Synthetic biology strategies for vascular diseases and cancer immunotherapy: hammerhead ribozymes and SNIPR orthogonal circuits

Recent advances in synthetic biology have progressively transformed biological systems from objects of observation into programmable platforms capable of sensing, processing and responding to pathological cues, opening new perspectives for next-generation therapeutics. This thesis explores two complementary strategies that converge on the control of gene expression across distinct biological scales: RNA-based post-transcriptional regulation and programmable immune cell engineering. We first investigated gene regulation at the molecular level through the development of catalytic hammerhead ribozymes (HHRzs) designed to selectively reduce the expression of CD93, a critical mediator of pathological angiogenesis, through targeted cleavage of its mRNA. To improve intracellular stability and catalytic performance, we designed a novel chimeric scaffold incorporating a pair of histone mRNA derived stem-loop elements. Guided by chemical probing, we further characterised the intracellular mRNA accessibility and screened a panel of chimeric HHRzs targeting distinct NUH↓ cleavage sites to assess the effects of CD93 modulation on endothelial cell migration and tube formation. To complement experimental observations, we generated three-dimensional models of HHRz-target interactions and refined them with molecular dynamics simulations, providing structural insights into catalytic mechanism. Altogether, these findings support the potential of engineered HHRzs as precise RNA-based modulators for anti-angiogenic therapies. Beyond the direct modulation of protein expression, we subsequently investigated a new generation of synthetic intramembrane proteolysis receptors (SNIPRs), designed to implement logic-gated control of gene expression in engineered T lymphocytes. We used this strategy to restrict the transcriptional activation of chimeric antigen receptors (CARs) to the presence of a specific tumour-associated marker, potentially reducing tonic signalling, exhaustion and off-target toxicities. By screening multiple SNIPR variants, we identified an architecture displaying robust ligand-dependent activation with minimal background signalling from the inducible promoter. Functional assays demonstrated selective CAR expression and efficient killing of tumour cells, validating the ability of SNIPR circuits to implement programmable IF/THEN logic in T cells and support context-dependent immune responses within complex tumour microenvironments. By integrating genetic engineering, RNA biology, cellular immunotherapy and computational modelling, this thesis highlights how synthetic biology is progressively redefining therapeutic design, moving towards adaptive and programmable systems with enhanced precision, controllability and translational potential.