Abstract
While generally referred to as “non-integrating” vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous) recombination. The present study provides a quantitative assessment of the potential integration frequency of adenovirus 5 (Ad5)-based vectors following intravenous injection in mice, a common route of administration in gene therapy applications particularly for transgene expression in liver. We examined the uptake level and persistence in liver of first generation (FG) and helper-dependent (HD) Ad5 vectors containing the mouse leptin transgene. As expected, the persistence of the HD vector was markedly higher than that of the FG vector. For both vectors, the majority of the vector DNA remained extrachromosomal and predominantly in the form of episomal monomers. However, using a quantitative gel-purification-based integration assay, a portion of the detectable vector was found to be associated with high molecular weight (HMW) genomic DNA, indicating potential integration with a frequency of up to ~44 and 7000 integration events per μg cellular genomic DNA (or ~0.0003 and 0.05 integrations per cell, respectively) for the FG and HD Ad5 vectors, respectively, following intravenous injection of 1 × 1011 virus particles. To confirm integration occurred (versus residual episomal vector DNA co-purifying with genomic DNA), we characterized nine independent integration events using Repeat-Anchored Integration Capture (RAIC) PCR. Sequencing of the insertion sites suggests that both of the vectors integrate randomly, but within short segments of homology between the vector breakpoint and the insertion site. Eight of the nine integrations were in intergenic DNA and one was within an intron. These findings represent the first quantitative assessment and characterization of Ad5 vector integration following intravenous administration in vivo in wild-type mice.
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References
Hitt MM, Addison CL, Graham FL. Human adenovirus vectors for gene transfer into mammalian cells. Adv Pharmacol. 1997;40:137–206.
Lai CM, Lai YK, Rakoczy PE. Adenovirus and adeno-associated virus vectors. DNA Cell Biol. 2002;21:895–913.
St George JA. Gene therapy progress and prospects: adenoviral vectors. Gene Ther. 2003;10:1135–41.
Ginn SL, Anais K, Amaya AK, Ian E, Alexander IE, Edelstein M, et al. Gene therapy clinical trials worldwide to 2017: an update. J Gene Med. 2018;20:e3015. https://doi.org/10.1002/jgm.3015.
Morsy MA, Gu M, Motzel S, Zhao J, Jing Lin J, Su Q, et al. An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene. Proc Natl Acad Sci USA. 1998;95:7866–71.
Morsy MA, Gu MC, Zhao JZ, Holder DJ, Rogers IT, Pouch WJ, et al. Leptin gene therapy and daily protein administration: a comparative study in the ob/ob mouse. Gene Ther. 1998;5:8–18.
European Medicines Agency. Expert Committee on Medicinal Products Gene Therapy. Report from the CPMP Gene Therapy Expert Group Meeting 26th-27th February 2004. EMEA/CPMP/1879/04/Final.
Mitani K, Kubo S. Adenovirus as an integrating vector. Curr Gene Ther. 2002;2:135–44.
Harui A, Suzuki S, Kochanek S, Mitani K. Frequency and stability of chromosomal integration of adenovirus vectors. J Virol. 1999;73:6141–6.
Hillgenberg M, Tonnies H, Strauss M. Chromosomal integration pattern of a helper-dependent minimal adenovirus vector with a selectable marker inserted into a 27.4-kilobase genomic stuffer. J Virol. 2001;75:9896–908.
Doerfler W. A new concept in (adenoviral) oncogenesis: integration of foreign DNA and its consequences. Biochim Biophys Acta. 1996;1288:F79–F99.
Stephen SL, Sivanandam VG, Kochanek S. Homologous and heterologous recombination between adenovirus vector DNA and chromosomal DNA. J Gene Med. 2008;10:1176–89.
Hilger-Eversheim K, Doerfler W. Clonal origin of adenovirus type 12-induced hamster tumors: nonspecific chromosomal integration sites of viral DNA. Cancer Res. 1997;57:3001–9.
Stephen SL, Montini E, Sivanandam VG, Al-Dhalimy M, Kestler HA, Finegold M, et al. Chromosomal integration of adenoviral vector DNA in vivo. J Virol. 2010;84:9987–94.
Nichols WW, Ledwith BJ, Manam SV, Troilo PJ. Potential DNA vaccine integration into host cell genome. Ann N Y Acad Sci. 1995;772:30–39.
Ledwith BJ, Manam S, Troilo PJ, Barnum AB, Pauley CJ, Griffiths TG II, et al. Plasmid DNA vaccines: assay for integration into host genomic DNA. Dev Biol (Basel). 2000;104:33–43.
Ledwith BJ, Manam S, Troilo PJ, Barnum AB, Pauley CJ, Griffiths TG II, et al. Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice. Intervirology. 2000;43:258–72.
Wang Z, Troilo PJ, Wang X, Griffiths TG II, Pacchione SJ, Barnum AB, et al. Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation. Gene Ther. 2004;11:711–21.
Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF, Agarwal P, et al. Initial sequencing and comparative analysis of the mouse genome. Nature. 2002;420:520–62.
Liu Q, Muruve DA. Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther. 2003;10:935–40.
Li H, Malani N, Hamilton SR, Schlachterman A, Bussadori G, Edmonson SE, et al. Assessing the potential for AAV vector genotoxicity in a murine model. Blood. 2011;117:3311–9.
Nowrouzi A, Penaud-Budloo M, Kaeppel C, Appelt U, Guiner CL, Moullier P, et al. Integration frequency and intermolecular recombination of rAAV vectors in non-human primate skeletal muscle and liver. Mol Ther. 2012;20:1177–86.
Kaeppel C, Beattie SG, Fronza R, Logtenstein RV, Salmon F, Schmidt S, et al. A largely random AAV integration profile after LPLD gene therapy. Nat Med. 2013;19:889.
Milholland B, Dong X, Zhang L, Hao X, Suh Y, Vijg JJ. Differences between germline and somatic mutation rates in humans and mice. Nat Commun. 2017;8:15183.
Cole J, Skopek TR. International Commission for Protection Against Environmental Mutagens and Carcinogens. Working paper no. 3. Somatic mutant frequency, mutation rates and mutational spectra in the human population in vivo. Mutat Res. 1994;304:33–105.
Willyard C. New human gene tally reignites debate. Nature. 2018;558:354–5.
Wurtele H, Little KC, Chartrand P. Illegitimate DNA integration in mammalian cells. Gene Ther. 2003;10:1791–9.
Nakai H, Montini E, Fuess S, Storm TA, Grompe M, Kay MA. AAV serotype 2 vectors preferentially integrate into active genes in mice. Nat Genet. 2003;34:297–302.
Schroder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell. 2002;110:521–9.
Wu X, Li Y, Crise B, Burgess SM. Transcription start regions in the human genome are favored targets for MLV integration. Science. 2003;300:1749–51.
McCormack MP, Rabbitts TH. Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency. N Engl J Med. 2004;350:913–22.
Kaiser J. Gene therapy. Panel urges limits on X-SCID trials. Science. 2005;307:1544–5.
Insertional mutagenesis and oncogenesis: update from non-clinical and clinical studies. Gene Therapy Expert Group of the Committee for Proprietary Medical Products (CPMP), European Agency for the Evaluation of Medical Products - June 2003 meeting. J Gene Med. 2004;6:127–9.
Cichutek K. Lessons learned from gene therapy concerning and the use of integrating vectors and the possible risk of insertional oncogenesis. Dev Biol (Basel). 2006;123:29–34.
Chandler RJ, Sands MS, Venditti CP. Recombinant adeno-associated viral integration and genotoxicity: insights from animal models. Human Gene Ther. 2017;28:314–22.
Nguyen GN, Everett JK, Kafle S, Roche AM, Raymond HE, Leiby J. A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells. Nat Biotech. 2021;39:47–55.
Ledwith BJ, Lanning CL, Gumprecht LA, Anderson CA, Coleman JB, Gatto NT, et al. Tumorigenicity assessments of Per.C6 cells and of an Ad5-vectored HIV-1 vaccine produced on this continuous cell line. Dev Biol (Basel). 2006;123:251–63.
WHO. Meeting Report. WHO informal consultation on characterization and quality aspect of vaccines based on live viral vectors. Geneva: World Health Organization; 2003. pp. 9–10.
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BJL supervised and conceived the project; ZW and PJT designed the experiments; ZW, PJT, TGG, LBH, ABB, SJP, and CJP conducted the experiments and collected the data; ZW drafted the manuscript; BJL, JAL, JW, and PJT edited the manuscript. All authors reviewed the paper.
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Wang, Z., Troilo, P.J., Griffiths, T.G. et al. Characterization of integration frequency and insertion sites of adenovirus DNA into mouse liver genomic DNA following intravenous injection. Gene Ther 29, 322–332 (2022). https://doi.org/10.1038/s41434-021-00278-2
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DOI: https://doi.org/10.1038/s41434-021-00278-2