Periodic Reporting for period 3 - Synthetic T-rEX (A synthetic biology approach for T cell exhaustion)
Reporting period: 2023-02-01 to 2024-07-31
The project Synthetic T-rEX aims at developing enhanced and more effective immune cell-based therapies by engineering T cells that are resistant to exhaustion. CD8+ T cells fight viral infection and cancer through the ability to “search and destroy” infected or abnormal cells. However, during chronic infections or when they enter a suppressive tumor microenvironment, they undergo exhaustion, a process by which they lose their immune activity. Exhaustion occurs both in natural and chimeric antigen receptors (CAR) T-based immune responses that have been proven successful for the treatment of blood tumors. The critical issue of exhausted T cells is impaired production of cytokines, loss of toxicity and proliferative capacity, meaning that they are not effective against tumors or infections anymore. Some strategies to overcome this problem have been implemented. Those targeting immune checkpoint blockade with anti-CTLA-4 and/or PD1 monoclonal antibodies, have provided clinical benefits to patients with advanced cancer. However, two main problems arise: 1) the efficacy and long-term effect of this approach depends on several factors, including the stage of the dysfunction and 2) adverse effects relative to high dosage, toxic metabolites, or mechanism-associated have been reported hampering the overall benefits. Synthetic T-rEX proposes to address the dysfunction of T cells by engineering them with sensors strategies to detect exhaustion at its onset and to activate a local response that neutralize the dysfunction (therapeutic intervention). This proposal has a strong medical relevance since exhaustion arises in cancers, chronic infections, and chronic inflammations under condition of antigen-persistence. Thus, this approach has a broad range of applications both towards understanding dysfunction in diseases and for development of therapeutic strategies. Furthermore, because T cell exhaustion is associated with better prognosis in autoimmune diseases, our findings and our system can shed light on mechanisms underlying this important group of disorders.
We engineer T cells with specific, self-contained genetic circuits (Objective 4) with minimal impact on normal cell physiology (objective 3) that sense (Objective 1) and process exhaustion-specific intracellular signals (i.e. genes and miRNAs differentially expressed in exhausted T cells), to rewire T cell activity (Objective 2) and restoring normal function.
We engineer T cells with specific, self-contained genetic circuits (Objective 4) with minimal impact on normal cell physiology (objective 3) that sense (Objective 1) and process exhaustion-specific intracellular signals (i.e. genes and miRNAs differentially expressed in exhausted T cells), to rewire T cell activity (Objective 2) and restoring normal function.
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.
In addition, within the block of objective 2, we have developed a computational tool that enables the automated selection of orthogonal gRNAs with minimized off target effects and promoter crosstalk. We next engineered a Lachnospiraceae bacterium Cas12a (dLbCas12a)-based repression system that downregulates target gene expression by means of steric hindrance of the cognate promoter. Finally, we generated a library of orthogonal synthetic dCas12a-repressed promoters and experimentally demonstrated it in HEK293FT, U2OS and H1299 cells lines. 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 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.
In addition, within the block of objective 2, we have developed a computational tool that enables the automated selection of orthogonal gRNAs with minimized off target effects and promoter crosstalk. We next engineered a Lachnospiraceae bacterium Cas12a (dLbCas12a)-based repression system that downregulates target gene expression by means of steric hindrance of the cognate promoter. Finally, we generated a library of orthogonal synthetic dCas12a-repressed promoters and experimentally demonstrated it in HEK293FT, U2OS and H1299 cells lines. 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).
The main breakthrough consists in the realization that resource-aware genetic circuits exhibit better performances and more predictable behavior. This has an impact for the entire Synthetic Biology community as well as for biomedical interventions, clearly going beyond the state of the art of genetic circuits design.
Further our work on a new automated tool for gRNA selection accompanied by a novel transcriptional system is a novel tool that can aid the design of complex network, which has fundamental importance in mammalian synthetic biology.
The project is tackling and providing solutions toward multiple key player of T cell exhaustion rather than simply modifying the expression of only one gene. This is a paradigm shift in the development of strategies against T cell exhaustion and a solid break-through towards enhanced natural and cell-based immunotherapy.
Further our work on a new automated tool for gRNA selection accompanied by a novel transcriptional system is a novel tool that can aid the design of complex network, which has fundamental importance in mammalian synthetic biology.
The project is tackling and providing solutions toward multiple key player of T cell exhaustion rather than simply modifying the expression of only one gene. This is a paradigm shift in the development of strategies against T cell exhaustion and a solid break-through towards enhanced natural and cell-based immunotherapy.