Use of Cloned Genetically Modified Human Fibroblasts to Assess Long-Term Survival In Vivo

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Abstract
Because human fibroblasts are easily brought to tissue culture conditions and can be stably transduced with retroviral vectors encoding transgenes ex vivo, genetically modified fibroblasts are frequently considered in strategies to correct disease with gene therapy. This enthusiasm has been dampened by studies showing that transgene expression by genetically modified fibroblasts diminishes with time in vivo, but not in vitro, for reasons that are unclear. We elected to study this problem using cloned human fibroblasts that had been cloned by limiting dilution and stably transduced with a retroviral vector encoding lacZ ex vivo. These were seeded onto a nonbiodegradable nylon matrix that was transplanted to nude mice. Transgene expression was followed prospectively by histologic exam. Data show that human fibroblasts can withstand the pressure of cloning by limiting dilution. In addition, they can be passaged from 10 to >20 times, and >1 × 1020 of genetically modified fibroblasts can be generated as progeny of one cell. Loss of transgene expression by the cloned genetically modified fibroblasts in vivo occurs in an orderly and progressive fashion, but is not complete by 4 months. Neither the loss nor the persistence of expression appear to be random. These observations are most compatible with the thesis that a major cause of the loss of transgene expression in vivo is secondary to apoptosis of the genetically modified fibroblast. Loss of expression of transgenes in senescent genetically modified fibroblasts occurs more rapidly than in their presenescent counterparts in the age-neutral, in vivo setting of the nude mouse. Although human fibroblasts can be easily and stably transduced with retroviral vectors encoding transgenes ex vivo, the loss of transgene expression in vivo, for reasons that are unclear, diminishes their clinical utility as a shuttle for gene therapy. In the system we have developed, all of the stably transduced fibroblasts have the same ancestry; that is, the transgene is integrated into the same site on the genome, and the matrix carrying the genetically modified fibroblast is a cassette that can be modulated in vitro, placed in vivo, removed from this setting with the genetically modified fibroblast intact, and analyzed. This system has been used to begin to understand more clearly the etiology of the loss of transgene expression in vivo. We show that loss of transgene expression or loss of genetically modified fibroblasts in vivo is an organized event that appears linked to the age of genetically modified human fibroblasts and apoptotic pathways inherent to the in vivo setting. These conclusions support the notion that gene therapy strategies need to be directed at altering the cell and its milieu, as well as developing new vectors, better promoters, and methods to deliver transgenes.