Abstract
Purpose :
Improving the efficacy of U1 snRNA (U1)-based therapeutic approaches applying a combination therapy with antisense oligonucleotides (AONs).
Methods :
We applied lentiviral shuttles to transduce patient-derived human skin fibroblasts with designed U1 splice factors. Six AONs were tested for their efficacy to block unwanted intron retention splice variants in BBS1 transcripts. Apoptosis assays were performed to evaluate safety of the AONs.
Results :
We previously identified a family with retinitis pigmentosa caused by a BBS1 splice donor site mutation (c.479G>A, p.R160Q). To correct the mutation-induced splice defects, we designed a U1 splice factor that efficiently binds to the mutated BBS1 splice donor site. The therapeutic effect of the designed U1 was tested in patient-derived and family control skin fibroblasts. Although exon skipping was efficiently reverted to normal BBS1 splicing applying the designed U1, intron retention was not corrected. We tested the efficacy of 6 different AONs to block the intron retention and identified 2 AONs which prevented the generation of the intron retained splice variant. The combination of designed U1 and AON treatments presented with the highest therapeutic efficacy and clearly increased the concentration of correctly spliced BBS1 transcripts. We did not detect elevated levels of apoptotic cell death in AON-treated patient-derived or control cell lines.
Conclusions :
Correcting disease-associated splice variants using a combination of designed U1 and AONs is an efficient therapeutic approach. Technologies to manipulate splice variants are well-established and provide promising possibilities in genetic therapy development.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.