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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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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

Authors
  1. Yi Lai
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  2. Yongping Yue
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  7. Dongsheng Duan
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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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