Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice
Open accessAbstract
Vectors derived from adeno-associated virus (AAV) are promising for human gene therapy, including treatment for retinal blindness. One major limitation of AAVs as vectors is that AAV cargo capacity has been considered to be restricted to 4.7 kb. Here we demonstrate that vectors with an AAV5 capsid (i.e., rAAV2/5) incorporated up to 8.9 kb of genome more efficiently than 6 other serotypes tested, independent of the efficiency of the rAAV2/5 production process. Efficient packaging of the large murine Abca4 and human MYO7A and CEP290 genes, which are mutated in common blinding diseases, was obtained, suggesting that this packaging efficiency is independent of the specific sequence packaged. Expression of proteins of the appropriate size and function was observed following transduction with rAAV2/5 carrying large genes. Intraocular administration of rAAV2/5 encoding ABCA4 resulted in protein localization to rod outer segments and significant and stable morphological and functional improvement of the retina in Abca4(-/-) mice. This use of rAAV2/5 may be a promising therapeutic strategy for recessive Stargardt disease, the most common form of inherited macular degeneration. The possibility of packaging large genes in AAV greatly expands the therapeutic potential of this vector system.
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Systematic screens to mutagenize AAV capsids and evaluate whether amino acid changes may improve the packaging abilities of AAVs have been conducted.
“However, systematic screens to mutagenize AAV capsids and evaluate whether amino acid changes may improve the packaging abilities of AAVs have been conducted [22].”
From Circumventing the packaging limit of AAV-mediated gene replacement therapy for neurological disorders (2022) · cites this paper as [22]
AAV5 vectors with supposedly 8.9 kb genomes resulted in expression of ABCA4, MYO7A, and CEP290 from genomes whose intact genome length would be 8.9, 8.1, and 8.9 kb, respectively.
“Nonetheless, AAV5vectorswith the supposedly 8.9kb large viral genomes clearly did result in expression of ABCA4, MYO7A, and CEP290 from genomes whose intact genome length would be 8.9, 8.1, and 8.9 kb in size, respectively.”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [29]
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“In 2008, Allocca et al. [2] attempted to package much larger genomes of up to 8.9 kb and given the positive outcomes of their strategy, assumed the packaging to have been successful.”
BackgroundInfluentialInnovative therapies for inherited retinal dystrophies: navigating DNA, RNA, and protein approaches
Gómez-Escribano, A. P., García-García, G., Pérez-Santamarina, E., Aller-Mañas, E., Vázquez-Manrique, R. P., & Millán-Salvador, J. M. (2025) · eBioMedicine · doi:10.1016/j.ebiom.2025.105751 · Cited by 6 · see citing works
“…vectors like rAAV2/5 (containing internal terminal repeats from AAV2 and the capsid from AAV5) can ef fi ciently package sequences of up to 8.9 kb. 26 Alternative strategies for delivering large genes involve dual-viral systems, which use the same or different viral vectors to package two gene…”
Influential
- Authors
- Allocca, M., Doria, M., Petrillo, M., Colella, P., Garcia-Hoyos, M., Gibbs, D., Kim, S. R., Maguire, A., Rex, T. S., Di Vicino, U., Cutillo, L., Sparrow, J. R., Williams, D. S., Bennett, J., & Auricchio, A.
- Affiliations
- Telethon Institute Of Genetics And Medicine (IT)
- University of California San Diego (US)
- Columbia University (US)
- University of Pennsylvania (US)
- Federico II University Hospital (IT)
- Corresponding author
- Auricchio, Alberto
- ORCID
- Venue
- Journal of Clinical Investigation
- Published
- 2008-05-01
- Volume
- 118
- Issue
- 5
- Pages
- 1955-1964
- ISSN
- 0021-9738 (ISSN-L)
- PMC
- PMC2298836
- OpenAlex
- W2087185853
- Cited by
- 292
- References
- 10
- Citations by year
- 2026: 5 · 2025: 8 · 2024: 9 · 2023: 10 · 2022: 6 · 2021: 17 · 2020: 13 · 2019: 10 · 2018: 13 · 2017: 11 · 2016: 12 · 2015: 26 · 2014: 23 · 2013: 17 · 2012: 23
- FWCI
- 16.72
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of Clinical Investigation (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- CINECA IRIS Institutial research information system (Parthenope University of Naples) (submittedVersion)
- Funders
- F. M. Kirby Foundation
- Research to Prevent Blindness
- James N. Kirby Foundation
- National Institutes of Health
- National Eye Institute (EY10820, R01EY012951, EY 12951, R01EY010820, R01EY015136, R01EY007042, EY015136, EY07042)
- Institute of Genetics
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- adeno-associated virus vectors, AAV serotypes, AAV2/5, gene therapy, retinal gene therapy, ABCA4, MYO7A, CEP290, Stargardt disease, intraocular administration, rod outer segments
- MeSH terms
- Myosin VIIa, Dependovirus, Animals, Antigens, Neoplasm, Cytoskeletal Proteins, Dyneins, Electroretinography, Genetic Vectors, Humans, Mice, Inbred BALB C, Molecular Sequence Data, Myosins, Neoplasm Proteins, Retina, Serotyping, Genetic Therapy, Gene Transfer Techniques, Mice, Knockout, ATP-Binding Cassette Transporters, Cell Cycle Proteins, Mice
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