Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase.
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.


CONCLUSIONS
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.
Moldt
Brian
B
Department of Human Genetics, University of Aarhus, Aarhus, Denmark. moldt@humgen.au.dk
Staunstrup
Nicklas H
NH
Jakobsen
Maria
M
Yáñez-Muñoz
Rafael J
RJ
Mikkelsen
Jacob G
JG
D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004270:DNA, Circular / Q000235:genetics*; D005656:Fungal Proteins / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D014162:Transfection / Q000379:methods*
D004270:DNA, Circular; D005656:Fungal Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase