Figure 3. Design considerations for preventing transgene silencing via selection of genetic elements, locus, and cell type of interest.

A. Genetic elements including insulators, promoters, and the combination of activating and repressive elements regulates gene expression. Promoters, enhancers, insulators, and CpG islands facilitate continuous gene expression. Elements such as low-density CpGs among GC-poor regions and viral sequences such as long-terminal repeats act as targets for transcriptional repression. Stability of transgene expression can be improved through the inclusion of activating elements, exclusion of repressive elements, and sequence-specific parameter optimization. B. Transgene circuit integration into heterochromatic, repressive genomic loci increases the likelihood of silencing. Targeted integration of transgenes into genomic safe harbors that remain ubiquitously euchromatic may reduce silencing. C. Stem cell differentiation induces genome-wide changes across CpG methylation, histone modification, and chromatin remodeling landscapes. Transgene expression depends on the epigenome of the differentiated lineage; expression might be safeguarded through CpG islands, tissue-specific enhancers, and transgene integration into ubiquitously open genomic safe harbors. Abbreviations: Gene of interest (GOI); Poly-adenylation signal (pA); Long terminal repeat (LTR).