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: 10080736
• 负责人: BARRY L. STODDARD
• 金额: $ 35.2万
• 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 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10080736
• 关键词: Affect; Affinity; Agriculture; Architecture; Basic Science; Behavior; Bibliography; Bioinformatics; Biological Models; Biophysics; CCR5 gene; CRISPR/Cas technology; Cell Line; Cell Lineage; Cell model; Cells; Charge; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; DNA Binding; DNA Modification Process; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA Sequence; Data; Development; Disease; Dissociation; Engineering; Engraftment; Enzymes; Equilibrium; Erythroid Cells; Fetal Hemoglobin; Funding; Gene Targeting; Gene-Modified; Generations; Genes; Genome; Genome engineering; Genomics; Globin; Half-Life; Hematopoietic stem cells; Hemoglobinopathies; Human; Individual; Industrialization; Insecta; Kinetics; Lead; Lesion; Letters; Medical; Modification; Monoamine Oxidase B; Open Reading Frames; Outcome; Pathway interactions; Performance; Phenotype; Problem Solving; Property; Proteins; Protocols documentation; Publications; Publishing; Reagent; Regulation; Repression; Research; Sequence Homologs; Severities; Site; Specificity; Structure; Surface; System; T-Lymphocyte; Technology; Testing; Text; Therapeutic; Thermodynamics; Transgenic Organisms; Transplantation; Up-Regulation; Viral; beta Globin; biophysical properties; chimeric antigen receptor T cells; engineered nucleases; fetal; gene correction; gene therapy; genome editing; genomic locus; improved; in vivo; mRNA delivery; monomer; nanoparticle; novel; nuclease; programmed cell death protein 1; programs; repaired; scaffold; stem cell engraftment; targeted nucleases; transcription activator-like effector nucleases; treatment optimization; zinc finger nuclease

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.

项目摘要 锌指核酸酶（“ZFN”）、TAL效应物核酸酶（“TALEN”）、CRISPR-Cas9核酸酶（“CRISPR”）和 大范围核酸酶/TAL效应子融合体（“MegaTAL”，这是本项目的重点）都是高度特异性的 可以在单个基因组基因座处产生单链或双链断裂的核酸酶。这一切成功都 核酸酶平台正在被开发用于各种各样的应用，包括基础研究、工业应用和生物技术应用。 以及农业基因组工程、细胞治疗（例如，CAR T细胞）和直接基因治疗。 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 用于治疗应用的性能不是一个解决的问题。对于临床使用，核酸酶性能是 由其包装和递送的容易性、其在活细胞中的活性和特异性以及其在细胞中的平衡来定义。 竞争DNA修复结果。MegaTAL核酸酶显示出用于这些目的的几种有利性质， 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突出端。我们已经产生了大量的工程MegaTAL核酸酶， 描述了它们在原代人细胞和转基因生物体中的离体和体内性能， 在本项目说明书全文中进行了总结。 虽然所有这四种平台都在研究中并用于基因治疗，但它们的优化仍然存在。 性质和行为（特别是通过同源性驱动的校正来驱动基因修饰，而不是通过 通过诱变末端连接的基因破坏）是一个重要的正在进行的优先事项。对于任何核酸酶， DNA结合、切割和解离（以及每个步骤中相应的亲和力和半衰期）可以改变DNA的结合、切割和解离。 DSB损伤的组成、结构和动力学行为可能会影响每个通路 不同.正如我们的初步数据所示，这可能导致修复结果的显著差异。 在这一更新申请中，我们建议利用我们的工程化核酸酶构建体和最近发表的 两个特定目的的结果：（1）确定核酸酶功能的生物物理和酶参数 最强烈地影响DNA修复结果并通过HDR增强基因修饰。整体 第一个目标的前提是单个DNA修复途径及其蛋白质因子具有独特的敏感性 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（尺寸减小，显示出改善的 活性和特异性）和相应的mRNA递送系统在基因组编辑中针对主要 造血干细胞（HSC）。第二个目标的总体前提是，高度可变（但相当 MegaTAL及其递送系统的性质特别适合于评估 基因组修饰的效率和基因编辑的原代细胞的后续持久性，无论是在培养物中还是在 在移植和植入后。