We implemented an experimental set-up of ex vivo induction of CD8+ T cell exhaustion by repeated stimulation with CD3/CD28 beads, TGF-beta and VEGF. We confirmed that the model recapitulates the exhausted T cells (Tex) features by immunophenotyping (flow cytometry analysis on markers such as PD-1, CTLA-4, Tim-3, Tox etc.) and functional assays (cytokine production, and degranulation, etc). Objective1: We performed RNA-seq on exhausted T cells (Tex) generated in vitro, and identified upregulated transcription factors, to build a library of cognate, resource aware, synthetic promoters (SPs). The SPs exhibit from 70x to 300x fold change in reporter activation in Jurkat T cells (patent filing ongoing), and we are currently testing their efficiency in integrated CD8+ T cells.
Further, we have recently performed microRNA sequencing and identified miRNA differentially expressed in exhausted T cells and we are currently engineering reporters that harbor in the 5’-UTR the target sites responding to the selected miRNA to tightly regulate the chosen reporter. Additionally, by reverse engineering approaches we are inferring and testing the network of dysregulated miRNA and responsive genes to shed light on the metabolic processes involved.
Objective2: We are working on the therapeutic output activation to counteract the T cell dysfunction. We designed genetically encoded nanobodies anti PD-L1/CTLA4, whereas to modulate epigenetic regulators of the exhaustion we designed new siRNAs (patent in preparation). Moreover, as TOX has recently emerged as master transcriptional regulator of exhaustion, in collaboration with Dr. Girotto (@IIT) and the Dr. Konig (University of Bonn) we are developing nanobodies against the catalytic pocket of the protein to interfere with its ability to tune genes that lead to exhaustion.
Collectively, this work enabled by the complementarity of different expertise, namely computational biology (provided by our collaborator at Imperial College London) and synthetic biology (provided by my lab). This system expands the toolkit of mammalian synthetic promoters with a new complementary and orthogonal CRISPRi-based system, ultimately enabling the design of synthetic promoter libraries for multiplex gene perturbation that facilitate the understanding of complex cellular phenotypes, which can be then used to control T cell reprogramming (Crone et al npj Systems Biology and Applications 2022).
Objective3: To gain a deeper understanding about the discrepancy between predicted and experimentally observed behavior of genetic circuits we have explored the effect of synthetic circuits on host cells. We identified competition for intracellular resources which we named ‘burden’, as a core problem for the poor predictability of gene expression circuits. This competition results in coupling of otherwise independent exogenous and endogenous genes, generating a divergence between intended and actual functions. In collaboration with the groups of Khammash (ETH, Switzerland) and Stan (Imperial College London-ICL, UK), we elucidated the contributions of the transcriptional and translational processes to burden, developed a mathematical model to design resource aware synthetic networks that account for limited resources, and we engineered microRNA-based incoherent feedforward (iFFL) circuits to effectively ensure robust circuits performance, independently of the host cell line. This work resulted in a publication (Frei T, Cella F et al Nature Communications 2020). Further we defined the role of microRNAs to regulate gene expression and mitigate resource competition, a research that resulted in a publication (Cella F et al Nucleic Acid Research 2023). To ensure improved transgene expression as part of objective 4 we integrated systems and synthetic biology to identify small molecules that resulted in robustness cell engineering (Pisani et al Nature Communications 2025).
Finally the design of novel sensors for specific T cell activation has been validated in primary CD8 T cells and in vivo, with a manuscript that is in revision in Nature Communications
