title: Synthetic intron improves transduction efficiency of trans-splicing adeno-associated viral vectors.
abstract: 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.
authors.affiliation: Department of Molecular Microbiology and Immunology, University of Missouri, Columbia 65212, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lai
authors.forename: Yongping
authors.initials: Y
authors.lastname: Yue
authors.forename: Mingju
authors.initials: M
authors.lastname: Liu
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Duan
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000592:standards / Q000627:therapeutic use*; D007438:Introns; D008297:Male; D051379:Mice; D018101:Mice, Inbred mdx; D018482:Muscle, Skeletal / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D020040:Trans-Splicing; D014161:Transduction, Genetic / Q000379:methods* / Q000592:standards
chemical_list: D016189:Dystrophin; D012322:RNA Precursors; D012333:RNA, Messenger