Circular Intermediates of Recombinant Adeno-Associated Virus Have Defined Structural Characteristics Responsible for Long-Term Episomal Persistence in Muscle Tissue
Open accessAbstract
Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
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?
During transduction, rAAV genomes form circular or linear (mostly) head-to-tail arrays of viral genomes.
“During transduction, rAAV genomes have been shown to form circular or linear (mostly) head-to-tail arrays of viral genomes, 14,50,51”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [14]
The Results and Discussion report head-to-tail circular rAAV concatemers in infected muscle tissue. The Results say that linear integrated concatemers had previously been hypothesized, whereas this study's data suggest the persistent genomes are circular and episomal. The claim's assertion that linear arrays have been shown to form is therefore not established by this paper. (LLM, conf=0.86)
Experimental — accuracy not yet measuredAAV 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 [14]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
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
In vivo model of adeno-associated virus vector persistence and rescue
Afione, S. A., Conrad, C. K., Kearns, W. G., Chunduru, S., Adams, R., Reynolds, T. C., Guggino, W. B., Cutting, G. R., Carter, B. J., & Flotte, T. R. (1996) · Journal of Virology · doi:10.1128/jvi.70.5.3235-3241.1996 · Cited by 223 · 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
Flagged first, then by citations
Research works printed but not linked: 5
- [12]12. Flannery, J. G., S. Zolotukhin, M. I. Vaquero, M. M. LaVail, N. Muzyczka, and W. W. Hauswirth. 1997. Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus. Proc. Natl. Acad. Sci. USA 94: 6916-6921.
- [15]15. Herzog, R. W., J. N. Hagstrom, S. H. Kung, S. J. Tai, J. M. Wilson, K. J. Fisher, and K. A. High. 1997. Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc. Natl. Acad. Sci. USA 94: 5804-5809.
- [19]19. Kessler, P. D., G. M. Podsakoff, X. Chen, S. A. McQuiston, P. C. Colosi, L. A. Matelis, G. J. Kurtzman, and B. J. Byrne. 1996. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic - protein. Proc. Natl. Acad. Sci. USA 93: 14082-14087.
- [20]20. Koeberl, D. D., I. E. Alexander, C. L. Halbert, D. W. Russell, and A. D. Miller. 1997. Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors. Proc. Natl. Acad. Sci. USA 94: 1426-1431.
- [22]22. Linden, R. M., E. Winocour, and K. I. Berns. 1996. The recombination signals for adeno-associated virus site-specific integration. Proc. Natl. Acad. Sci. USA 93: 7966-7972.
Cited by
Sorted by citations
Progress and problems with the use of viral vectors for gene therapy
Thomas, C. E., Ehrhardt, A., & Kay, M. A. (2003) · Nature Reviews Genetics · doi:10.1038/nrg1066 · Cited by 2,501 · see citing works
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
Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy
Gao, G.-P., Alvira, M. R., Wang, L., Calcedo, R., Johnston, J., & Wilson, J. M. (2002) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.182412299 · Cited by 1,520 · see citing works
Viral vector platforms within the gene therapy landscape
Bulcha, J. T., Wang, Y., Ma, H., Tai, P. W. L., & Gao, G. (2021) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-021-00487-6 · Cited by 1,404 · see citing works
Foust, K. D., Wang, X., McGovern, V. L., Braun, L., Bevan, A. K., Haidet, A. M., Le, T. T., Morales, P. R., Rich, M. M., Burghes, A. H. M., et al. (2010) · Nature Biotechnology · doi:10.1038/nbt.1610 · Cited by 688 · see citing works
RetractedInagaki, K., Fuess, S., Storm, T. A., Gibson, G. A., Mctiernan, C. F., Kay, M. A., & Nakai, H. (2006) · Molecular Therapy · doi:10.1016/j.ymthe.2006.03.014 · Cited by 610 · 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
Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus
Duan, D., Yue, Y., Yan, Z., Yang, J., & Engelhardt, J. F. (2000) · Journal of Clinical Investigation · doi:10.1172/jci8317 · Cited by 391 · see citing works
Self-citationIn vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels
Rothgangl, T., Dennis, M. K., Lin, P. J. C., Oka, R., Witzigmann, D., Villiger, L., Qi, W., Hruzova, M., Kissling, L., Lenggenhager, D., et al. (2021) · Nature Biotechnology · doi:10.1038/s41587-021-00933-4 · Cited by 378 · see citing works
Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy
Yan, Z., Zhang, Y., Duan, D., & Engelhardt, J. F. (2000) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.97.12.6716 · Cited by 305 · see citing works
Self-citationEffective 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
Genetic 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
Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues
Schnepp, B. C., Jensen, R. L., Chen, C.-L., Johnson, P. R., & Clark, K. R. (2005) · Journal of Virology · doi:10.1128/jvi.79.23.14793-14803.2005 · Cited by 231 · 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
Yan, Z., Zak, R., Luxton, G. W. G., Ritchie, T. C., Bantel-Schaal, U., & Engelhardt, J. F. (2002) · Journal of Virology · doi:10.1128/jvi.76.5.2043-2053.2002 · Cited by 222 · see citing works
Self-citationJüttner, J., Szabo, A., Gross-Scherf, B., Morikawa, R. K., Rompani, S. B., Hantz, P., Szikra, T., Esposti, F., Cowan, C. S., Bharioke, A., et al. (2019) · Nature Neuroscience · doi:10.1038/s41593-019-0431-2 · Cited by 218 · 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
Kruzik, A., Fetahagic, D., Hartlieb, B., Dorn, S., Koppensteiner, H., Horling, F. M., Scheiflinger, F., Reipert, B. M., & de la Rosa, M. (2019) · Molecular Therapy - Methods & Clinical Development · doi:10.1016/j.omtm.2019.05.014 · Cited by 182 · see citing works
rAAV immunogenicity, toxicity, and durability in 255 clinical trials: A meta-analysis
Shen, W., Liu, S., & Ou, L. (2022) · Frontiers in Immunology · doi:10.3389/fimmu.2022.1001263 · Cited by 173 · see citing works
PEG conjugation moderately protects adeno‐associated viral vectors against antibody neutralization
Lee, G. K., Maheshri, N., Kaspar, B., & Schaffer, D. V. (2005) · Biotechnology and Bioengineering · doi:10.1002/bit.20562 · Cited by 169 · see citing works
Production of adeno-associated virus vectors for in vitro and in vivo applications
Kimura, T., Ferran, B., Tsukahara, Y., Shang, Q., Desai, S., Fedoce, A., Pimentel, D. R., Luptak, I., Adachi, T., Ido, Y., et al. (2019) · Scientific Reports · doi:10.1038/s41598-019-49624-w · Cited by 155 · see citing works
Cordier, L., Gao, G.-P., Hack, A. A., McNally, E. M., Wilson, J. M., Chirmule, N., & Sweeney, H. L. (2001) · Human Gene Therapy · doi:10.1089/104303401750061267 · Cited by 152 · see citing works
Silencing and un-silencing of tetracycline-controlled genes in neurons
Zhu, P., Aller, M. I., Baron, U., Cambridge, S., Bausen, M., Herb, J., Sawinski, J., Cetin, A., Osten, P., Nelson, M. L., et al. (2007) · PLoS ONE · doi:10.1371/journal.pone.0000533 · Cited by 136 · 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
Haberman, R. P., McCown, T. J., & Samulski, R. J. (2000) · Journal of Virology · doi:10.1128/jvi.74.18.8732-8739.2000 · Cited by 139 · see citing works
Design and Packaging of Adeno-Associated Virus Gene Targeting Vectors
Hirata, R. K., & Russell, D. W. (2000) · Journal of Virology · doi:10.1128/jvi.74.10.4612-4620.2000 · Cited by 133 · see citing works
The Mre11/Rad50/Nbs1 complex limits adeno-associated virus transduction and replication
Schwartz, R. A., Palacios, J. A., Cassell, G. D., Adam, S., Giacca, M., & Weitzman, M. D. (2007) · Journal of Virology · doi:10.1128/jvi.01523-07 · Cited by 130 · 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 targeting with viral vectors
Hendrie, P. C., & Russell, D. W. (2005) · Molecular Therapy · doi:10.1016/j.ymthe.2005.04.006 · Cited by 126 · see citing works
Immune responses to AAV-vectors, the Glybera example from bench to bedside
Ferreira, V., Petry, H., & Salmon, F. (2014) · Frontiers in Immunology · doi:10.3389/fimmu.2014.00082 · Cited by 120 · see citing works
Schwartz, R. A., Carson, C. T., Schuberth, C., & Weitzman, M. D. (2009) · Journal of Virology · doi:10.1128/jvi.00318-09 · Cited by 115 · 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
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
Dosage thresholds and influence of transgene cassette in adeno-associated virus–related toxicity
Khabou, H., Cordeau, C., Pacot, L., Fisson, S., & Dalkara, D. (2018) · Human Gene Therapy · doi:10.1089/hum.2018.144 · Cited by 93 · 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
Casazza, J. P., Cale, E. M., Narpala, S., Yamshchikov, G. V., Coates, E. E., Hendel, C. S., Novik, L., Holman, L. A., Widge, A. T., Apte, P., et al. (2022) · Nature Medicine · doi:10.1038/s41591-022-01762-x · Cited by 91 · see citing works
Xin, K.-Q., Mizukami, H., Urabe, M., Toda, Y., Shinoda, K., Yoshida, A., Oomura, K., Kojima, Y., Ichino, M., Klinman, D., et al. (2006) · Journal of Virology · doi:10.1128/jvi.00890-06 · Cited by 81 · see citing works
Gene therapy for inherited retinal disease: Long-term durability of effect
Leroy, B. P., Fischer, M. D., Flannery, J. G., MacLaren, R. E., Dalkara, D., Scholl, H. P. N., Chung, D. C., Spera, C., Viriato, D., & Banhazi, J. (2023) · Ophthalmic Research · doi:10.1159/000526317 · Cited by 70 · 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-citationMarrone, L., Marchi, P. M., & Azzouz, M. (2022) · Expert Opinion on Biological Therapy · doi:10.1080/14712598.2022.2012148 · Cited by 56 · see citing works
“correlating with long-term transgene expression [34].”
Xu, Z., Yue, Y., Lai, Y., Ye, C., Qiu, J., Pintel, D. J., & Duan, D. (2004) · Human Gene Therapy · doi:10.1089/hum.2004.15.896 · Cited by 55 · see citing works
Self-citationRational 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-citationChandler, L., Yusuf, I., McClements, M., Barnard, A., MacLaren, R., & Xue, K. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21144972 · Cited by 49 · see citing works
Surace, E. M., Auricchio, A., Reich, S. J., Rex, T., Glover, E., Pineles, S., Tang, W., O'Connor, E., Lyubarsky, A., Savchenko, A., et al. (2003) · Journal of Virology · doi:10.1128/jvi.77.14.7957-7963.2003 · Cited by 46 · see citing works
Kinetics and durability of transgene expression after intrastriatal injection of AAV9 vectors
Hollidge, B. S., Carroll, H. B., Qian, R., Fuller, M. L., Giles, A. R., Mercer, A. C., Danos, O., Liu, Y., Bruder, J. T., & Smith, J. B. (2022) · Frontiers in Neurology · doi:10.3389/fneur.2022.1051559 · Cited by 45 · 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
Methods for in vitro CRISPR/CasRx-Mediated RNA Editing
Chuang, Y.-F., Wang, P.-Y., Kumar, S., Lama, S., Lin, F.-L., & Liu, G.-S. (2021) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2021.667879 · Cited by 21 · see citing works
- Authors
- Duan, D., Sharma, P., Yang, J., Yue, Y., Dudus, L., Zhang, Y., Fisher, K. J., & Engelhardt, J. F.
- Affiliations
- University of Iowa (US)
- Tulane University (US)
- Venue
- Journal of Virology
- Publisher
- American Society for Microbiology
- Published
- 1998-11-01
- Volume
- 72
- Issue
- 11
- Pages
- 8568-8577
- ISSN
- 0022-538X (ISSN-L)
- 1098-5514
- PMC
- PMC103904
- OpenAlex
- W2106378095
- Cited by
- 522
- References
- 3
- Citations by year
- 2026: 11 · 2025: 19 · 2024: 16 · 2023: 13 · 2022: 23 · 2021: 24 · 2020: 15 · 2019: 24 · 2018: 16 · 2017: 14 · 2016: 13 · 2015: 14 · 2014: 19 · 2013: 14 · 2012: 11
- FWCI
- 12.99
- Type
- journal article
- Language
- en
- Update notices
- correction (1999-01-01)
- Open access
- bronze
- doi.org
- Locations
- Journal of Virology (publishedVersion)
- PubMed Central (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Heart, Lung, and Blood Institute (R01HL058340)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK/HL58340)
- 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
- adeno-associated virus, muscle tissue, inverted terminal repeats, shuttle vector, long-term transgene expression, non-viral gene delivery
- MeSH terms
- Dependovirus, Animals, Base Sequence, DNA, Viral, Genetic Vectors, HeLa Cells, Humans, Luminescent Proteins, Mice, Inbred C57BL, Molecular Weight, Plasmids, Recombination, Genetic, Time Factors, Transfection, Gene Expression, Genome, Viral, DNA Primers, Muscle, Skeletal, Terminal Repeat Sequences, Green Fluorescent Proteins, 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
