title: Effective retrovirus-mediated gene transfer in normal and mutant human melanocytes.
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 increased 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, including 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 pigmentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the presence 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 endogenous 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 wildtype 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, human melanocytes with nonmitogenic genes.
authors.affiliation: TIGEM, Telethon Institute of Genetics and Medicine, 20132 Milan, Italy. schiaffino.mariavittoria@hsr.it
authors.forename: Maria Vittoria
authors.initials: MV
authors.lastname: Schiaffino
authors.forename: Elena
authors.initials: E
authors.lastname: Dellambra
authors.forename: Katia
authors.initials: K
authors.lastname: Cortese
authors.forename: Cinzia
authors.initials: C
authors.lastname: Baschirotto
authors.forename: Sergio
authors.initials: S
authors.lastname: Bondanza
authors.forename: Maurizio
authors.initials: M
authors.lastname: Clementi
authors.forename: Paolo
authors.initials: P
authors.lastname: Nucci
authors.forename: Andrea
authors.initials: A
authors.lastname: Ballabio
authors.forename: Carlo
authors.initials: C
authors.lastname: Tacchetti
authors.forename: Michele
authors.initials: M
authors.lastname: De Luca
mesh_terms: D015415:Biomarkers; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D016368:Frameshift Mutation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008297:Male; D008544:Melanocytes / Q000378:metabolism*; D020460:Melanosomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D018895:Microsatellite Repeats; D010375:Pedigree; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic
chemical_list: D015415:Biomarkers; D005136:Eye Proteins; C095276:GPR143 protein, human; D008562:Membrane Glycoproteins