Identification of liver-specific enhancer–promoter activity in the 3′ untranslated region of the wild-type AAV2 genome

Abstract

Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications1,2. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC)4 has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety5,6,7,8,9. Here we describe and functionally characterize a previously unknown liver-specific enhancer–promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes4 and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC10.

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Figure 1: An AAV vector lacking a heterologous promoter drives transgene expression in vitro and in vivo.
Figure 2: Mapping of the 3′ UTR of the cap gene in WT AAV2 for transcriptional activity.
Figure 3: The 2/1-105wt element has ITR-independent enhancer–promoter activity.
Figure 4: The 2/1-105wt element from WT AAV2 operates in a cell-type-specific manner and drives transgene expression in mouse and human hepatocytes in vivo.

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Acknowledgements

We thank M. Latham for assistance in manuscript preparation, M. Grompe (Oregon Stem Cell Center, Oregon Health and Science University) for the FRG mice, J. Laurence (University of Sydney) for the HuH-7 cells, S. Goss (University of Oxford) for the BWTG3 cells and G. Sharbeen (University of New South Wales) for the MiaPaCa2 pancreatic carcinoma cell line. This work was supported by an Australian National Health and Medical Research Council (NHMRC) Postgraduate Research Scholarship (477110; A.P.D.), Children's Medical Research Institute (CMRI) Ph.D. stipends (A.P.D., A.K.A. and M.C.-C.), a University of Sydney International Scholarship (A.K.A.), an Institute of Child Health, London stipend (M.C.-C.) and NHMRC project grants APP1008021 (I.E.A.), APP1022498 (I.E.A.) and APP1080330 (I.E.A.).

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G.J.L. conceived, designed and performed experiments, analyzed data and wrote the manuscript; A.P.D. conceived and performed experiments; C.V.H. and E.E.W. analyzed data; C.M.S., A.K.A., E.Z., N.K., S.L.G., S.H.Y.L., S.C.C., N.S. and M.C.-C. performed experiments; P.P.L.T. jointly supervised the research; D.W.R. and L.L. conceived experiments; and I.E.A. jointly supervised the research, conceived and designed experiments, analyzed data and wrote the manuscript.

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Correspondence to Ian E Alexander.

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Logan, G., Dane, A., Hallwirth, C. et al. Identification of liver-specific enhancer–promoter activity in the 3′ untranslated region of the wild-type AAV2 genome. Nat Genet 49, 1267–1273 (2017). https://doi.org/10.1038/ng.3893

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