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Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes

Gene Therapy volume 8, pages 1323–1332 (2001)Cite this article

Abstract

This study compared a range of mammalian CNS expression cassettes in recombinant adeno-associated virus (AAV-2) vectors using strong endogenous promoter sequences, with or without a strong post-regulatory element and polyadenylation signal. Changes in these elements led to transgene expression varying by over three orders of magnitude. In experiments conducted in primary cell culture and in >100 stereotactically injected rats, we observed highly efficient and stable (>15 months) gene expression in neurons and limited expression in glia; the highest expression occurred with endogenous, nonviral promoters such as neuron-specific enolase and β-actin. The packaging size of AAV-2 was maximized at 5.7 kb without impairing gene expression, as judged by direct comparison with a number of smaller AAV-2 constructs. The genomic insert size and titer were confirmed by Southern blot and quantitative PCR, and infectivity was tested by particle titer using ELISA with a conformation-dependent epitope that requires the full intact capsid. A packaging and purification protocol we describe allows for high-titer, high-capacity AAV-2 vectors that can transduce over 2 × 105 neurons in vivo per microliter of vector, using the strongest expression cassette.

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Acknowledgements

We would like to thank the following individuals for kindly providing gene constructs and plasmids: the human enkephalin promoter was provided by Dr M Comb; the NF promoters were donated by Dr J-P Julien; the human nAChR promoter was donated by Drs A Bessis and J-P Changeux at Institut Pasteur; and the human EF promoter was donated by Dr A Shibui. We would also like to thank S McPhee for his help with the digital images.

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  1. R Xu and CG Janson: These authors contributed equivalently

Authors and Affiliations

  1. Department of Molecular Medicine, University of Auckland Medical School, Auckland, New Zealand

    R Xu, M Mastakov, P Lawlor, D Young, A Mouravlev, H Fitzsimons, K-L Choi, H Ma, M Dragunow, Q Chen, B Dicker & MJ During

  2. Yale University School of Medicine, Molecular Pharmacology and Neurogenetics Laboratory, New Haven, CT, USA

    CG Janson, P Leone & MJ During

  3. Jefferson Medical College, CNS Gene Therapy Center, Philadelphia, PA, USA

    R Xu, CG Janson, M Mastakov, P Lawlor, D Young, A Mouravlev, H Fitzsimons, P Leone & MJ During

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Xu, R., Janson, C., Mastakov, M. et al. Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes. Gene Ther 8, 1323–1332 (2001). https://doi.org/10.1038/sj.gt.3301529

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