Gene addition therapy depends on efficient and long-term transgene expression. Both terminally differentiated and progenitor cells can be targeted for therapeutic gene transfer. After delivery with viral or non-viral vectors, the transgenes can integrate into the chromosomal DNA, replicate episomally or persist as episomal elements in non-dividing cells. However, the introduced genes are subject to the pre-existing and developing gene expression patterns in target cells, which can override the signals from the transgenes’ own regulatory elements and, thus, can cause transgene expression shutdown. The shutdown can either be uniform through the target cell population and permanent in time (silencing) or occur only in some cells of the target cell population, with a tendency to be epigenetically inherited in cell divisions (variegation). Stable long-term transgene expression in differentiating cells is particularly challenging. At the transcriptional level, the changing scenery of transcription initiation factors, chromatin re-modelling and DNA methylation events during differentiation contribute to the transiency of transgene expression. Silencing can also occur through the reduced efficiency of mRNA transport from nuclei, mRNA degradation, alternative splicing and RNA editing. Unfavourably for gene therapy, mammalian cells harbour machinery for the silencing of foreign DNA, which is still poorly understood but is known to be triggered by multimeric DNA and some bacterial plasmid DNA sequences. In this review we analyse epigenetic interferences to successful transgene expression and the vectorology stratagems, which can be employed to avoid or counter-act undesired transgene silencing.