Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes
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 beta-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 x 10(5) neurons in vivo per microliter of vector, using the strongest expression cassette.
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WPRE contributes to strong expression of transgenes in AAV systems.
“WPRE is a 600 bp long tripartite element containing gamma, alpha, and beta elements, in the given order [16] and contributes to the strong expression of transgenes in AAV systems [11-13].”
From Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons (2014) · cites this paper as [13]
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- Authors
- Xu, R., Janson, C., Mastakov, M., Lawlor, P., Young, D., Mouravlev, A., Fitzsimons, H., Choi, K.-L., Ma, H., Dragunow, M., Leone, P., Chen, Q., Dicker, B., & During, M.
- Affiliations
- University of Auckland (NZ)
- Yale University (US)
- Venue
- Gene Therapy
- Published
- 2001-09-01
- Volume
- 8
- Issue
- 17
- Pages
- 1323-1332
- ISSN
- 0969-7128 (ISSN-L)
- 1476-5462
- OpenAlex
- W2321725396
- Cited by
- 174
- References
- 4
- Citations by year
- 2025: 4 · 2024: 3 · 2023: 6 · 2022: 2 · 2021: 7 · 2020: 7 · 2019: 7 · 2018: 6 · 2017: 5 · 2016: 4 · 2015: 4 · 2014: 7 · 2013: 5 · 2012: 6
- FWCI
- 6.88
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Gene Therapy (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
- Keywords
- adeno-associated virus type 2, brain-specific expression, neuron-specific enolase promoter, beta-actin promoter, post-transcriptional regulation, polyadenylation signal, vector titer, vector purification, vector infectivity, stereotactic injection, transgene stability, neuronal transduction, glial expression
- MeSH terms
- Dependovirus, Animals, Brain, Cells, Cultured, Genetic Vectors, Injections, Luciferases, Blotting, Southern, Blotting, Western, Genetic Therapy, Gene Expression, Rats, Wistar, Rats
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