Regulation of Targeted Gene Correction

靶向基因校正调控

• 批准号: 7415046
• 负责人: Eric B. Kmiec
• 金额: $ 21.04万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7415046
• 关键词: Acids; Activity Cycles; Address; Adopted; Affect; Amino Acids; Animal Model; Annexins; Antibodies; Antineoplastic Agents; Apoptosis; Apoptotic; Base Pair Mismatch; Base Pairing; Behavior; Binding; Biological Assay; Biological Models; Blood; Cell Cycle; Cell Cycle Arrest; Cell Line; Cell division; Cells; Characteristics; Chromosomal Breaks; Chromosomes; Cloning; Complement; DNA; DNA Damage; DNA Repair; DNA Sequence; DNA Sequence Rearrangement; DNA biosynthesis; Data; Daughter; Development; Disease; Doctor of Philosophy; Endothelium; Event; Experimental Designs; Frequencies; Gene Mutation; Gene Targeting; General Practices; Generations; Genes; Genetic Recombination; Genome; Genomics; Goals; Gold; Grant; Growth; HPRT1 gene; Hemophilia A; Hepatocyte; Hereditary Disease; Human; Hypoxanthine Phosphoribosyltransferase; Induction of Apoptosis; Inherited; Injection of therapeutic agent; Injury; Laboratories; Labyrinth fenestration; Lead; Lesion; Liver; Liver diseases; Location; Malignant Neoplasms; Malignant neoplasm of liver; Mammalian Cell; Measures; Methods; Modeling; Molecular; Monitor; Mucopolysaccharidosis VII; Mus; Mutagenesis; Mutation; Natural regeneration; Necrosis; Nucleotides; Numbers; Oligonucleotides; Organ; Pathway interactions; Pattern; Phase; Phenotype; Point Mutation; Population; Positioning Attribute; Process; Processed Genes; Proteins; Protocols documentation; Range; Rate; Reaction; Regulation; Relative (related person); Reporter; Reporter Genes; Resected; Resistance; Sampling; Screening procedure; Single-Stranded DNA; Site; Small Interfering RNA; Sorting - Cell Movement; Specificity; Staging; Staining method; Stains; Standards of Weights and Measures; Stimulus; Stream; Stretching; Synthetic Genes; System; Tail; Target Populations; TdT-Mediated dUTP Nick End Labeling Assay; Techniques; Terminator Codon; Testing; Therapeutic Effect; Therapeutic Uses; Thioguanine; Time; Transformed Cell Line; Veins; Week; Width; Work; Yeasts; alpha 1-Antitrypsin Deficiency; base; cancer genetics; cell type; clinical application; clinically relevant; design; gene correction; gene repair; gene therapy; homologous recombination; in vivo; insight; mutant; preference; repaired; research study; response; restriction enzyme; success; tumor

DESCRIPTION (provided by applicant): The ultimate form of gene therapy for inherited diseases is to reverse the phenotype by correcting the genetic mutation at its endogenous location in the chromosome. We have been developing a gene repair strategy that relies on DNA oligonucleotides to enter the cell, hybridize to the mutant sequence and direct single base exchanges in the target gene. During the initial grant period, we created several model systems in yeast and mammalian cells that enabled the elucidation of pathways that control the frequency of gene correction. The data indicate that the gene repair process is controlled by the activation of homologous recombination and rate at which DNA replication takes place. We now propose to transition from model systems to a clinically relevant cell type that is likely to serve as a target in the initial clinical application. Results from several laboratories indicate that liver cells, particularly hepatocytes, are highly responsive to this technique and enable gene correction to take place at robust levels. We shall target a integrated, mutant eGFP gene and the endogenous HPRT gene in clonal isolates of HepG2 and THLE cells, two established hepatocytic cell lines that have been used in the development phase of therapies aimed at liver diseases. Guided by the results of our first grant term, we will focus on the activation of homologous recombination as a means to support enhanced levels of correction in a reproducible and sustainable fashion. The experiments outlined in this grant will address the following questions; 1) are random ds breaks required for attaining high levels of gene correction?; 2) do lesions at replication forks or stalled forks themselves provide enough stimulus for elevating the levels of gene correction in the absence of DNA damage; 3) is the process of gene repair itself mutagenic at non-targeted sites and are cells undergoing gene repair more prone to genome rearrangement?; 4) how does the cell respond to the intemalization of the ssODN in terms of DNA damage response pathways. The key to developing this technique in the long term, even for liver disease and cancer, lies in the ability to regulate, predict and reliably attain correction efficiencies that have therapeutic effects. These goals support the choice of liver as a target for clinical applications of gene repair but there are other important reasons for focusing on hepatocvtes: they are the target cell for gene therapy for Alpha-1 Antitrypsin Deficiency. Crigler-Naiiar. OTC. MPSVII. Hemophilia A and B and many lysosomal storage disorders among others. Our work will uncover restrictions or limitations for gene repair in hepatocvtes with the goal of treating hepatic cancer and genetic diseases of the liver.

描述（由申请人提供）：遗传疾病基因治疗的最终形式是通过纠正其内源性位置的染色体中的遗传突变来逆转表型。我们一直在制定一种基因修复策略，该策略依赖于DNA寡核苷酸进入细胞，与突变序列杂交并直接在靶基因中直接单基碱基交换。在最初的赠款期间，我们在酵母和哺乳动物细胞中创建了几个模型系统，这些系统能够阐明控制基因校正频率的途径。数据表明，基因修复过程由同源重组和发生DNA复制的速率的激活控制。现在，我们建议从模型系统过渡到临床相关的细胞类型，该细胞类型可能在初始临床应用中作为目标。几个实验室的结果表明，肝细胞，尤其是肝细胞，对该技术有很高的反应，并使基因校正能够在稳健的水平下进行。我们将靶向HEPG2和THLE细胞克隆分离株中的集成，突变的EGFP基因和内源性HPRT基因，这是两种已建立的肝细胞细胞系，这些细胞系已在针对肝病的疗法的发育阶段使用。在我们第一个赠款术语的结果的指导下，我们将重点关注同源重组的激活，以此作为支持可重复且可持续的方式增强校正水平的手段。该赠款中概述的实验将解决以下问题； 1）获得高水平的基因校正需要随机DS断裂吗？ 2）在复制叉或失速的叉子本身时，在没有DNA损伤的情况下提供了足够的刺激，以提高基因校正水平； 3）基因修复本身在非靶向位点的诱变过程，并且经过基因修复的细胞是否更容易重排基因组重排？ 4）细胞如何根据DNA损伤响应途径对SSODN的幻想反应。从长远来看，即使是肝病和癌症，也可以开发这种技术的关键，即具有调节，预测和可靠达到具有治疗作用的校正效率的能力。这些目标支持选择肝脏作为基因修复的临床应用的靶标，但还有其他重点关注肝的原因：它们是α-1抗胰蛋白酶缺乏症基因治疗的靶细胞。 crigler-noiar。 OTC。 mpsvii。血友病A和B以及许多溶酶体储存障碍。我们的工作将发现肝脏中基因修复的限制或局限性，目的是治疗肝癌和肝脏的遗传疾病。