Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase
Open accessAbstract
Background: Circular forms of viral genomic DNA are generated during infection of cells with retroviruses like HIV-1. Such circles are unable to replicate and are eventually lost as a result of cell division, lending support to the prevalent notion that episomal retroviral DNA forms are dead-end products of reverse transcription. Results: We demonstrate that circular DNA generated during transduction with HIV-1-based lentiviral vectors can be utilized as substrate for gene insertion directed by nonviral recombinases co-expressed in the target cells. By packaging of lentiviral genomic RNA in integrase-defective lentiviral vectors, harboring an inactive form of the viral integrase, the normal pathway for viral integration is blocked and circular vector DNA accumulates in transduced cells as a result. We find that the amount of DNA circles is increased 4-fold in cells transduced with integration-defective vectors relative to cells treated with integrase-proficient vectors. By transduction of target cells harboring engineered recognition sites for the yeast Flp recombinase with integration-defective lentiviral vectors containing an ATG-deficient hygromycin B selection gene we demonstrate precise integration of lentiviral vector-derived DNA circles in a drug-selective approach. Moreover, it is demonstrated that trans-acting Flp recombinase can be delivered by Flp-encoding transfected plasmid DNA or, alternatively, by co-transduced integrase-defective lentiviral vectors carrying a Flp expression cassette. Conclusion: Our data provide proof-of-principle that nonviral recombinases, like Flp, produced by plasmid DNA or non-integrating lentiviral vectors can gain access to circular viral recombination substrates and facilitate site-directed genomic insertion of such episomal DNA forms. Replacement of the normal viral integration machinery with nonviral mediators of integration represents a new platform for creation of lentiviral vectors with an altered integration profile.
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Non-viral yeast Flpx9 recombinase produced by NILV can enter circular viral recombination substrates and facilitate site-directed genomic insertion.
“Another study explained that non-viral yeast Flpx9 recombinase produced by NILV can enter the circular viral recombination substrates and facilitate the site-directed genomic insertion [111].”
From Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through (2022) · cites this paper as [111]
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- Authors
- Moldt, B., Staunstrup, N. H., Jakobsen, M., Yáñez-Muñoz, R. J., & Mikkelsen, J. G.
- Affiliations
- Aarhus University (DK)
- Corresponding author
- Mikkelsen, Jacob G
- Venue
- BMC Biotechnology
- Published
- 2008-08-09
- Volume
- 8
- Issue
- 1
- Pages
- 60-60
- ISSN
- 1472-6750 (ISSN-L)
- PMC
- PMC2538528
- OpenAlex
- W2115065031
- Cited by
- 44
- References
- 3
- Citations by year
- 2024: 1 · 2023: 1 · 2022: 4 · 2021: 1 · 2020: 2 · 2018: 1 · 2017: 1 · 2016: 1 · 2015: 2 · 2014: 2 · 2013: 4 · 2012: 1
- FWCI
- 4.37
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- BMC Biotechnology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Royal Holloway Digital Repository (Royal Holloway University of London) (submittedVersion, cc-by)
- Royal Holloway Digital Repository (Royal Holloway University of London) (submittedVersion)
- Funders
- Aarhus Universitet
- Kræftens Bekæmpelse
- Sundhed og Sygdom, Det Frie Forskningsråd
- University College London
- École Polytechnique Fédérale de Lausanne
- Novo Nordisk
- Novo Nordisk Fonden
- University of California, San Francisco
- Medical Research Council
- 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
- lentiviral vectors, circular DNA, episomal DNA, Flp recombinase, viral integration, hygromycin B selection, HIV-1
- MeSH terms
- DNA Nucleotidyltransferases, DNA, Circular, Fungal Proteins, Genetic Vectors, Transfection, Lentivirus, Mutagenesis, Insertional, Genome, Viral
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