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. 2024 Dec 4;32(12):4256-4271.
doi: 10.1016/j.ymthe.2024.09.035. Epub 2024 Oct 5.

AAV-mediated genome editing is influenced by the formation of R-loops

Francesco Puzzo  1 , Magdalena P Crossley  2 , Aranyak Goswami  1 , Feijie Zhang  1 , Katja Pekrun  1 , Jada L Garzon  2 , Karlene A Cimprich  2 , Mark A Kay  3
Affiliations

AAV-mediated genome editing is influenced by the formation of R-loops

Francesco Puzzo et al. Mol Ther. .

Abstract

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, may promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

Keywords: AAV; R-loops; gene editing; homologous recombination; in vivo DRIP-seq; liver gene therapy; vector integration.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Update of

References

    1. Li C., Samulski R.J. Engineering adeno-associated virus vectors for gene therapy. Nat. Rev. Genet. 2020;21:255–272. doi: 10.1038/S41576-019-0205-4. - DOI - PubMed
    1. Mendell J.R., Al-Zaidy S.A., Rodino-Klapac L.R., Goodspeed K., Gray S.J., Kay C.N., Boye S.L., Boye S.E., George L.A., Salabarria S., et al. Current Clinical Applications of In Vivo Gene Therapy with AAVs. Mol. Ther. 2021;29:464–488. doi: 10.1016/j.ymthe.2020.12.007. - DOI - PMC - PubMed
    1. Nakai H., Fuess S., Storm T.A., Meuse L.A., Kay M.A. Free DNA ends are essential for concatemerization of synthetic double-stranded adeno-associated virus vector genomes transfected into mouse hepatocytes in vivo. Mol. Ther. 2003;7:112–121. doi: 10.1016/s1525-0016(02)00034-5. - DOI - PubMed
    1. Wang L., Wang H., Bell P., McMenamin D., Wilson J.M. Hepatic gene transfer in neonatal mice by adeno-associated virus serotype 8 vector. Hum. Gene Ther. 2012;23:533–539. doi: 10.1089/hum.2011.183. - DOI - PMC - PubMed
    1. Barzel A., Paulk N.K., Shi Y., Huang Y., Chu K., Zhang F., Valdmanis P.N., Spector L.P., Porteus M.H., Gaensler K.M., Kay M.A. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature. 2015;517:360–364. doi: 10.1038/nature13864. - DOI - PMC - PubMed

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