A novel approach to gene therapy of albino hair in histoculture with a retroviral streptomyces tyrosinase gene.

Abstract

In order to induce melanin production in mammalian cells with pigment disorders such as albino hair, a recombinant retrovirus containing the mel locus of Streptomyces antibioticus was constructed. The S. antibioticus mel locus, which consists of the open reading frame (ORF)-438 and the tyrosinase gene, was specifically derived by polymerase chain reaction (PCR) from Streptomyces plasmid pIJ702. The ORF-438 is required for the transfer of copper to apotyrosinase, which is essential for tyrosinase enzymatic activity. The tyrosinase gene was inserted into the XhoI/BamHI cloning site of the pLXSN retroviral vector to obtain pLtyrSN. An internal ribosome entry site (IRES) suitable for mammalian cell expression was obtained from the pLXIN retroviral vector by PCR. The ORF-438 and IRES DNA fragments were inserted into the pLtyrSN vector to obtain the tyrosinase-expression retroviral vector pLmelSN. The expression vector was amplified in murine PT67 packaging cells, where the ORF-438 and tyrosinase genes were also co-expressed as determined by reverse transcription-PCR. In order to evaluate the vector's ability to restore pigment production in cells with a pigment disorder, albino-mouse skins were histocultured and then infected with the pLmelSN retrovirus. Six days after infection, melanin granules were observed in approximately 60% of albino-mouse hair follicles in the histocultured skin. These results demonstrated that the S. antibioticus mel operon could express an active tyrosinase and produce melanin in the albino-mouse hair follicles. This novel gene therapy approach, using a small and simple tyrosinase operon in a high-expression vector, has a potentially wide application for therapy of pigment disorders in hair follicles.