Recombinant adeno-associated viruses (rAAVs) are well-established vectors for delivering therapeutic genes. However, previous reports have suggested that wild-type AAV is linked to hepatocellular carcinoma, raising concern with the safety of rAAVs. In addition, a recent long-term follow-up study in canines, which received rAAVs for factor VIII gene therapy, demonstrated vector integration into the genome of liver cells, reviving the uncertainty between AAV and cancer. To further explore this relationship, we performed large-scale molecular epidemiology of AAV in resected tumor samples and non-lesion tissues collected from 413 patients, reflecting nine carcinoma types: breast carcinoma, rectal cancer, pancreas carcinoma, brain tumor, hepatoid adenocarcinoma, hepatocellular carcinoma, gastric carcinoma, lung squamous, and adenocarcinoma. We found that over 80% of patients were AAV-positive among all nine types of carcinoma examined. Importantly, the AAV sequences detected in patient-matched tumor and adjacent non-lesion tissues showed no significant difference in incidence, abundance, and variation. In addition, no specific AAV sequences predominated in tumor samples. Our data shows that AAV genomes are equally abundant in tumors and adjacent normal tissues, but lack clonality. The finding critically adds to the epidemiological profile of AAV in humans, and provides insights that may assist rAAV-based clinical studies and gene therapy strategies.
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Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019;18:358–78.
Ogden PJ, Kelsic ED, Sinai S, Church GM, Comprehensive AAV. Capsid fitness landscape reveals a viral gene and enables machine-guided design. Science. 2019;366:1139–43.
Duan D, Sharma P, Yang J, Yue Y, Dudus L, Zhang Y, et al. Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue. J Virol. 1998;72:8568–77.
Schnepp BC, Jensen RL, Chen CL, Johnson PR, Clark KR. Characterization of adeno-associated virus genomes isolated from human tissues. J Virol. 2005;79:14793–803.
Nakai H, Montini E, Fuess S, Storm TA, Meuse L, Finegold M, et al. Helper-independent and AAV-ITR-independent chromosomal integration of double-stranded linear DNA vectors in mice. Mol Ther. 2003;7:101–11.
Berns KI. The unusual properties of the AAV inverted terminal repeat. Hum Gene Ther. 2020;31:518–23.
Kotin RM, Menninger JC, Ward DC, Berns KI. Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter. Genomics .1991;10:831–4.
Donsante A, Miller DG, Li Y, Vogler C, Brunt EM, Russell DW, et al. AAV vector integration sites in mouse hepatocellular carcinoma. Science .2007;317:477.
Wang PR, Xu M, Toffanin S, Li Y, Llovet JM, Russell DW. Induction of hepatocellular carcinoma by in vivo gene targeting. Proc Natl Acad Sci USA. 2012;109:11264–9.
Pachori AS, Melo LG, Zhang L, Loda M, Pratt RE, Dzau VJ. Potential for germ line transmission after intramyocardial gene delivery by adeno-associated virus. Biochem Biophys Res Commun. 2004;313:528–33.
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.
Zhong L, Malani N, Li M, Brady T, Xie J, Bell P, et al. Recombinant adeno-associated virus integration sites in murine liver after ornithine transcarbamylase gene correction. Hum Gene Ther. 2013;24:520–5.
Chandler RJ, LaFave MC, Varshney GK, Trivedi NS, Carrillo-Carrasco N, Senac JS, et al. Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy. J Clin Invest. 2015;125:870–80.
Rosas LE, Grieves JL, Zaraspe K, La Perle KM, Fu H, McCarty DM. Patterns of scAAV vector insertion associated with oncogenic events in a mouse model for genotoxicity. Mol Ther. 2012;20:2098–110.
Gil-Farina I, Schmidt M. Interaction of vectors and parental viruses with the host genome. Curr Opin Virol. 2016;21:35–40.
Nault JC, Datta S, Imbeaud S, Franconi A, Mallet M, Couchy G, et al. Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas. Nat Genet. 2015;47:1187–93.
La Bella T, Imbeaud S, Peneau C, Mami I, Datta S, Bayard Q, et al. Adeno-associated virus in the liver: natural history and consequences in tumour development. Gut 2020;69:737–47.
Berns KI, Byrne BJ, Flotte TR, Gao G, Hauswirth WW, Herzog RW, et al. Adeno-associated virus type 2 and hepatocellular carcinoma? Hum Gene Ther. 2015;26:779–81.
Buning H, Schmidt M. Adeno-associated vector toxicity—to be or not to be? Mol Ther. 2015;23:1673–5.
Nault JC, Mami I, La Bella T, Datta S, Imbeaud S, Franconi A, et al. Wild-type AAV insertions in hepatocellular carcinoma do not inform debate over genotoxicity risk of vectorized AAV. Mol Ther. 2016;24:660–1.
Hanlon KS, Kleinstiver BP, Garcia SP, Zaborowski MP, Volak A, Spirig SE, et al. High levels of AAV vector integration into CRISPR-induced DNA breaks. Nat Commun. 2019;10:4439.
Miller DG, Petek LM, Russell DW. Adeno-associated virus vectors integrate at chromosome breakage sites. Nat Genet. 2004;36:767–73.
Nguyen GN, Everett JK, Kafle S, Roche AM, Raymond HE, Leiby J. et al. A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells. Nat Biotechnol. 2021;39:47–55.
Calcedo R, Vandenberghe LH, Gao G, Lin J, Wilson JM. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis. 2009;199:381–90.
Huser D, Khalid D, Lutter T, Hammer EM, Weger S, Hessler M. et al. High prevalence of infectious adeno-associated virus (AAV) in human peripheral blood mononuclear cells indicative of T Lymphocytes as Sites of AAV Persistence. J Virol. 2017;91:e02137–16.
Chen CL, Jensen RL, Schnepp BC, Connell MJ, Shell R, Sferra TJ, et al. Molecular characterization of adeno-associated viruses infecting children. J Virol. 2005;79:14781–92.
Boutin S, Monteilhet V, Veron P, Leborgne C, Benveniste O, Montus MF, et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther. 2010;21:704–12.
Halbert CL, Miller AD, McNamara S, Emerson J, Gibson RL, Ramsey B, et al. Prevalence of neutralizing antibodies against adeno-associated virus (AAV) types 2, 5, and 6 in cystic fibrosis and normal populations: implications for gene therapy using AAV vectors. Hum Gene Ther. 2006;17:440–7.
Gao G, Alvira MR, Somanathan S, Lu Y, Vandenberghe LH, Rux JJ, et al. Adeno-associated viruses undergo substantial evolution in primates during natural infections. Proc Natl Acad Sci USA. 2003;100:6081–6.
Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Cech M, et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018;46:W537–W44.
Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics .2010;26:2460–1.
Chandler RJ, LaFave MC, Varshney GK, Burgess SM, Venditti CP. Genotoxicity in mice following AAV gene delivery: a safety concern for human gene therapy? Mol Ther. 2016;24:198–201.
Logan GJ, Dane AP, Hallwirth CV, Smyth CM, Wilkie EE, Amaya AK, et al. Identification of liver-specific enhancer-promoter activity in the 3’ untranslated region of the wild-type AAV2 genome. Nat Genet. 2017;49:1267–73.
Ling C, Wang Y, Feng YL, Zhang YN, Li J, Hu XR, et al. Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions. J Integr Med. 2015;13:341–6.
Liu Q, Huang W, Zhang H, Wang Y, Zhao J, Song A, et al. Neutralizing antibodies against AAV2, AAV5 and AAV8 in healthy and HIV-1-infected subjects in China: implications for gene therapy using AAV vectors. Gene Ther. 2014;21:732–8.
Liu Q, Huang W, Zhao C, Zhang L, Meng S, Gao D, et al. The prevalence of neutralizing antibodies against AAV serotype 1 in healthy subjects in China: implications for gene therapy and vaccines using AAV1 vector. J Med Virol. 2013;85:1550–6.
Kramer CJH, Vangangelt KMH, van Pelt GW, Dekker TJA, Tollenaar R, Mesker WE. The prognostic value of tumour-stroma ratio in primary breast cancer with special attention to triple-negative tumours: a review. Breast Cancer Res Treat. 2019;173:55–64.
Mesker WE, Junggeburt JM, Szuhai K, de Heer P, Morreau H, Tanke HJ, et al. The carcinoma-stromal ratio of colon carcinoma is an independent factor for survival compared to lymph node status and tumor stage. Cell Oncol. 2007;29:387–98.
This work was supported by grants from the University of Massachusetts Medical School (an internal grant) and by the NIH (R01NS076991-01, P01AI100263-01, P01HL131471-02, R01AI121135, UG3HL147367-01, R01HL097088, and U19AI149646-01).
Conflict of interest
GG work has been funded by the NIH, and is a scientific co-founder of Voyager Therapeutics and Aspa Therapeutics, and holds equity in these companies. GG is an inventor on patents with potential royalties licensed to Voyager Therapeutics, Aspa Therapeutics, and other biopharmaceutical companies. The remaining authors declare no competing interests.
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Qin, W., Xu, G., Tai, P.W.L. et al. Large-scale molecular epidemiological analysis of AAV in a cancer patient population. Oncogene 40, 3060–3071 (2021). https://doi.org/10.1038/s41388-021-01725-5