HUMAN GENE THERAPY 13:947–957 (May 20, 2002) © Mary Ann Liebert, Inc. Effective Retrovirus-Mediated Gene Transfer in Normal and Mutant Human Melanocytes MARIA VITTORIA SCHIAFFINO, 1,2 ELENA DELLAMBRA, 3 KATIA CORTESE, 4 CINZIA BASCHIROTTO, 1 SERGIO BONDANZA, 3 MAURIZIO CLEMENTI, 5 PAOLO NUCCI, 6 ANDREA BALLABIO, 1,7,8 CARLO TACCHETTI, 4 and MICHELE DE LUCA 3 ABSTRACT Melanocytes represent the second most important cell type in the skin and are primarily responsible for the pigmentation of skin, hair, and eyes. Their function may be affected in a number of inherited and acquired disorders, characterized by hyperpigmentation or hypopigmentation, consequent aesthetic problems, and in- creased susceptibility to sun-mediated skin damage and photocarcinogenesis. Nevertheless, the possibility of genetically manipulating human melanocytes has been hampered so far by a number of limitations, includ- ing their resistance to retroviral infection. To address the problem of human melanocyte transduction, we generated a melanocyte culture from a patient affected with ocular albinism type 1 (OA1), an X-linked pig- mentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the pres- ence of macromelanosomes in skin melanocytes and retinal pigment epithelium (RPE). The cultured patient melanocytes displayed a significant impairment in replication ability and showed complete absence of en- dogenous OA1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure. To correct the genetic defect in these cells, we used a retroviral vector carrying the OA1 cDNA and exploited a melanocyte–keratinocyte coculturing approach. Despite their lower replication rate with respect to wild- type cells, the patient melanocytes were efficiently transduced and readily selected in vitro, and were found to express, process, and properly sort large amounts of recombinant OA1 protein. These results indicate the feasibility of efficiently and stably transducing in vitro not only normal neonatal, but also mutant adult, hu- man melanocytes with nonmitogenic genes. 947 OVERVIEW SUMMARY The skin represents one of the most attractive target tissues for ex vivo gene therapy, not only for its accessibility, but also for the availabilityof advanced skin culture and surgery tech- niques. Moreover, we previously showed that the main cell type in the skin, the keratinocyte and its precursor stem cells, can be efficiently and stably transduced by retroviral vectors and selected in vitro. However, no effective gene transfer pro- cedure has been developed yet for the melanocytes, despite the fact that these cells represent the second physiologically most important cell type in the skin. We now report the set- ting up of an improved gene transfer method for normal and mutant human melanocytes, allowing the expression of non- mitogenic recombinant proteins in high amounts and in a uniform and a stable manner. Our findings indicate the fea- sibility of an ex vivo gene therapy approach for the treatment of inherited and acquired pigmentation disorders. 1 TIGEM, Telethon Institute of Genetics and Medicine, 20132 Milan, Italy. 2 Present address: DIBIT, Scientific Institute San Raffaele, 20132 Milan, Italy. 3 Laboratory of Tissue Engineering, IDI IRCCS, Istituto Dermopatico dell’Immacolata, 00040 Pomezia (Rome), Italy. 4 Department of Experimental Medicine, Anatomy Section, University of Genoa, 16132 Genoa, Italy. 5 Medical Genetics, Department of Pediatrics, University of Padua, 35128 Padua, Italy. 6 Department of Ophthalmology, San Raffaele Hospital, 20132 Milan, Italy. 7 San Raffaele Faculty of Medicine, 20132 Milan, Italy. 8 Present address: TIGEM, Telethon Institute of Genetics and Medicine, and Second University of Naples, 80131 Naples, Italy.