Ribozyme-Mediated Repair of Sickle Beta-Globin RNA and DNA

这一建议的总体目标是探索第一组和第二组内含子修复的能力 突变的β-珠蛋白基因和转录本，并评估这些分子在人类细胞中的潜在用途。这些内含子引起了极大的科学兴趣，因为它们能够执行催化作用，而且这些RNA酶的一个亚类可以作为可移动的遗传元件。此外，它们通过正向和反向剪接反应修改RNA和DNA序列的能力使翻译研究人员对这些内含子特别感兴趣。在此之前，我们证明了反式剪接第一组核酶可以将镰刀状β-珠蛋白编码的mRNAs转化为伽玛珠蛋白编码的转录本，然后将核酶瞬时转染到来自镰状细胞病患者的红细胞前体细胞。此外，我们已经证明，在293细胞中，这种RNA修复可以以低到中等的效率进行(高达50%的修复)，这些细胞与核酶和镰刀状β-珠蛋白表达盒共转染。最近，我们已经证明了乳酸乳球菌第二组内含子可以反向剪接并将其自身特异性地插入到所需的DNA靶标序列中。这些概念证明研究表明，这种催化RNA可能代表可以用来修改遗传指令的分子，以达到治疗镰状细胞疾病和其他遗传疾病的目的。这些研究还强调了进一步评估和优化这些催化RNA的必要性，如果它们要成为治疗有用的。在这里，我们建议对人类细胞中第一组和第二组内含子的活性进行更详细的分析，重点是突变的β-珠蛋白转录本和基因的修复。这些研究的完成将建立必要的实验基础，在此基础上进行治疗镰状细胞疾病和其他遗传疾病的治疗第一组和第二组核酶的合理开发。

The overall goal of this proposal is to explore the ability of group I and group II introns to repair mutant beta-globin genes and transcripts and assess the potential utility of these molecules in human cells. These introns have been of great scientific interest because they are able to perform catalysis and because a subclass of these RNA enzymes can act as mobile genetic elements. Moreover, their ability to modify RNA and DNA sequences through forward and reverse-splicing reactions makes these introns of particular interest to translational researchers. Previously, we demonstrated that trans-splicing group I ribozymes can convert sickle beta-globin encoding mRNAs into gamma-globin encoding transcripts following transient transfection of the ribozyme into erythrocyte precursors derived from patients with sickle cell disease. In addition, we have demonstrated that such RNA repair can proceed with low to moderate efficiency (up to 50% repair) in 293 cells cotransfected with ribozyme and sickle beta-globin expression cassettes. More recently, we have demonstrated that the Lactococcus lactis group II intron can reverse-splice and site specificallv insert itself into desired DNA target sequences in transfected human cells. These proof of concept studies suggest that such catalytic RNAs may represent molecules that can be employed to modify genetic instructions for therapeutic ends to treat sickle cell disease and other genetic disorders. These studies also underscore the necessity for further evaluation and optimization of these catalytic RNAs if they are to become therapeutically useful. Here we propose to perform more detailed analyses of group I and group II intron activity in human ceils focusing upon repair of mutant beta-globin transcripts and genes. The completion of these studies will establish the needed experimental foundation from which the logical development of therapeutic group I and group II ribozymes for the treatment of sickle cell disease and other genetic disorders can proceed.