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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：
8629497
负责人：
BARRY L. STODDARD
金额：
$ 33.44万
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 2018-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8629497
关键词：
Address Animal Model Biological Models Cell Line Cellular Assay Cleaved cell Clinical Clustered Regularly Interspaced Short Palindromic Repeats Cystic Fibrosis DNA DNA Repair DNA Sequence Deoxyribonucleases Development Disease Engineering Epithelium Gene Conversion Gene Delivery Gene Targeting Gene-Modified Genes Genetic Recombination Genome engineering Genomics Globin Hematopoietic Hemoglobinopathies Hereditary Disease Homing Human Human Cell Line Human Genetics Human Genome In Vitro Individual Laboratories Letters Lung Medical Methods Modeling Modification Molecular Mutagenesis Outcome Pathology Patients Preparation Property Proteins Protocols documentation Reagent Recruitment Activity Repetitive Sequence Research Science Site Solid Specificity Structure Subfamily lentivirinae System Technology Testing Therapeutic Tissues Toxic effect Transfection Work Zinc Fingers arm biological systems endonuclease falls gene correction gene therapy genome-wide human disease in vivo meetings nuclease programs public health relevance repaired scaffold therapeutic target

项目摘要

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.

项目总结