summary
The development of trans-splicing vectors opens the door for delivering a large therapeutic gene with adeno-associated viral vectors (AAV). One potential application is to deliver the 6 kb mini-dystrophin gene for Duchenne muscular dystrophy (DMD) gene therapy. However, early attempts have been very disappointing because of low transduction efficiency. We have recently identified mRNA accumulation as a critical barrier for the trans-splicing AAV vectors. This barrier can be overcome by rational selection of the gene splitting site. Here we outline a detailed RNase protection assay-based strategy to determine the optimal gene splitting site for the mini-dystrophin gene. We also provide methods to evaluate transduction efficiency of the mini-dystrophin trans-splicing vectors in mdx mouse, a model for DMD.
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Acknowledgments
We thank Dr. Jeffrey Chamberlain for providing the p\(\Delta\)H2-R19 mini-dystrophin plasmid. We also thank Dr. A. Dusty Miller for the AAV-6 packaging plasmids (pMT-Rep2 and pCMVCap6). This work is supported by grants from the National Institutes of Health (AR-49419, DD) and the Muscular Dystrophy Association (DD). We thank Mr. Arka Ghosh for optimizing AAV-6 preparation protocol. We thank Ms. Chun Long for animal care and Mr. Brian Bostick for helpful discussion.
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Lai, Y., Li, D., Yue, Y., Duan, D. (2008). Design of Trans-Splicing Adeno-Associated Viral Vectors for Duchenne Muscular Dystrophy Gene Therapy. In: Gene Therapy Protocols. Methods in Molecular Biology™, vol 433. Humana Press. https://doi.org/10.1007/978-1-59745-237-3_16
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DOI: https://doi.org/10.1007/978-1-59745-237-3_16
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