Evidence for the Failure of Adeno-associated Virus Serotype 5 to Package a Viral Genome ≥8.2 kb
Open accessAbstract
Limited packaging capacity hinders adeno-associated virus (AAV) gene therapy. A recent study seems to have provided a solution to this problem. Allocca et al. reported that AAV-5 could package an 8.9 kb vector genome. Here we tested whether this approach can be used to deliver a large genome for Duchenne muscular dystrophy (DMD) gene therapy. We first evaluated AAV-5 packaging of an 8.2 kb genome. This vector carries two independent reporter gene cassettes, one for alkaline phosphatase (AP) and another for LacZ. Viral yield was log-fold lower than that of a regular AAV-5. Nevertheless, both AP and LacZ genes were detected in purified virus. Injection to dystrophic muscle resulted in both AP and LacZ expression. On electron microscopy, virion structure appeared normal. Surprisingly, we did not find the full-length single-stranded viral genome by alkaline gel electrophoresis. Neither did we see the full-length double-stranded replication forms in adenovirus coinfected cells. We suspect that AP and LacZ expression may have come from partially packaged 5' or 3'-half of the genome. Additional studies revealed failure of AAV-5 to package and express an 8.7 kb minidystrophin gene cassette. In summary, our results do not support the extraordinary packaging capacity of AAV-5.
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AAV has a strict size cutoff of 5.2 kb.
“other studies [6-8] indicated that intact genomes were not, in fact, packaged in that case [3], and further suggested a strict size cutoff of 5.2 kb.”
From Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons (2014) · cites this paper as [8]
The oversized AAV virus stock was in fact a mixture of partially packaged viruses.
“further molecular characterization found that the oversized virus stock was in fact a mixture of partially packaged viruses.”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [30]
No full-length viral genomes of approximately 8.2 kb could be detected by alkaline gel Southern blot.
“no full-length viral genomes of * 8.2 kb could be detected by alkaline gel Southern blot. 30”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [30]
The finding that AAV5 capsids could package intact genomes of up to 8.9 kb in length was not supported by further studies; such genomes were truncated and the packaging capacity of AAV was still limited to genomes substantially smaller than 8.9 kb.
“However, the finding that AAV5 capsids could package intact genomes of up to 8.9 kb in length was not supported by further studies, which found that such genomes were truncated and that the packaging capacity of AAV was still limited to genomes substantially smaller than 8.9 kb. 30-32”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [30]
Southern blot hybridization revealed that purportedly oversized packaged genomes were in fact truncated, and the authors suggested that the truncated fragments could assemble to restore the full-length expression cassette.
“Southern blot hybridization revealed that purportedly oversized packaged genomes were in fact truncated, and the authors suggested that the truncated fragments could assemble thus restoring the full-length expression cassette.”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [30]
Both AP expression and LacZ expression were detected in mdx muscle of adult mice injected with the oversized AAV vector preparations.
“both AP expression and LacZ expression were detected in mdx muscle of adult mice injected with such AAV vector preparations.”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [30]
In a slot blot with probes against AP and LacZ open reading frames, both probes hybridized to viral DNA from purified rAAV.
“in a slot blot with probes directed against the AP and the LacZ open reading frames (ORFs), both probes hybridized to viral DNA from purified rAAV.”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [30]
rAAV genome-packaging capacity is approximately 5 kb.
“One limitation of rAAVs is that their genome-packaging capacity is only * 5kb.”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [30]
Cites
Safety and Efficacy of Gene Transfer for Leber's Congenital Amaurosis
Maguire, A. M., Simonelli, F., Pierce, E. A., Pugh, E. N., Mingozzi, F., Bennicelli, J., Banfi, S., Marshall, K. A., Testa, F., Surace, E. M., et al. (2008) · New England Journal of Medicine · doi:10.1056/nejmoa0802315 · Cited by 2,145 · see citing works
Effect of gene therapy on visual function in Leber’s congenital amaurosis
Bainbridge, J. W. B., Smith, A. J., Barker, S. S., Robbie, S., Henderson, R., Balaggan, K., Viswanathan, A., Holder, G. E., Stockman, A., Tyler, N., et al. (2008) · New England Journal of Medicine · doi:10.1056/nejmoa0802268 · Cited by 1,924 · see citing works
Effect of genome size on AAV vector packaging
Wu, Z., Yang, H., & Colosi, P. (2010) · Molecular Therapy · doi:10.1038/mt.2009.255 · Cited by 1,025 · see citing works
Grieger, J. C., & Samulski, R. J. (2005) · Journal of Virology · doi:10.1128/jvi.79.15.9933-9944.2005 · Cited by 419 · see citing works
Characterization of Genome Integrity for Oversized Recombinant AAV Vector
Dong, B., Nakai, H., & Xiao, W. (2010) · Molecular Therapy · doi:10.1038/mt.2009.258 · Cited by 230 · see citing works
Halbert, C. L., Allen, J. M., & Miller, A. D. (2002) · Nature Biotechnology · doi:10.1038/nbt0702-697 · Cited by 121 · see citing works
Wang, X. S., Ponnazhagan, S., & Srivastava, A. (1996) · Journal of Virology · doi:10.1128/jvi.70.3.1668-1677.1996 · Cited by 104 · see citing works
Ghosh, A., Yue, Y., & Duan, D. (2006) · The Journal of Gene Medicine · doi:10.1002/jgm.835 · Cited by 69 · see citing works
Self-citationWang, X. S., Qing, K., Ponnazhagan, S., & Srivastava, A. (1997) · Journal of Virology · doi:10.1128/jvi.71.4.3077-3082.1997 · Cited by 61 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
McIntosh, J., Lenting, P. J., Rosales, C., Lee, D., Rabbanian, S., Raj, D., Patel, N., Tuddenham, E. G. D., Christophe, O. D., McVey, J. H., et al. (2013) · Blood · doi:10.1182/blood-2012-10-462200 · Cited by 278 · see citing works
Effective delivery of large genes to the retina by dual AAV vectors
Trapani, I., Colella, P., Sommella, A., Iodice, C., Cesi, G., de Simone, S., Marrocco, E., Rossi, S., Giunti, M., Palfi, A., et al. (2014) · EMBO Molecular Medicine · doi:10.1002/emmm.201302948 · Cited by 269 · see citing works
A comprehensive review of retinal gene therapy
Boye, S. E., Boye, S. L., Lewin, A. S., & Hauswirth, W. W. (2013) · Molecular Therapy · doi:10.1038/mt.2012.280 · Cited by 262 · see citing works
AAV Capsid Structure and Cell Interactions
Agbandje-McKenna, M., & Kleinschmidt, J. (2012) · Methods in Molecular Biology · doi:10.1007/978-1-61779-370-7_3 · Cited by 215 · see citing works
Choi, J.-H., Yu, N.-K., Baek, G.-C., Bakes, J., Seo, D., Nam, H. J., Baek, S. H., Lim, C.-S., Lee, Y.-S., & Kaang, B.-K. (2014) · Molecular Brain · doi:10.1186/1756-6606-7-17 · Cited by 197 · see citing works
Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids
Chamberlain, K., Riyad, J. M., & Weber, T. (2016) · Human Gene Therapy Methods · doi:10.1089/hgtb.2015.140 · Cited by 141 · see citing works
Horowitz, E. D., Rahman, K. S., Bower, B. D., Dismuke, D. J., Falvo, M. R., Griffith, J. D., Harvey, S. C., & Asokan, A. (2013) · Journal of Virology · doi:10.1128/jvi.03017-12 · Cited by 139 · see citing works
Dyka, F. M., Boye, S. L., Chiodo, V. A., Hauswirth, W. W., & Boye, S. E. (2014) · Human Gene Therapy Methods · doi:10.1089/hgtb.2013.212 · Cited by 128 · see citing works
Gene therapy and genome surgery in the retina
DiCarlo, J. E., Mahajan, V. B., & Tsang, S. H. (2018) · Journal of Clinical Investigation · doi:10.1172/jci120429 · Cited by 127 · see citing works
Monahan, P. E., Lothrop, C. D., Sun, J., Hirsch, M. L., Kafri, T., Kantor, B., Sarkar, R., Tillson, D. M., Elia, J. R., & Samulski, R. J. (2010) · Molecular Therapy · doi:10.1038/mt.2010.170 · Cited by 125 · see citing works
Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus
Lopes, V. S., Boye, S. E., Louie, C. M., Boye, S., Dyka, F., Chiodo, V, Fofo, H., Hauswirth, W. W., & Williams, D. S. (2013) · Gene Therapy · doi:10.1038/gt.2013.3 · Cited by 112 · see citing works
Methods for Gene Transfer to the Central Nervous System
Kantor, B., Bailey, R. M., Wimberly, K., Kalburgi, S. N., & Gray, S. J. (2014) · Advances in Genetics · doi:10.1016/b978-0-12-800149-3.00003-2 · Cited by 109 · see citing works
Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors
Colella, P., Trapani, I., Cesi, G., Sommella, A., Manfredi, A., Puppo, A., Iodice, C., Rossi, S., Simonelli, F., Giunti, M., et al. (2014) · Gene Therapy · doi:10.1038/gt.2014.8 · Cited by 103 · see citing works
AAV.Dysferlin Overlap Vectors Restore Function in Dysferlinopathy Animal Models
Sondergaard, P. C., Griffin, D. A., Pozsgai, E. R., Johnson, R. W., Grose, W. E., Heller, K. N., Shontz, K. M., Montgomery, C. L., Liu, J., Clark, K. R., et al. (2015) · Annals of Clinical and Translational Neurology · doi:10.1002/acn3.172 · Cited by 99 · see citing works
Structural insights into adeno-associated virus serotype 5
Govindasamy, L., DiMattia, M. A., Gurda, B. L., Halder, S., McKenna, R., Chiorini, J. A., Muzyczka, N., Zolotukhin, S., & Agbandje-McKenna, M. (2013) · Journal of Virology · doi:10.1128/jvi.00867-13 · Cited by 95 · see citing works
Ghosh, A., Yue, Y., & Duan, D. (2011) · Human Gene Therapy · doi:10.1089/hum.2010.122 · Cited by 95 · see citing works
Self-citationAdvances in gene therapy for diseases of the eye
Petit, L., Khanna, H., & Punzo, C. (2016) · Human Gene Therapy · doi:10.1089/hum.2016.040 · Cited by 82 · see citing works
Grose, W. E., Clark, K. R., Griffin, D., Malik, V., Shontz, K. M., Montgomery, C. L., Lewis, S., Brown, R. H., Janssen, P. M. L., Mendell, J. R., et al. (2012) · PLoS ONE · doi:10.1371/journal.pone.0039233 · Cited by 76 · see citing works
Gene therapy for Stargardt disease associated with ABCA4 gene
Han, Z., Conley, S. M., & Naash, M. I. (2014) · Advances in Experimental Medicine and Biology · doi:10.1007/978-1-4614-3209-8_90 · Cited by 74 · see citing works
Adeno-Associated Viral Vectors as a Tool for Large Gene Delivery to the Retina
Trapani, I. (2019) · Genes · doi:10.3390/genes10040287 · Cited by 73 · see citing works
Suppression of Choroidal Neovascularization by AAV-Based Dual-Acting Antiangiogenic Gene Therapy
Askou, A. L., Alsing, S., Benckendorff, J. N. E., Holmgaard, A., Mikkelsen, J. G., Aagaard, L., Bek, T., & Corydon, T. J. (2019) · Molecular Therapy - Nucleic Acids · doi:10.1016/j.omtn.2019.01.012 · Cited by 64 · see citing works
Oversized AAV Transductifon Is Mediated via a DNA-PKcs-independent, Rad51C-dependent Repair Pathway
Hirsch, M. L., Li, C., Bellon, I., Yin, C., Chavala, S., Pryadkina, M., Richard, I., & Samulski, R. J. (2013) · Molecular Therapy · doi:10.1038/mt.2013.184 · Cited by 62 · see citing works
Specific tools for targeting and expression in Müller glial cells
Pellissier, L. P., Hoek, R. M., Vos, R. M., Aartsen, W. M., Klimczak, R. R., Hoyng, S. A., Flannery, J. G., & Wijnholds, J. (2014) · Molecular Therapy - Methods & Clinical Development · doi:10.1038/mtm.2014.9 · Cited by 58 · see citing works
Marrone, L., Marchi, P. M., & Azzouz, M. (2022) · Expert Opinion on Biological Therapy · doi:10.1080/14712598.2022.2012148 · Cited by 56 · see citing works
Hohman, T. C. (2016) · Handbook of Experimental Pharmacology · doi:10.1007/164_2016_91 · Cited by 56 · see citing works
Rational engineering of a functional CpG-free ITR for AAV gene therapy
Pan, X., Yue, Y., Boftsi, M., Wasala, L. P., Tran, N. T., Zhang, K., Pintel, D. J., Tai, P. W. L., & Duan, D. (2022) · Gene Therapy · doi:10.1038/s41434-021-00296-0 · Cited by 53 · see citing works
Self-citationNon-viral therapeutic approaches to ocular diseases: An overview and future directions
Zulliger, R., Conley, S. M., & Naash, M. I. (2015) · Journal of Controlled Release · doi:10.1016/j.jconrel.2015.10.007 · Cited by 50 · see citing works
Nicolson, S. C., Li, C., Hirsch, M. L., Setola, V., & Samulski, R. J. (2016) · Journal of Virology · doi:10.1128/jvi.02953-15 · Cited by 48 · see citing works
Zhou, Q., Tian, W., Liu, C., Lian, Z., Dong, X., & Wu, X. (2017) · Scientific Reports · doi:10.1038/s41598-017-04054-4 · Cited by 23 · see citing works
Inhibition of antigen presentation during AAV gene therapy using virus peptides
Shao, W., Chen, X., Samulski, R. J., Hirsch, M. L., & Li, C. (2018) · Human Molecular Genetics · doi:10.1093/hmg/ddx427 · Cited by 17 · see citing works
McClements, M. E., Charbel Issa, P., Blouin, V., & MacLaren, R. E. (2016) · Journal of Genetic Syndromes & Gene Therapy · doi:10.4172/2157-7412.1000311 · Cited by 13 · see citing works
- Authors
- Lai, Y., Yue, Y., & Duan, D.
- Affiliations
- University of Missouri (US)
- Corresponding author
- Duan, Dongsheng
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2010-01
- Volume
- 18
- Issue
- 1
- Pages
- 75-79
- ISSN
- 1525-0016 (ISSN-L)
- PMC
- PMC2839223
- OpenAlex
- W2090289970
- Cited by
- 166
- References
- 9
- Citations by year
- 2026: 5 · 2025: 2 · 2024: 2 · 2023: 4 · 2022: 11 · 2021: 12 · 2020: 11 · 2019: 10 · 2018: 4 · 2017: 8 · 2016: 9 · 2015: 20 · 2014: 26 · 2013: 11 · 2012: 12
- FWCI
- 6.01
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Therapy (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR49419, R01AR049419)
- 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
- Health Sciences › Medicine › Genetics › Neurogenetic and Muscular Disorders Research
- Keywords
- Duchenne muscular dystrophy, alkaline phosphatase reporter, lacZ reporter, virus yield
- MeSH terms
- Dependovirus, Alkaline Phosphatase, Animals, Genetic Vectors, Lac Operon, Male, Transduction, Genetic, Blotting, Southern, Genetic Therapy, Genome, Viral, Muscular Dystrophy, Duchenne, Microscopy, Electron, Transmission, Mice
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