Biologic Aspects of Expression of Stably Integrated Transgenes in Cells of the Skin In Vitro and In Vivo

Abstract

The observation that transgenes can be stably integrated into the genome of fibroblasts using recombinant retroviruses enhanced interest in using these cells as a vector for gene therapy. This enthusiasm has lessened during the past 8 years, not because skin has lost the features that make it attractive for gene therapy, but rather because stable transgene expression in vivo has not been achieved. All investigators who have used genetically modified fibroblasts to study in vivo aspects of gene therapy have shown a decrease in transgene expression with time. This contrasts with transgene expression 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 transgenes in an in vivo setting. Experiments described permit the following conclusions. Expression by and survival of genetically modified fibroblasts a) requires 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 observations support the notion that prolonged expression of transgenes by fibroblasts can be achieved in vivo and that gene therapy utilizing fibroblasts and other cells of the skin has clinical utility.

A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy

“…Fibroblasts are easy to obtain, cultivate, and store, and the delivery of these robust cells via intradermal injections is practical. In view of regulatory and/or gene therapy vector issues, it would be advantageous that the cells synthesize sufficient amounts of the therapeutic protein yet not require genetic modification, because preliminary studies have shown that genetically modified fibroblasts (29,30) may be effective, but can lose the transgene expression within a relatively short time (38).…”

Endothelial cell–based systemic gene therapy of metastatic melanoma

“…55 -59 However, infusion of lymphocytes in patients has been associated with serious side effects, 32,57 and fibroblasts have been reported to be incapable of maintaining long -term survival or expression of exogenous genes in vivo in the absence of a matrix scaffold. 55 The results of the present study demonstrate for the first time that targeted delivery of therapeutic gene -expressing endothelial cells to sites of tumor-associated angiogenesis is a feasible, efficient, safe, and potentially effective strategy to treat established tumor metastases in the lungs.…”

Gene Transfer into Human Keloid Tissue with Adeno-Associated Virus Vector

“…Furthermore, stable transgene expression in vivo has not yet been achieved by retrovirus. 38 Plasmid DNA that contains the desired gene can be injected intradermally, and it is rapidly absorbed and expressed by the epidermis. However, gene expression after plasmid injection is only transient.…”