CLONAL ANALYSIS OF STABLY TRANSDUCED HUMAN EPIDERMAL STEM-CELLS IN CULTURE

We have transduced normal human keratinocytes with retroviral construc ts expressing a bacterial beta-galactosidase (beta-gal) gene or a huma n interleukin-6 (hIL-6) cDNA under control of a long terminal repeat. Efficiency of gene transfer averaged approximately 50% and 95% of clon ogenic keratinocytes for beta-gal and hIL-6, respectively. Both genes were stably integrated and expressed for more than 150 generations. Cl onal analysis showed that both holoclones and their transient amplifyi ng progeny expressed the transgene permanently. Southern blot analysis on isolated clones showed that many keratinocyte stem cells integrate d multiple proviral copies in their genome and that the synthesis of t he exogenous gene product in vitro was proportional to the number of p roviral integrations. When cohesive epidermal sheets prepared from ste m cells transduced with hIL-6 were grafted on athymic animals, the ser um Levels of hIL-6 were strictly proportional to the rate of secretion in vitro and therefore to the number of proviral integrations. The po ssibility of specifying the level of transgene expression and its perm anence in a homogeneous clone of stem cell origin opens new perspectiv es in the long-term treatment of genetic disorders.