Engineering Intracellular Protein Sensors in Mammalian Cells

The study and modulations of cellular responses via controllable engineered gene circuits are major goals in synthetic biology^1,2,3 for the development of prospective tools with relevant biological or medical applications in cancer⁴, infections⁵,  metabolic diseases⁶, and immunology⁷.

Reprogramming cell functions in response to specific signals requires the design of smart interfaces that link sensing of extracellular or intracellular dynamic changes (input) to downstream processing, triggering specific output either for diagnostic purposes (i.e., reporter genes) or to rewire cell response (therapeutics). The inputs detected by the sensing module can be small analytes⁸, proteins^9,10,11 or microRNAs^11,12,13, specific for the onset or progression of a disease. Moreover, complex circuit regulation can be achieved by multiple input information processing, increasing the tight control over transgene expression in response to the defined conditions^14,15,16. For example, microRNA-based sensors can identify specific cell types, such as cancer cells, inducing their clearance with the expression of an apoptotic gene¹³. Since microRNAs are easily implementable in synthetic circuits in a modular manner, they represent a widely used input for genetically encoded biosensors^12,13,17. Proteins are also a valid biomarker for genetic mutations, cancer and infections, and indeed a number of extracellular protein-sensing devices have been reported^18,19.

Many of the circuits that detect extracellular proteins rely on the use of engineered receptors, which tether a transcription factor (TF) to the membrane, fused to a TEVp-responsive cleavage site (TCS). A major advantage of the TEVp is the specificity of the cleavage and lack of interference with endogenous protein processing. In these systems, TEVp is fused to a second peptide that interacts with the engineered receptor upon binding of the extracellular molecules. Thus, the external inputs induce TEVp-mediated cleavage and TF release. Systems that function with this mechanism are Tango/TEVp¹⁸, light-induced²⁰ and Modular Extracellular Sensor Architecture (MESA)¹⁹. Despite progress in detecting extracellular proteins, the technology for sensing intracellular proteins in a modular fashion was never realized before, with the limitation of going through many build-and-test-iterations for single devices responsive to a specific protein.

Our system is the first platform for intracellular protein sensing²¹. The modularity is guaranteed by the use of intrabodies that define the specificity to the target, whereas the cell-reprogramming is TEVp-mediated. Specifically, one intrabody is membrane bound and fused to the C-terminal to a fluorescent protein mKate, a TCS and a TF (fusion protein 1); the second intrabody is fused to the TEVp and located in the cytosol (fusion protein 2) (Figure 1).

Thus, the interaction between two intrabodies and the target protein occurs in the cytoplasm and leads to TCS cleavage by TEVp, resulting in TF translocation into the nucleus to activate functional output. The sensing—actuating device was successfully tested for four intracellular disease-specific proteins: NS3 serine protease expressed by the HCV virus²², Tat and Nef proteins from HIV infection^23,24, and mutated huntingtin (HTT) of the Huntington’s disease²⁵. Output expression includes fluorescent reporters, apoptotic gene (hBax)²⁶ and immunomodulators (XCL-1)²⁷. We demonstrate that the system can also impair pathological functionality of its targets. For instance, the Nef-responsive device interferes with the spreading of viral infection by sequestering the target protein and reverting the downmodulation of HLA-I receptor on infected T cells²⁴. The described sensing-actuating platform is the first of this kind for the detection of intracellular proteins and can be potentially implemented to sense abnormal protein expression, post-translational or epigenetic modifications, for diagnostic and therapeutic purposes²⁰.