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.
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