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Cluster assembled nanostructured TiO2 film mediates efficient and safe retroviral gene transduction in primary adult human melanocytes for ex-vivo gene therapy

R. Carbone, I. Marangi, A. Zanardi, L. Lanfrancone, S. Minucci, P.G. Pelicci, L. Giorgetti, E. Clerici, G. Bongiorno, P. Piseri and P. Milani
European Institute of Oncology, IT

nanostructured TiO2 film, primary human melanocytes, retroviral infection, gene therapy

Primary human melanocytes represent the precursor cells to melanoma and also specific targets of inherited and acquired pigmentation disorders (1,3). The possibility to genetically manipulate these cells for biological studies or therapy has not been fully exploited for the lack of efficient and safe stable gene transfer techniques (4). We have developed a new gene transfer technology on primary adult human melanocytes by means of retroviral infection in absence of any toxic polycations currently employed to improve infection efficency (i.e. polybrene). We demonstrate that this polycation induce apoptosis and DNA damage through ATM phosphorylation, causing a further genetic perturbation to cells. We have cultured primary human melanocytes on a biocompatible nanostructured TiO2 film, obtained by the deposition of a supersonic beam of titania clusters (5, 7), coated by retroviral vectors expressing GFP. By means of a “reverse infection “ mechanism we achieved 80% of infection in absence of any toxic effect. This high efficiency is related to the interaction of retroviral vectors and cells with the nanoparticle-assembled substrates. We expect that such technology based on nanostructured surfaces will allow efficient and safe genetic manipulation of primary cells for ex-vivo gene therapy.

1.Sober A.J. et al. (1991). Epidemiology of cutaneous melanoma. An update. Dermatol. Clin. 9, 617-629.
2. King R.A. et al. (1995) Albinism. In the metabolic and molecular bases of Inherited disease C.R. Scriver, A.L. Beaudel, W.S. Sly, and A. Valle, eds (McGraw-Hill, New York) pp. 4353-4392
3.Incerti B. et al. (2000) OA1 Knock-out: new insight on the pathogenesis of ocular albinism type 1. Hum. Mol. Genet. 9, 2781-2788.
4. Schiaffino M.V. et al. Effective retrovirus Mediated Gene transfer in Normal and Mutant Human Melanocytes Hum. Gene Ther. 13; 947-957
5. T. Mazza et al. Libraries of cluster-assembled titania films for chemical sensing, Appl. Phys. Lett. 87, 103108 (2005)
6. Milani, P. and Iannotta, S. Cluster beam synthesis of nanostructured materials. Springer (Berlin), 1999.
7. Carbone R. et al (2005 ) submitted to Biomaterials

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