Mechanisms of Transgene Silencing in Neural Cells -Implications for Ex Vivo Gene Therapy to the Brain

Abstract: The use of genetically modifi ed neural stem and progenitor cells could possibly be a future strategy for repairing a brain suffering from neurodegeneration. However, so far it has proven diffi cult to maintain sufficient levels of a transferred gene in suitable cell types, after transplantation. In the present thesis I studied the silencing of transgenes induced upon differentiation of neural progenitor cell lines when grafted to the adult rat CNS. The silencing could be induced in vitro by initiating differentiation and this silencing followed the same temporal pattern as detected upon grafting. In this in vitro model, mechanisms important for transgene silencing in these cells could be studied and we found that inhibition of histone deacetylation could reactivate transgene expression. However, when analyzing the actual level of histone acetylation, the levels were not different in differentiated cells compared to levels detected in proliferating cells. This indicates although histone deacetylase inhibitors could boost transgene expression, this mechanism is not the major player in the silencing induced by differentiation. In the proliferating state transgene expression was stable but did variegate in the cells. The transgene expression did not refl ect proviral content, but was correlated to the level of histone3-acetylation. This suggests that positional effects regulate transgene activity. Although inhibition of histone deacetylation dramatically increased transgene expression and the expression was tightly correlated to histone3- acetylation, the inhibition could not override the positional effects. Data in this thesis propose that DNA methylation could be one of the mechanisms mediating a more static repression in these cells. By fl anking the transgene by insulator sequences, known to form boundaries in chromatin protecting sequences from condensation we detected a partial prevention of transgene silencing both after in vitro and in vivo differentiation. This prevention was also detected by inhibiting histone deacetylation and DNA methylation, prior to grafting. In vivo, cells exhibiting immature morphology were mainly affected whereas differentiated cells were rarely found to express the transgene. Further indicating other mechanisms to be important for the silencing taking place upon differentiation.