Correction of globin gene mutations by gene targeting

通过基因打靶纠正珠蛋白基因突变

• 批准号: 6904426
• 负责人: Zhi Hong Lu
• 金额: $ 5.35万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6904426
• 关键词: Lentivirus; biotechnology; bone marrow; cell line; complementary DNA; embryonic stem cell; gene mutation; gene targeting; gene therapy; globin; hematopoietic stem cells; hypoxanthine phosphoribosyltransferase; laboratory mouse; postdoctoral investigator; sickle cell anemia; thalassemia; transfection /expression vector

DESCRIPTION (provided by applicant): Beta-thalassaemia and sickle cell anemia are severe illnesses that result in considerable morbidity and early mortality for most patients. Conventional therapy prolongs life, but does not cure these disorders. Gene therapy has not yet been effective, due to large variations in the outputs of beta -globin genes when they are randomly integrated into host cell genomes. Furthermore, this approach causes random mutations in the genome, which could potentially lead to the disregulation of oncogenes. For these reasons, the specific correction of mutant beta -globin genes may represent the optimal means for gene therapy. Subtle mutations can be corrected with homologous recombination, where wild type DNA sequences can replace mutant genes in a specific locus. This process has not been used in gene therapy trials to-date, because it is extremely inefficient. Recent experiments in our lab suggest that embryonic stem cells might perform homologous recombination more efficiently if they are given extra amounts of the proteins important for the homologous recombination process. In this proposal, we will determine whether any of the homologous recombination proteins are rate-limiting for this process in embryonic stem cells, and we will extend that information to primary bone marrow cells. We hope to ultimately determine whether this approach could be used to perform homologous recombination-mediated gene "correction" of mutant human beta -globin genes.

描述（由申请人提供）：β-丘脑贫血和镰状细胞性贫血是严重疾病，导致大多数患者的发病率和早期死亡率相当大。常规疗法延长了寿命，但不能治愈这些疾病。由于基因疗法在β-珠蛋白基因的输出中发生较大差异时，尚未有效。此外，这种方法在基因组中导致随机突变，这可能导致癌基因的脱离。由于这些原因，突变β-珠蛋白基因的特定校正可能代表基因治疗的最佳手段。微妙的突变可以通过同源重组来校正，其中野生型DNA序列可以替代特定基因座中的突变基因。该过程尚未用于迄今为止的基因治疗试验，因为它极低效率。我们实验室中的最新实验表明，如果给出额外量的蛋白质对同源重组过程很重要，则胚胎干细胞可能会更有效地进行同源重组。在此提案中，我们将确定任何同源重组蛋白在胚胎干细胞中是否限制了速率，我们将将该信息扩展到原代骨髓细胞。我们希望最终确定该方法是否可以用于执行突变人β-珠蛋白基因的同源重组介导的基因“校正”。