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Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo

Gene Therapy volume 10, pages 2112–2118 (2003)Cite this article

Abstract

An important limitation of recombinant adeno-associated virus (rAAV) vector efficiency is the requirement of hostcell-mediated synthesis of double-stranded DNA from the single-stranded genome. We have bypassed this step in a specialized self-complementary rAAV (scAAV) vector, by utilizing the tendency of AAV to package DNA dimers when the replicating genome is half the length of the wild type (wt). To produce these vectors efficiently, we have deleted the terminal resolution site (trs) from one rAAV TR, preventing the initiation of replication at the mutated end. These constructs generate single-stranded, inverted repeat genomes, with a wt TR at each end, and a mutated TR in the middle. After uncoating, the viral DNA folds through intramolecular base pairing within the mutant TR, which then proceeds through the genome to form a double-stranded molecule. We have used the scAAV to investigate barriers to rAAV transduction in the mouse liver, muscle and brain. In each tissue, scAAV was characterized by faster onset of gene expression and higher transduction efficiency. This study confirms earlier predictions that complementary-strand DNA synthesis is the primary barrier to rAAV-2 transduction. The scAAV is unaffected by this barrier, and provides an extremely efficient vector for gene transfer into many types of cells in vivo.

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Acknowledgements

We thank Jenifer Naspinski for technical assistance. This work was supported by NIH Grants PO1-HL66973-01 and PO1-HL51818-06. PEM was supported in part by NIH HL 03960.

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Authors and Affiliations

  1. Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    D M McCarty & C E Toulson

  2. Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    H Fu & P E Monahan

  3. Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    D M McCarty, P E Monahan, P Naik & R J Samulski

  4. Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    R J Samulski

  5. Department of Orthopedic Surgery, Johns Hopkins University, Baltimore, MD, USA

    C E Toulson

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  1. D M McCarty
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  2. H Fu
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  3. P E Monahan
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  4. C E Toulson
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  5. P Naik
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  6. R J Samulski
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McCarty, D., Fu, H., Monahan, P. et al. Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Ther 10, 2112–2118 (2003). https://doi.org/10.1038/sj.gt.3302134

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