TRANCERs: Engineering enhancers into autonomous tissue-specific expression cassettes

The process by which a foreign gene is introduced into a cell and translated into a functional protein is referred to as transgene expression. Understanding and optimising transgene expression is crucial for a wide variety of applications which range from investigating basic biology, synthetic biology, recombinant protein production to gene therapy. However, precise control over the timing and location of transgene expression remains a key limitation, particularly in therapeutic contexts where specificity and compactness are critical.

To address limitations of traditional promoter-based transgene construct approaches, we developed TRANCERs (TRanscriptionally Autonomous enhaNCERs), a modular and compact system that transforms cell type-specific enhancers into standalone sources of productive transcription. To accomplish this, we leveraged basic facets of enhancer function as well as known and novel principles of transcript stability and nuclear export.

We demonstrate that TRANCERs can drive lineage-restricted expression of both reporter and therapeutic genes across multiple mammalian cell types both in vitro and in vivo , and that this expression pattern faithfully mirrors the activity of the enhancer element used. Enhancer elements are the most cell type specific element in the mammalian genome and are used to drive development, differentiation and cellular responses to extrinsic signals. Therefore, the TRANCER system allows for the generation of specific stable expression constructs for all conceivable situations for which a native enhancer exists with the required activity pattern. Furthermore, we show that the TRANCER system is compatible with previously published machine learning derived artificial enhancer sequences suggesting the possibility even for specific bespoke expression patterns that do not exist in nature. This, combined with a significantly reduced vector footprint ideal for size-constrained delivery systems such as lentiviral vectors, means that the TRANCER system represent a significant step forward in our ability to control gene expression for both basic biology and therapeutic applications.