1. ¹Molecular Immunology Unit, Institute of Child Health, University College London, London, UK
2. ²Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany
3. ³Cell Biology and Development Director, Gene Therapy Program Institute of Human Genetics, Minneapolis, Minnesota, USA

Correspondence: Waseem Qasim, Molecular Immunology Unit, Institute of Child Health, University College London, 30 Guilford Street, London, WC1N 1EH, United Kingdom. E-mail: W.Qasim@ICH.UCL.AC.UK

Received 22 December 2008; Accepted 3 April 2009; Published online 5 May 2009.

Abstract

Lentiviral vectors enter cells with high efficiency and deliver stable transduction through integration into host chromosomes, but their preference for integration within actively transcribing genes means that insertional mutagenesis following disruption of host proto-oncogenes is a recognized concern. We have addressed this problem by combining the efficient cell and nuclear entry properties of HIV-1–derived lentiviral vectors with the integration profile benefits of Sleeping Beauty (SB) transposase. Importantly, this integration enzyme does not exhibit a preference for integration within active genes. We generated integrase-deficient lentiviral vectors (IDLVs) to carry SB transposon and transposase expression cassettes. IDLVs were able to deliver transient transposase expression to target cells, and episomal lentiviral DNA was found to be a suitable substrate for integration via the SB pathway. The hybrid vector system allows genomic integration of a minimal promoter-transgene cassette flanked by short SB inverted repeats (IRs) but devoid of HIV-1 long terminal repeats (LTRs) or other virus-derived sequences. Importantly, integration site analysis revealed redirection toward a profile mimicking SB-plasmid integration and away from integration within transcriptionally active genes favored by integrase-proficient lentiviral vectors (ILVs).