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: 10312783
• 负责人: BARRY L. STODDARD
• 金额: $ 7.53万
• 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 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312783
• 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（“ ZFNS”），TAL效应核'（Talens'），CRISPR-CAS9核（“ CRISPRS”）和 大核酸酶/TAL效应器融合（“ Megatals”，这是该项目的重点）都是非常具体的 可以在单个基因组基因座上产生单链断裂的核酸酶。每个 正在为各种应用程序开发核酸酶平台，包括基础研究，工业 以及农业基因组工程，细胞疗法（例如，T细胞）和直接基因治疗。 尽管CRISPR核武器现在是几乎所有基因组工程，效用和 治疗应用的性能不是解决问题。为了临床使用，核酸酶的性能是 通过其包装和交付的便捷性，活细胞中的活动和特异性以及 竞争DNA修复结果。巨核核武器出于此目的显示出几种有利的特性， 包括单体结构，小尺寸，高活动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工程的巨核，并且 将它们在原代人类细胞和转基因生物中描述为体内和体内性能， 在此项目描述的全文中总结。 虽然所有这四个平台都在研究并用于基因治疗，但优化了它们 属性和行为（部分是通过同源驱动的校正来驱动基因修饰，而不是 通过诱变末端结合而造成的基因破坏是一个重要的优先级。对于任何核酸酶， DNA结合，裂解和解离（以及相应的亲和力和半衰期）可以改变 DSB病变的组成，结构和动态行为可能会影响每种途径 对不同。如我们的初步数据所示，这可能导致修复结果的显着差异。 在此续订应用中，我们建议利用我们的工程核酸酶结构并最近发表 两个特定目的的结果：（1）确定核酸酶功能的生物物理和酶促参数 最强烈影响DNA修复结果并通过HDR增强基因修饰。总体 第一个目的的前提是单个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”大型脚手架（尺寸降低并且似乎显示出改进的 活性和特异性）和针对主要的基因组编辑中的相应mRNA输送系统 造血干细胞（HSC）。第二个目标的总体前提是高度可变（但是 可控制的）大型属性及其输送系统的特性特别适合评估 基因组修饰的效率和随后基因编辑的原代细胞在培养和 移植和植入后。