测定核酸酶功能的生物物理参数和酶参数 这对DNA修复结果影响最大，并通过HDR加强基因修饰。整体而言 第一个目标的前提是个体dna修复途径及其蛋白质因子是唯一敏感的。 产生DSB的酶的机制和生物物理行为的差异。(2)优化 我们的‘第二代’MegaTAL支架(尺寸缩小了，看起来更好了 活性和特异性)以及针对初级基因组编辑的相应的信使核糖核酸递送系统 造血干细胞(HSC)。第二个目标的总体前提是高度可变(但相当 可控性)MegaTALs及其输送系统的性质特别适合于评估 基因组修改的效率和随后基因编辑的原代细胞的持久性，在培养和 在移植和嫁接时。

Project Summary Zinc finger nucleases ('ZFNs'), TAL effector nucleases '(TALENs'), CRISPR-Cas9 nucleases (‘CRISPRs’) and meganuclease/TAL effector fusions ('MegaTALs', which are the focus of this project) are all highly specific nucleases that can generate single- or double-strand breaks at individual genomic loci. Each of these nuclease platforms is being developed for a wide variety of applications, including basic research, industrial and agricultural genome engineering, cellular therapeutics (for example, CAR T-cells), and direct gene therapy. Although CRISPR nucleases are now the system of choice for almost all genome engineering, their utility and performance for therapeutic applications is not a solved problem. For clinical use, nuclease performance is defined by the ease of its packaging and delivery, its activity and specificity in a living cell, and the balance of competing DNA repair outcomes. MegaTAL nucleases display several favorable properties for such purposes, including monomeric structures, small size, high activity and specificity, and unique cleavage mechanisms that produce 3' DNA overhangs. We have generated a large number of engineered MegaTAL nucleases and have described their ex vivo and in vivo performance in primary human cells and transgenic organisms, as summarized in the full text of this project description. While all these four of these platforms are being studied and used for gene therapy, optimization of their properties and behaviors (particularly to drive gene modification via homology-driven correction, rather than gene disruption via mutagenic end-joining) is an important ongoing priority. For any nuclease, the kinetics of DNA binding, cleavage and dissociation (and the corresponding affinity and half-life at each step) can alter the composition, structure and dynamic behavior of the DSB lesion in a manner that might affect each pathway differently. This can lead to significant differences in repair outcomes, as illustrated via our preliminary data. In this renewal application, we propose to leverage our engineered nuclease constructs and recently published results for two Specific Aims: (1) Determine the biophysical and enzymatic parameters of nuclease function that most strongly influence DNA repair outcomes and enhance gene modification via HDR. The overall premise for the first aim is that individual DNA repair pathways and their protein factors are uniquely sensitive to differences in the mechanisms and biophysical behaviors of the enzymes that generate a DSB. (2) Optimize our '2nd generation' of MegaTAL scaffolds (that are reduced in size and that appear to display improved activity and specificity) and corresponding mRNA delivery systems in genome editing directed towards primary hematopoietic stem cells (HSCs). The overall premise for the second aim is that the highly variable (but quite controllable) properties of MegaTALs and their delivery systems are particularly appropriate for assessing the efficiency of genome modification and subsequent persistence of gene edited primary cells, both in culture and upon transplantation and engraftment.