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Y. Ikeda, T. Tabata, K. Mitomo, M. Miyazaki, R.-I. Kohno, Y. Murakami, Y. Yonemitsu, M. Inoue, M. Hasegawa, T. Ishibashi; Preclinical Safety Study of Simian Immunodeficiency Virus (SIV)-Based Lentiviral Vector for Retinal Gene Therapy: Characterization of the Integration Sites. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3029.
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Recently, we have demonstrated the therapeutic outcome in some animal models of retinal degeneration using the recombinant simian immunodeficiency virus (SIV)-based lentiviral vector carrying neurotrophic factors. To prevent adverse events in clinical gene therapy trials, it is important to precisely evaluate the potential safety issues surrounding the gene transfer vectors themselves. In this preclinical safety study, we evaluated integration pattern of our SIV vector into host-cell chromosomes, because they have a chance of causing insertional mutagenesis.
We examined targeting of our SIV vector carrying human pigment epithelium-derived factor (SIV-hPEDF) in human retinal pigment epithelium (RPE)-derived cell line, ARPE-19. ARPE-19 was grown on plastic culture dishes in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium with 3 mM L-glutamine and 10% fetal bovine serum (FBS). Gene transfer was performed by adding 4x106 TU (transducing units) of SIV-hPEDF to the culture media of the RPE cells (1x106 cells). After 3 days incubation, RPE cells were collected and RNA solutions were extracted. We analyzed the integration through linear amplification-mediated PCR (LAM-PCR) method.
Up to 747 different sites of the SIV integration were sequenced and mapped in human genome. SIV-hPEDF favored gene-dense regions and transcription units of the genome for integration. Integration in cancer-related genes or that near their promoters was observed, however, there was no particular preference for the integration to them.
Our data indicate that the integration site preferences of our SIV-hPEDF show much the same tendency of other lentiviral vectors, which were previously reported.
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