1063. Optimizing Intron Splicing Signal Overcomes the mRNA Accumulation Barrier in Trans-Splicing AAV Vectors

Trans-splicing adeno-associated viral (AAV) vectors hold great promise in many gene therapy applications. We have recently shown that rational selection of the gene splitting site and the endogenous intron sequence can lead to extremely efficient trans-splicing vectors compatible to that of a single intact AAV vector (Lai et al Nature Biotechnology 23:1435, 2005). A key factor in constructing effective trans-splicing vectors is the mRNA production from the reconstituted viral genome. In the dystrophin gene (a therapeutic gene for Duchenne muscular dystrophy), the 60/60/61 (exon/intron/exon) junction yields the highest mRNA level while the 63/63/64 junction is the most efficiently spliced junction when tested in the reconstituted trans-splicing viral genome. However, only vectors based on the 60/60/61 junction can produce therapeutic level protein. Many endogenous introns carry sub-optimal splicing signals. It is not clear whether optimizing intron sequence can further improve trans-splicing AAV vectors. In this study, we evaluated the effect of replacing the endogenous intron with a synthetic intron that matches perfectly with the conserved motif. We first generated two additional constructs p60/synthetic/61 and p63/synthetic/64. In these constructs, the endogenous introns were replaced by the synthetic intron in p60/60/61 and p63/63/64, respectively. Similar to the parent constructs, the newly synthesized constructs also carried the double- D inverted terminal repeat junction in the middle of the intron. RNase protection assay (RPA) was used to determine the level of unspliced and spliced RNA, and the relative splicing efficiency. At 48 hrs after transfection, RNA was extracted for RPA. The splicing indices in intron 60 are quite compatible to those of the conserved motif. Substitution of the endogenous intron with the synthetic intron resulted in marginal but statistically not significant improvement. The mRNA level in p60/60/61 was 236.5 |[plusmn]| 132.5 (relative unit) and it was 300.2 |[plusmn]| 86.6 in p60/synthetic/61 (p >0.05). However, the same replacement in the 63/64 exon junction led to a three-fold increase in accumulated mRNA from 124.3 |[plusmn]| 5.8 to 362.4 |[plusmn]| 32.3 (p < 0.05). We next examined the relative splicing efficiency. Interestingly, the synthetic intron did not augment the already efficient splicing in the 63/63/64 junction. However, the relatively poor splicing in p60/60/61 was improved. The ratio of spliced to unspliced RNA increased from 2.8 |[plusmn]| 0.3 in p60/60/61 to 7.7 |[plusmn]| 1.9 in p60/synthetic/61. The percentage of splicing also increased from 72.6 % to 85.2 %.To further explore the molecular mechanisms, we quantified the pre-mRNA stability. At 48 hrs after transfection, actinomycin D was added to cell culture to stop denovo RNA synthesis. RNA was extracted at 0, 0.5, 1 and 3 hrs after actinomycin D treatment. In these studies, we observed increased pre- mRNA stability in both 60/synthetic/61 and 63/synthetic/64 transcripts. Taken together, our results suggest that optimizing intron sequence may boost the transduction efficiency of trans-splicing AAV vectors.