Biologic aspects of expression of stably integrated transgenes in cells ofthe skin in vitro and in vivo

The observation that transgenes can be stably integrated into the genome of fibroblasts using recombinant retroviruses enhanced interest in using thes e cells as a vector for gene therapy. This enthusiasm has lessened during t he past 8 years, not because skin has lost the features that make it attrac tive for gene therapy, but rather because stable transgene expression in vi vo has not been achieved. All investigators who have used genetically modif ied fibroblasts to study in vivo aspects of gene therapy have shown a decre ase in transgene expression with time. This contrasts with transgene expres sion in similarly transduced fibroblasts in vitro, where expression is not lost or is lost very slowly. We have initiated an approach to bring further understanding to the biology of transgene expression by fibroblasts carrying stably integrated transgen es in an in vivo setting. Experiments described permit the following conclu sions. Expression by and survival of genetically modified fibroblasts a) re quires a persistent matrix scaffold in in vivo settings; b) is prolonged if the matrix is allowed to mature in vitro; c) is enhanced if the matrix is partially sequestered behind a coating of normal fibroblasts; and d) can be substantively prolonged in vivo by immortalizing the cells. These observat ions support the notion that prolonged expression of transgenes by fibrobla sts can be achieved in vivo and that gene therapy utilizing fibroblasts and other cells of the skin has clinical utility.