Key result
Synthetic intron-based AAV vectors outperform endogenous vectors for RNA production in mdx mouse skeletal muscle.
Why the study?
The original trans-splicing AAV vector strategy requires screening endogenous introns to overcome the mRNA accumulation barrier, which is labor-intensive.
Does the use of a synthetic intron improve RNA production and transduction efficiency of trans-splicing AAV vectors in dystrophin-null mdx mouse skeletal muscle compared to endogenous introns?
Does the use of a synthetic intron improve RNA production and transduction efficiency of trans-splicing AAV vectors in dystrophin-null mdx mouse skeletal muscle compared to endogenous introns?
Optimizing intron sequences with synthetic introns can boost the transduction efficiency of trans-splicing AAV vectors, potentially improving gene therapy applications.
Answers are grounded in this paper and cite their sources.
May boost split AAV efficiency in mdx muscle; hypothesis-generating for DMD gene therapy, no practice change.
Trans-splicing adeno-associated viral (AAV) vectors hold great promise in many gene therapy applications. We have shown that rational selection of the gene-splitting site in a therapeutic target gene can lead to extremely efficient trans-splicing vectors [Lai, Y., Yue, Y., Liu, M., Ghosh, A., Engelhardt, J.F., Chamberlain, J.S., and Duan, D. (2005). Nat. Biotechnol. 23, 1435–1439]. Our original strategy requires the screening of endogenous introns that are capable of overcoming the mRNA accumulation barrier. To further develop transsplicing vectors, we have tested whether the use of a generic synthetic intron can bypass the labor-intensive intron-screening process. Two previously characterized exon/intron/exon junctions (60/60/61 and 63/63/64, respectively) in the 6 kb minidystrophin gene were used as templates to represent highly efficient (60/60/61) and relatively poor (63/63/64) gene-splitting sites. We compared RNA production from the reconstituted viral genome and transduction efficiency of the trans-splicing vectors in dystrophin-null mdx mouse skeletal muscle. Our results suggest that a synthetic intron can successfully overcome the mRNA accumulation barrier at the exon 63/64 junction. Furthermore, when the gene was split at the exon 63/64 junction, the synthetic intronbased vectors performed better than the endogenous intron-based vectors. When the gene was split at the exon 60/61 junction, we observed only nominal improvement in mRNA production. Nevertheless, vectors based on the exon 60/61 junction remain the best set in transduction efficiency. Taken together, our results suggest that optimizing intron sequence may boost the transduction efficiency of trans-splicing AAV vectors.
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Lai et al. (2006) studied Duchenne muscular dystrophy (dystrophin-null mdx mouse model). Synthetic intron-based trans-splicing AAV vectors vs. Endogenous intron-based vectors was evaluated on RNA production and transduction efficiency. Synthetic intron-based trans-splicing AAV vectors performed better than endogenous intron-based vectors when the gene was split at the exon 63/64 junction in mdx mouse skeletal muscle.
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