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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要锌指核酸酶（'ZFN'）、TAL效应核酸酶'（TALENs'）、CRISPR-Cas9核酸酶（'CRISPRs'）和大范围核酸酶/TAL 效应子融合（“MegaTAL”，这是该项目的重点）都是高度特异性的可以在各个基因组位点产生单链或双链断裂的核酸酶。核酸酶平台正在开发用于各种应用，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，但它们的实用性和对于临床应用来说，核酸酶的性能是一个尚未解决的问题。其定义是其包装和运输的难易程度、其在活细胞中的活性和特异性，以及竞争性 DNA 修复结果。 MegaTAL 核酸酶显示出多种有利的特性，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 突出端我们已经生成了大量的工程 MegaTAL 核酸酶，并已获得。描述了它们在原代人类细胞和转基因生物中的离体和体内表现，如总结在本项目描述的全文中。虽然所有这四个平台都正在研究并用于基因治疗，但它们的优化属性和行为（特别是通过同源驱动的校正来驱动基因修饰，而不是对于任何核酸酶来说，通过诱变末端连接破坏基因）是一个重要的持续优先事项。 DNA 结合、切割和解离（以及每个步骤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个通路正如我们的初步数据所示，这可能会导致修复结果出现显着差异。在此更新申请中，我们建议利用我们的工程核酸酶构建体和最近发表的两个具体目标的结果：（1）确定核酸酶功能的生物物理和酶学参数最强烈地影响 DNA 修复结果并通过 HDR 增强基因修饰。第一个目标的前提是个体 DNA 修复途径及其蛋白质因子具有独特的敏感性 (2)优化我们的“第二代”MegaTAL 支架（尺寸减小，并且似乎表现出改进活性和特异性）以及针对初级基因组编辑的相应 mRNA 传递系统第二个目标的总体前提是造血干细胞（HSC）的高度可变（但相当大）。 MegaTAL 及其递送系统的可控）特性特别适合评估基因组修饰的效率以及随后基因编辑原代细胞在培养物和移植和植入后。