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Characterization of integration frequency and insertion sites of adenovirus DNA into mouse liver genomic DNA following intravenous injection


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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Fig. 1: FG-leptin and HD-leptin structures and vector primers for RAIC PCR.
Fig. 2: Southern analysis to examine the in vivo state of adenoviral DNA.
Fig. 3: Pulsed-field gel to remove free adenovirus vector DNA from HMW genomic DNA.
Fig. 4: Strategy of the RAIC PCR assay.
Fig. 5: Integrations in the FG-leptin-treated mouse liver DNA identified by RAIC PCR.
Fig. 6: Integration FG22 contained a rearrangement of both the mouse genomic and the vector DNA.
Fig. 7: Integrations in the FG-leptin-treated mouse liver DNA identified by RAIC PCR.
Fig. 8: Integrations in the HD-leptin-treated mouse liver DNA identified by RAIC PCR.


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Authors and Affiliations



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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Correspondence to Zhibin Wang.

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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).

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