High-Level Human Adenosine Deaminase Expression in Dog Skin Fibroblasts Is Not Sustained Following Transplantation

Abstract

Primary skin fibroblasts are an attractive target tissue for retroviral-mediated gene therapy; however, transient expression of therapeutic genes has been a recurrent problem in several rodent models. The gradual decrease in gene expression could be tissue or species specific. To investigate the phenomenon further, human adenosine deaminase (ADA) expression was monitored in genetically modified skin fibroblasts transplanted in beagle dogs. In culture, transduced canine fibroblasts expressed high levels of human ADA activity (33.6 μmoles adenosine metabolized per hour per milligram of soluble protein) in comparison to canine ADA in untreated control cells (1.3 μmole/hr · mg protein) and for 2 weeks following transplantation, the graft contained up to four-fold more enzyme activity from human ADA than the endogenous canine enzyme. However, by 10 weeks, human ADA expression was undetectable. At the time when human ADA expression was greatly reduced, DNA analysis showed the presence of vector sequences. These results directly parallel those observed in rodent models and suggest retroviral vector inactivation is a tissue-specific rather than species-specific mechanism. A major question in human gene therapy protocols is how long the transferred gene will remain active in the target cells. Unfortunately, many constructs that appear to maintain stable gene expression in cultured cells do not do so in vivo in mice. Ramesh et al. have studied this phenomenon in primary skin fibroblasts transplanted into dogs using the adenosine deaminase gene. They report that the same loss of gene expression occurs in the canine in vivo as occurs in rodents in vivo.