Correction of globin gene mutations by gene targeting

描述（由申请人提供）：β-地中海贫血和镰状细胞贫血是严重的疾病，导致相当大的发病率和早期死亡的大多数患者。传统的治疗方法可以延长生命，但不能治愈这些疾病。基因治疗尚未有效，这是由于当β-珠蛋白基因随机整合到宿主细胞基因组中时，它们的输出变化很大。此外，这种方法会导致基因组中的随机突变，这可能会导致致癌基因的失调。由于这些原因，突变β-珠蛋白基因的特异性校正可能代表基因治疗的最佳手段。细微的突变可以通过同源重组来纠正，其中野生型DNA序列可以取代特定基因座中的突变基因。迄今为止，这个过程还没有用于基因治疗试验，因为它效率极低。我们实验室最近的实验表明，如果给予胚胎干细胞额外数量的对同源重组过程重要的蛋白质，它们可能会更有效地进行同源重组。在这个提议中，我们将确定是否有任何同源重组蛋白质是胚胎干细胞中这一过程的限速蛋白，我们将把这一信息扩展到原代骨髓细胞。我们希望最终确定这种方法是否可以用于执行同源重组介导的基因“校正”突变的人类β-珠蛋白基因。

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.