Adeno-associated virus Type 2 Rep proteins mediate integration of lentiviral vectors

Aims: Adeno-associated virus type 2 (AAV2) is a naturally defective human parvovirus that is being developed as a gene therapy vector. In dividing cells, AAV2 DNA persists by integration into the host chromosomes. AAV2 is unique among mammalian viruses in its ability to integrate preferentially into a particular locus within human chromosome 19, designated AAVS1(also known as Mbs 85). The AAV2 Rep68 and Rep78 proteins mediate this integration. Recent data suggest that Rep68 and Rep78 can mediate integration of nonAAV2 DNA with free ends. To test this hypothesis, we targeted insertion of different lentiviral vectors to AAVS1. Methods: Cells were co-infected with wild-type AAV2, and integraseproficient or integrase-deficient lentivirus vectors. A highly specific PCR-based assay was used to detect lentivirus integration at AAVS1. Similar experiments were performed using lentiviral vectors containing the AAV2 rep gene. Results: All lentiviral vectors tested integrated at AAVS1, if the rep gene was present either within the lentiviral vector or supplied in trans. All that was required for integration at AAVS1 was the amino acid sequence shared between Rep68 and Rep78. The results were similar with integrase-proficient or integrasedeficient lentiviral vectors. Conclusions: The inclusion of the rep gene with lentiviral vectors may produce more predictable integration patterns.