Programmable multi-input transcription factor based circuit for the identification of specific cell types/physiological states

Confining a transgene activity to specific mammalian cell type, tissues or physiological state has been one of the major goals in genetic engineering with applications in basic research and gene therapy. The classic approach to restrict a transgene is to place it under the upstream region of an endogenous gene known for its specific expression in the tissue of interest. Many tissue- and cell type-specific promoters or enhancers have been used to this purpose, driving specific protein and microRNAs expression.

This approach ultimately relies on the existence of a natural promoter that is active only in the cell type or state of interest: while this assumption proves to be reasonable and successful in the case of specialized tissues or cell types, that need to express proteins with highly specific functions, usually doesn’t hold for less specialized cells in a certain lineage or for pathological conditions. Pathological states in particular usually involve a dysfunctional protein or wide changes in expression patterns and rarely arise from (or result in) the organismal specific expression of a single gene.

Here we propose a scalable and programmable gene circuit able to sense transcription factors (TFs) activity in a modular fashion and integrating their levels, triggering a response only in case the inputs match a specific predetermined pattern. In particular we show a general strategy to sense, amplify and efficiently transduce the activity level of endogenous transcriptional activators. This robust transduction is then used to implement different Boolean logic functions.

As an initial proof of concept we apply this process to a small subset of TFs. Using a limited panel of NCI60 cells as a model system we show how a programmable logic integration of transcriptional regulators can be used to distinguish different lineage and potentially differentiate physiological and pathological profiles. Given the modular nature of the circuit this component can be used as a template to design similar sensor with different specificities and can be combined in larger pattern-sensing circuits using the same principles.

The specific conditional response of this system can be used for precise and specific manipulation of cell physiology: not only the final output can express an effector when all the criteria are met but each branch can be designed to interact with the cell through a miRNA or downstream transactivation in the presence of it’s respective TF.