The long term goal of this project is application of antisense oligonucleotides and antisense vectors in treatment of thalassemia. The project is based on a unique application of antisense oligonucleotides in which these compounds are used to restore correct functioning of a defective gene rather than to inhibit the expression of an undesirable gene, as commonly applied. Specifically, antisense oligonucleotides are used to reverse aberrant splicing of human beta-globin pre-mRNA by blocking splice sites activated by the mutations in intron 2 of Beta- globin gene which cause thalassemia. The oligonucleotides not only inhibit aberrant splicing but, by forcing the spliceosome to form on the adjacent correct splice sites, restore correct splicing of beta-globin pre-mRNA and in consequence restore translation of beta-globin polypeptide. In the previous granting period the effective correction of splicing in a number of thalassemic mutants was accomplished both in the cell free splicing extracts and in model cell lines which expressed thalassemic beta-globin transcripts. The main thrust of the work proposed in this application will be to establish the conditions for antisense treatment of thalassemic erythroid cells in vitro and in vivo in animal model.
The specific aims to accomplish these goals are: 1) To optimize the correction of splicing of human thalassemic beta-globin pre-mRNAs by antisense oligonucleotides in K562 cells expressing IVS2-654, IVS2-705 and IVS2-745 mutants. 2) To optimize the effects of antisense oligonucleotides on human beta-globin expression in bone marrow of the IVS2-654 mouse. 3) To treat the IVS2-654 thalassemic mice with antisense oligonucleotides either in a free form or with the aid of delivery agents. 4) To test in K-562 cell lines the correction of splicing of thalassemic beta-globin pre-mRNA by antisense vectors targeted to the aberrant splice sites. 5) To test the antisense snRNA vectors developed in Specific Aim 4 in IVS2-654 mice. To provide high level of expression and to explore the possibility of tissue tropic delivery of antisense sequences the snRNA genes will be incorporated into adeno-associated virus.
