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J. Neidhardt, G. Korner, E. Glaus, W. Berger; Novel Gene Therapeutic Approach to Rescue Splice Defects. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3113.
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Retinal degenerations and other genetic diseases are often caused by splice mutations. Their frequency is estimated to be approximately 20% of all mutations. Previously we reported a highly efficient rescue of splice defects caused by a mutation of the last base of an exon. Our approach was the adaptation of the splice factor U1snRNA (Tanner et al., Hum Mutat. 2009). As presented here, we addressed questions of more general applicability of our approach by studying the therapeutic potential of U1snRNA for other splice donor site mutations.
Nine different minigenes were generated that contain single mutations at each position of a splice donor site within the Bardet Biedl syndrome 1 (BBS1) gene. The splice factor U1snRNA was adapted to match the minigene mutations. Co-transfection in cell lines and RT-PCR were used to analyze RNA splice patterns.
We found that the majority of splice donor site mutations can be rescued by adaptation of U1snRNA. The splice defects are reverted to normal splicing, thus circumventing the fatal consequences of the mutations. In addition, we show that sequence alterations at highly conserved positions are more difficult to treat and may cause irreversible splice defects. Apparent side effects on transcripts of the wild type minigene have not been observed.
The adaptation of U1snRNA to rescue mutations at splice donor sites is a promising gene therapeutic approach that has the potential to treat patients with inherited eye diseases and other splice defect-associated disorders.
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