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PreprintCited by 4Virus-based gene therapy researchCRISPR and Genetic EngineeringRNA Interference and Gene Delivery

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

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Puzzo, Francesco, Crossley, Magdalena P., Goswami, Aranyak, Zhang, Feijie, Pekrun, Katja, Garzon, Jada L., Cimprich, Karlene A., Kay, Mark A.·2024·Molecular Therapy
DOI 10.1101/2024.05.07.592855PMC PMC11638834

Preprint — published as 10.1016/j.ymthe.2024.09.035 · Paper page

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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, 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.

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Metadata

Authors
Puzzo, F., Crossley, M. P., Goswami, A., Zhang, F., Pekrun, K., Garzon, J. L., Cimprich, K. A., & Kay, M. A.
Affiliations
  • Stanford Medicine (US)
  • Stanford University (US)
Corresponding author
Kay, Mark A.
ORCID
Venue
Molecular Therapy
Publisher
openRxiv
Published
2024-05-09
Volume
32
Issue
12
Pages
4256-4271
ISSN
1525-0016 (ISSN-L)
DOI
10.1101/2024.05.07.592855
PMC
PMC11638834
OpenAlex
W4403117174
Cited by
4
References
1
Citations by year
2026: 3 · 2024: 1
FWCI
1.06
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Molecular Therapy (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Funders
  • National Science Foundation (2146755)
  • Glenn Foundation for Medical Research
  • Fondation pour la Recherche Médicale
  • National Institutes of Health (1s10od018220-01, 1s10od021763-01)
  • National Heart, Lung, and Blood Institute (R01HL064272, R01HL064274)
  • National Institute of General Medical Sciences (GM119334, T32GM136631, R01GM119334)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
recombinant adeno-associated virus vectors, R-loops, homologous recombination, genome editing, AAV integration, albumin gene, hepatoma cells, liver, transcribed regions, gene therapy safety

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