title: Lentiviral protein transduction with genome-modifying HIV-1 integrase-I-PpoI fusion proteins: studies on specificity and cytotoxicity.
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.
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland ; FinVector Vision Therapies Oy, 70210 Kuopio, Finland.
authors.forename: Vesa
authors.identifier: 0000-0001-9626-1966
authors.initials: V
authors.lastname: Turkki
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland.
authors.forename: Diana
authors.initials: D
authors.lastname: Schenkwein
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland.
authors.forename: Oskari
authors.identifier: 0000-0002-6317-6260
authors.initials: O
authors.lastname: Timonen
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland.
authors.forename: Tiia
authors.identifier: 0000-0001-7536-451X
authors.initials: T
authors.lastname: Husso
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland ; FKD Therapies Oy, 70210 Kuopio, Finland.
authors.forename: Hanna P
authors.initials: HP
authors.lastname: Lesch
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland ; Department of Medicine, University of Eastern Finland, 70120 Kuopio, Finland ; Gene Therapy Unit, Kuopio University Hospital, 70120 Kuopio, Finland.
authors.forename: Seppo
authors.identifier: 0000-0001-7593-2708
authors.initials: S
authors.lastname: Ylä-Herttuala
mesh_terms: D002470:Cell Survival / Q000187:drug effects / Q000235:genetics*; D004706:Endodeoxyribonucleases / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome / Q000235:genetics*; D019427:HIV Integrase / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D017393:RNA Editing / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000633:toxicity*; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D011993:Recombinant Fusion Proteins; D019427:HIV Integrase; D004706:Endodeoxyribonucleases; C064579:I-Ppo endonuclease; C514801:p31 integrase protein, Human immunodeficiency virus 1