Recruitment of Single-Stranded Recombinant Adeno-Associated Virus Vector Genomes and Intermolecular Recombination Are Responsible for Stable Transduction of Liver In Vivo
Open accessAbstract
Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial Pae R7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
AAV vectors with two complementary ITRs can form circular concatamers.
“AAV vectors with two complementary ITRs can form circular concatamers. 13-16”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [15]
Cites
Ferrari, F. K., Samulski, T., Shenk, T., & Samulski, R. J. (1996) · Journal of Virology · doi:10.1128/jvi.70.5.3227-3234.1996 · Cited by 761 · see citing works
Recombinant Adeno-Associated Viral Vectors Mediate Long-Term Transgene Expression in Muscle
Clark, K. R., Sferra, T. J., & Johnson, P. R. (1997) · Human Gene Therapy · doi:10.1089/hum.1997.8.6-659 · Cited by 153 · see citing works
Adeno‐associated virus vectors and hematology
Russell, D. W., & Kay, M. A. (1999) · Blood · doi:10.1182/blood.v94.3.864.415k34_864_874 · Cited by 104 · see citing works
Self-citationStructural Analysis of Adeno-Associated Virus Transduction Circular Intermediates
Duan, D., Yan, Z., Yue, Y., & Engelhardt, J. F. (1999) · Virology · doi:10.1006/viro.1999.9821 · Cited by 100 · cited 1× · see citing works
“Although they assumed that ds rAAV circles were formed by second-strand synthesis (9), there was no definitive experimental evidence to support this.”
Background
Flagged first, then by citations
Cited by
Sorted by citations
Adeno-associated virus vector as a platform for gene therapy delivery
Wang, D., Tai, P. W. L., & Gao, G. (2019) · Nature Reviews Drug Discovery · doi:10.1038/s41573-019-0012-9 · Cited by 2,444 · see citing works
McCarty, D., Monahan, P., & Samulski, R. (2001) · Gene Therapy · doi:10.1038/sj.gt.3301514 · Cited by 735 · see citing works
Self-complementary AAV vectors; advances and applications
McCarty, D. M. (2008) · Molecular Therapy · doi:10.1038/mt.2008.171 · Cited by 515 · see citing works
Nakai, H., Yant, S. R., Storm, T. A., Fuess, S., Meuse, L., & Kay, M. A. (2001) · Journal of Virology · doi:10.1128/jvi.75.15.6969-6976.2001 · Cited by 478 · see citing works
Self-citationGenetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle
Schnepp, B. C., Clark, K. R., Klemanski, D. L., Pacak, C. A., & Johnson, P. R. (2003) · Journal of Virology · doi:10.1128/jvi.77.6.3495-3504.2003 · Cited by 262 · see citing works
Large‐scale molecular characterization of adeno‐associated virus vector integration in mouse liver
Nakai, H., Wu, X., Fuess, S., Storm, T. A., Munroe, D., Montini, E., Burgess, S. M., Grompe, M., & Kay, M. A. (2005) · Journal of Virology · doi:10.1128/jvi.79.6.3606-3614.2005 · Cited by 190 · see citing works
Self-citationWu, Z., Sun, J., Zhang, T., Yin, C., Yin, F., Van Dyke, T., Samulski, R. J., & Monahan, P. E. (2008) · Molecular Therapy · doi:10.1038/sj.mt.6300355 · Cited by 153 · see citing works
Favre, D., Blouin, V., Provost, N., Spisek, R., Porrot, F., Bohl, D., Marmé, F., Chérel, Y., Salvetti, A., Hurtrel, B., et al. (2002) · Journal of Virology · doi:10.1128/jvi.76.22.11605-11611.2002 · Cited by 150 · 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
Journey to the Center of the Cell: Tracing the Path of AAV Transduction
Dhungel, B. P., Bailey, C. G., & Rasko, J. E. J. (2021) · Trends in Molecular Medicine · doi:10.1016/j.molmed.2020.09.010 · Cited by 122 · see citing works
Reimsnider, S., Manfredsson, F. P., Muzyczka, N., & Mandel, R. J. (2007) · Molecular Therapy · doi:10.1038/sj.mt.6300227 · Cited by 125 · see citing works
Strategies to circumvent humoral immunity to adeno-associated viral vectors
Tse, L. V., Moller-Tank, S., & Asokan, A. (2015) · Expert Opinion on Biological Therapy · doi:10.1517/14712598.2015.1035645 · Cited by 61 · see citing works
Nakai, H., Montini, E., Fuess, S., Storm, T. A., Meuse, L., Finegold, M., Grompe, M., & Kay, M. A. (2003) · Molecular Therapy · doi:10.1016/s1525-0016(02)00023-0 · Cited by 52 · see citing works
Wang, Z., Lisowski, L., Finegold, M. J., Nakai, H., Kay, M. A., & Grompe, M. (2012) · Molecular Therapy · doi:10.1038/mt.2012.157 · Cited by 44 · see citing works
Self-citationGenome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction
Mano, M., Ippodrino, R., Zentilin, L., Zacchigna, S., & Giacca, M. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1503607112 · Cited by 39 · see citing works
Adeno-associated virus of a single-polarity DNA genome is capable of transduction in vivo
Zhou, X., Zeng, X., Fan, Z., Li, C., McCown, T., Samulski, R. J., & Xiao, X. (2008) · Molecular Therapy · doi:10.1038/sj.mt.6300397 · Cited by 41 · see citing works
Hendrie, P. C., Hirata, R. K., & Russell, D. W. (2003) · Journal of Virology · doi:10.1128/jvi.77.24.13136-13145.2003 · Cited by 33 · see citing works
Bell, P., Wang, L., Chen, S.-J., Yu, H., Zhu, Y., Nayal, M., He, Z., White, J., Lebel-Hagan, D., & Wilson, J. M. (2016) · Human Gene Therapy Methods · doi:10.1089/hgtb.2016.039 · Cited by 26 · see citing works
Gene therapy for the leukodystrophies: From preclinical animal studies to clinical trials.
Metovic, J., Li, Y., Gong, Y., & Eichler, F. (2024) · Neurotherapeutics · doi:10.1016/j.neurot.2024.e00443 · Cited by 16 · 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
“Within the host nucleus these have been predicted to regenerate the original oversized transgene through homologous recombination [7] or annealing of plus and minus strand complementary regions prior to second-strand synthesis [8], Trapani et al. have shown success with the fAAV approach in HEK293…”
Background
- Authors
- Nakai, H., Storm, T. A., & Kay, M. A.
- Affiliations
- Stanford Medicine (US)
- Venue
- Journal of Virology
- Publisher
- American Society for Microbiology
- Published
- 2000-10-15
- Volume
- 74
- Issue
- 20
- Pages
- 9451-9463
- ISSN
- 0022-538X (ISSN-L)
- 1098-5514
- PMC
- PMC112374
- OpenAlex
- W1964241088
- Cited by
- 194
- References
- 4
- Citations by year
- 2026: 2 · 2025: 3 · 2024: 3 · 2023: 2 · 2022: 4 · 2021: 2 · 2020: 7 · 2019: 5 · 2018: 6 · 2017: 8 · 2016: 7 · 2015: 13 · 2014: 5 · 2013: 8 · 2012: 10
- FWCI
- 6.22
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of Virology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- National Heart, Lung, and Blood Institute (R0I HL53682, R01HL053682, HL64274, R01HL064274)
- 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 › Epidemiology › Hepatitis B Virus Studies
- Keywords
- recombinant adeno-associated virus vectors, intermolecular recombination, second-strand synthesis, rolling circle replication, stable transduction, hepatocyte transduction, chromosomal integration, Dam methyltransferase, in vivo gene delivery
- MeSH terms
- Dependovirus, Animals, DNA, DNA Repair, Female, Genetic Vectors, Liver, Mice, Inbred C57BL, Recombination, Genetic, Genome, Viral, Gene Transfer Techniques, Base Pair Mismatch, Mice
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
