Correction of globin gene mutations by gene targeting

描述(申请人提供)：贝塔地中海贫血症和镰状细胞性贫血是一种严重的疾病，对大多数患者来说会导致相当大的发病率和早期死亡。传统疗法可以延长生命，但不能治愈这些疾病。基因治疗尚未见效，因为当β-珠蛋白基因随机整合到宿主细胞基因组中时，它们的输出会有很大的差异。此外，这种方法会导致基因组的随机突变，这可能会导致癌基因的失调。出于这些原因，对突变的β-珠蛋白基因的特异性纠正可能代表着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，在这种重组中，野生型DNA序列可以取代特定位点上的突变基因。到目前为止，这个过程还没有被用于基因治疗试验，因为它的效率非常低。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至关重要的蛋白质，它们可能会更有效地进行同源重组。在这项提案中，我们将确定在胚胎干细胞中是否有任何同源重组蛋白对这一过程具有限速作用，并将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对突变的人β-珠蛋白基因进行同源重组介导的基因“校正”。

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.