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Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors

Nature Biotechnology volume 23, pages 1435–1439 (2005)Cite this article

Abstract

Although adeno-associated virus (AAV)-mediated gene therapy has been hindered by the small viral packaging capacity of the vector, trans-splicing AAV vectors are able to package twice the size of the vector genome. Unfortunately, the efficiency of current trans-splicing vectors is very low. Here we show that rational design of the gene splitting site has a profound influence on trans-splicing vector-mediated gene expression. Using mRNA accumulation as a guide, we generated a set of efficient trans-splicing vectors and achieved widespread expression of the 6-kb ΔH2-R19 mini-dystrophin gene in skeletal muscle of mdx mice, a model for Duchenne muscular dystrophy. The dystrophic phenotype was ameliorated in both adult and aged mice. This demonstrates the use of trans-splicing vectors to efficiently express a large therapeutic structural protein. This strategy should be applicable to other large therapeutic genes or large transcription regulatory elements.

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Figure 1: Endogenous splicing signal screening.
Figure 2: ΔH2 mini-dystrophin expression from two independent sets of trans-splicing AAV vectors.
Figure 3: Widespread expression of mini-dystrophin in AV.Donor.60 and AV.Acceptor.60 co-infected mdx muscle attenuates dystrophic pathology.

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References

  1. Duan, D., Yue, Y. & Engelhardt, J.F. in Lung Biology in Health and Disease, Gene Therapy in Lung Disease (ed. Albelda, S.M.) 51–92 (Marcel Dekker Inc., New York, NY, 2002).

    Book  Google Scholar 

  2. Kay, M.A. et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 24, 257–261 (2000).

    Article  CAS  Google Scholar 

  3. Carter, B.J. Adeno-associated virus vectors in clinical trials. Hum. Gene Ther. 16, 541–550 (2005).

    Article  CAS  Google Scholar 

  4. Dong, J.Y., Fan, P.D. & Frizzell, R.A. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum. Gene Ther. 7, 2101–2112 (1996).

    Article  CAS  Google Scholar 

  5. Duan, D. et al. Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long term episomal persistence in muscle. J. Virol. 72, 8568–8577 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Duan, D., Yan, Z., Yue, Y. & Engelhardt, J.F. Structural analysis of adeno-associated virus transduction intermediates. Virology 261, 8–14 (1999).

    Article  CAS  Google Scholar 

  7. Yue, Y. & Duan, D. Double strand interaction is the predominant pathway for intermolecular recombination of adeno-associated viral genomes. Virology 313, 1–7 (2003).

    Article  CAS  Google Scholar 

  8. Yan, Z., Zhang, Y., Duan, D. & Engelhardt, J.F. Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. Proc. Natl. Acad. Sci. USA 97, 6716–6721 (2000).

    Article  CAS  Google Scholar 

  9. Sun, L., Li, J. & Xiao, X. Overcoming adeno-associated virus vector size limitation through viral DNA heterodimerization. Nat. Med. 6, 599–602 (2000).

    Article  CAS  Google Scholar 

  10. Nakai, H., Storm, T.A. & Kay, M.A. Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors. Nat. Biotechnol. [see comments] 18, 527–532 (2000).

    Article  CAS  Google Scholar 

  11. Duan, D., Yue, Y. & Engelhardt, J.F. Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison. Mol. Ther. 4, 383–391 (2001).

    Article  CAS  Google Scholar 

  12. Chao, H., Sun, L., Bruce, A., Xiao, X. & Walsh, C.E. Expression of human factor VIII by splicing between dimerized AAV vectors. Mol. Ther. 5, 716–722 (2002).

    Article  CAS  Google Scholar 

  13. Reich, S.J. et al. Efficient trans-splicing in the retina expands the utility of adeno-associated virus as a vector for gene therapy. Hum. Gene Ther. 14, 37–44 (2003).

    Article  CAS  Google Scholar 

  14. Yan, Z., Zak, R., Zhang, Y. & Engelhardt, J.F. Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes. J. Virol. 79, 364–379 (2005).

    Article  CAS  Google Scholar 

  15. Xu, Z. et al. Trans-splicing adeno-associated viral vector-mediated gene therapy is limited by the accumulation of spliced mRNA but not by dual vector coinfection efficiency. Hum. Gene Ther. 15, 896–905 (2004).

    Article  CAS  Google Scholar 

  16. England, S.B. et al. Very mild muscular dystrophy associated with the deletion of 46% of dystrophin. Nature 343, 180–182 (1990).

    Article  CAS  Google Scholar 

  17. Harper, S.Q. et al. Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat. Med. 8, 253–261 (2002).

    Article  CAS  Google Scholar 

  18. Sironi, M. et al. Analysis of splicing parameters in the dystrophin gene: relevance for physiological and pathogenetic splicing mechanisms. Hum. Genet. 109, 73–84 (2001).

    Article  CAS  Google Scholar 

  19. Chao, D.S. et al. Selective loss of sarcolemmal nitric oxide synthase in Becker muscular dystrophy. J. Exp. Med. 184, 609–618 (1996).

    Article  CAS  Google Scholar 

  20. Cartegni, L., Chew, S.L. & Krainer, A.R. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. 3, 285–298 (2002).

    Article  CAS  Google Scholar 

  21. Ibrahimel, C., Schaal, T.D., Hertel, K.J., Reed, R. & Maniatis, T. Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers. Proc. Natl. Acad. Sci. USA 102, 5002–5007 (2005).

    Article  Google Scholar 

  22. Li, X. & Manley, J.L. Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability. Cell 122, 365–378 (2005).

    Article  CAS  Google Scholar 

  23. Halbert, C.L., Allen, J.M. & Miller, A.D. Adeno-associated virus type 6 (aav6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of aav2 vectors. J. Virol. 75, 6615–6624 (2001).

    Article  CAS  Google Scholar 

  24. Duan, D., Yue, Y., Yan, Z. & Engelhardt, J.F. A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation. Nat. Med. 6, 595–598 (2000).

    Article  CAS  Google Scholar 

  25. Liu, M. et al. Adeno-associated virus-mediated micro-dystrophin expression protects young mdx muscle from contraction-induced injury. Mol. Ther. 11, 245–256 (2005).

    Article  CAS  Google Scholar 

  26. Yue, Y. et al. Microdystrophin gene therapy of cardiomyopathy restores dystrophin-glycoprotein complex and improves sarcolemma integrity in the mdx mouse heart. Circulation 108, 1626–1632 (2003).

    Article  CAS  Google Scholar 

  27. Yue, Y., Skimming, J.W., Liu, M., Strawn, T. & Duan, D. Full-length dystrophin expression in half of the heart cells ameliorates beta-isoproterenol-induced cardiomyopathy in mdx mice. Hum. Mol. Genet. 13, 1669–1675 (2004).

    Article  CAS  Google Scholar 

  28. Ervasti, J.M. & Campbell, K.P. Membrane organization of the dystrophin-glycoprotein complex. Cell 66, 1121–1131 (1991).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Dusty Miller for the AAV-6 packaging plasmids. We thank Scott Q. Harper and Zhuping Xu for helpful discussion and technical assistance. This work was supported by grants from the National Institutes of Health (AR-49419, D.D.) and the Muscular Dystrophy Association (D.D. and J.S.C.).

Author information

Authors and Affiliations

  1. Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, 1 Hospital Dr., Room M610G, MSB, Columbia, 65212, Missouri, USA

    Yi Lai, Yongping Yue, Mingju Liu, Arkasubhra Ghosh & Dongsheng Duan

  2. Department of Anatomy and Cell Biology, University of Iowa, 51 Newton Rd., Room 1-111, BSB, Iowa City, 52242, Iowa, USA

    John F Engelhardt

  3. Department of Neurology, University of Washington School of Medicine, 1959 NE Pacific St., Room K2438 HSB, Seattle, 98195, Washington, USA

    Jeffrey S Chamberlain

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  1. Yi Lai
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  7. Dongsheng Duan
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Corresponding author

Correspondence to Dongsheng Duan.

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Supplementary information

Supplementary Fig. 1

Adult mdx mice used in this study displayed more severe muscle pathology. (PDF 312 kb)

Supplementary Fig. 2

Mini-dystrophin expression is observed only in AV.Donor.60 and AV.Acceptor.60 co-infected muscle but not in single vector infected muscle. (PDF 283 kb)

Supplementary Fig. 3

Absence of CD4+ and CD8+ T cells in AV.Donor.60 and AV.Acceptor.60 co-infected muscle. (PDF 230 kb)

Supplementary Fig. 4

Mini-dystrophin expression restores the entire DGC complex but not nNOS to the sarcolemma. (PDF 488 kb)

Supplementary Fig. 5

AV.Donor.60 and AV.Acceptor.60 co-infection in aged mdx EDL muscle does not reduce muscle pathology neither does it increase specific force. (PDF 593 kb)

Supplementary Fig. 6

AAV-6 infects all muscle fiber types, but with a trend towards preferential transduction of fast fibers, in dystrophic mdx limb muscle. (PDF 806 kb)

Supplementary Methods (PDF 144 kb)

Supplementary Note (PDF 57 kb)

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Lai, Y., Yue, Y., Liu, M. et al. Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors. Nat Biotechnol 23, 1435–1439 (2005). https://doi.org/10.1038/nbt1153

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