Figure 1. Definition and impact of transgene silencing in mammalian cells.

A. Mammalian cells engineered to express a transgene often undergo silencing. A variety of host-cell mechanisms contribute to transgene silencing which correlates with changes in chromatin structure at the site of the integrated transgene. Over time, transgene silencing generates a proportion of the engineered cell population that does not express the gene of interest (GOI). Transgene silencing is often observed as a bimodal distribution of cells that express the transgene (are in the ON state) or do not express the transgene (are in the OFF state) as shown ^14,15. Transgene silencing may also be observed as a decrease in the relative levels of transgene expression rather than a complete loss of expression. B. Diverse applications in biotechnology rely on stable expression of transgenes in engineered mammalian cells. In biomanufacturing, silencing of mammalian cells engineered to produce a product of interest results in a decrease in product produced over time ^21,22. Similarly, silencing of theranostic circuits in mammalian cells engineered ex vivo or engineered in vivo via gene therapies leads to waning efficacy over time ²³. In cellular reprogramming and differentiation, cells engineered to express a gene or circuit of interest often undergo silencing as they change cell fates. In particular, differentiation of induced pluripotent stem cells into mature cell types often generates the desired cell type with a low proportion of cells that retain expression of the GOI ⁴⁷. C. Gene circuits often require robust expression of multiple transgenes. Silencing of any individual transgene may limit the performance of stably integrated genetic circuits ²⁴. In the example shown, a cascade of inducible transgenes regulates expression of the GOI. Silencing of any of the transgenes will result in failure to express the GOI.