The long term goal of this project is to assess the potential of antisense oligonucleotides in treatment of genetic defects. A novel application of antisense oligonucleotides is proposed, in which these compounds are used to restore correct functioning of a defective gene rather than for inhibition of expression of an undesirable gene, as is commonly used. Specifically, they are used to reverse aberrant splicing caused by mutations seen in a number of genetic diseases and in certain cancers. In this granting period the specific experimental system to be studied will be a subset of mutations in human beta-globin gene which cause thalassemia via-aberrant splicing of beta-globin pre-mRNA. The proposed approach stems from our recent observations that antisense 2'-O-methyl- ribooligonucleotides are able to inhibit aberrant splicing induced by thalassemic mutations and restore correct splicing in cell free splicing extracts.
The specific aims are intended to extend these results towards the long term goal of the project: 1) To optimize the effects of antisense oligonucleotides on aberrant splicing in vitro, in cell free extracts. The exact position of the targeted oligonucleotides as well as their length will be varied to obtain the highest efficacy of restoration of correct splicing. The targeted thalassemic mutations will include those studied recently i.e., IVS1-110 and IVS2-654 as well as others, such as those causing aberrant splicing at the 5' splice site (IVS1-5, IVS1-6). 2) To test the most effective antisense oligonucleotides identified in Specific Aim 1 in tissue culture cells. These will include HeLa or CHO cells stably transfected with thalassemic globin genes cloned under the CMV promoter and human x mouse hybrids expressing appropriate thalassemic beta-globin pre-mRNA. The oligonucleotides that will be most effective in reversal of aberrant splicing (presumably due to their efficient uptake by the cells and/or optimal targets) will be identified. 3) To test whether oligonucleotides other than 2'-O-methyl-oligonucleotides have better ability to restore correct splicing in vitro and in vivo. Initially 2'-O- methyl-phosphorothioates and methylphosphonates will be used. These modifications may improve the stability and/or the efficiency of cellular uptake of the oligonucleotides and will result in their increased activity.
