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BioMed Research International
Volume 2014, Article ID 379340, 11 pages
http://dx.doi.org/10.1155/2014/379340
Research Article

Lentiviral Protein Transduction with Genome-Modifying HIV-1 Integrase-I-PpoI Fusion Proteins: Studies on Specificity and Cytotoxicity

1Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland
2FinVector Vision Therapies Oy, 70210 Kuopio, Finland
3FKD Therapies Oy, 70210 Kuopio, Finland
4Department of Medicine, University of Eastern Finland, 70120 Kuopio, Finland
5Gene Therapy Unit, Kuopio University Hospital, 70120 Kuopio, Finland

Received 26 January 2014; Accepted 12 March 2014; Published 22 April 2014

Academic Editor: Brian Oliver

Copyright © 2014 Vesa Turkki et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Rare-cutting endonucleases, such as the I-PpoI, can be used for the induction of double strand breaks (DSBs) in genome editing and targeted integration based on homologous recombination. For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques. For its applications, the endonuclease needs to be transported into the target cell nucleus, where the mechanism of transport may affect its function. Here, we have studied the lentiviral protein transduction of the integrase (IN)-PpoI fusion protein using the cis-packaging method. In genome-wide interaction studies, IN-fusion proteins were verified to bind their target sequence containing 28S ribosomal RNA (rRNA) genes with a 100-fold enrichment, despite the well-documented behavior of IN to be tethered into various genomic areas by host-cell factors. In addition, to estimate the applicability of the method, DSB-induced cytotoxic effects with different vector endonuclease configurations were studied in a panel of cells. Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA. In cell studies, certain cancerous cell lines were especially prone to DSBs in rRNA genes, which led us to test the protein transduction in a tumour environment in an in vivo study. In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases.