Castling, a novel therapeutic concept for rewiring pathological gene-expression networks, enabled by the TRIPLE technology

Antony, D.; Roman Azcona, M. S.; Kalinski, H.; Pultar, M.; Adamsky, S.; Nachum, S. A.; Shalom, E.; Rhiel, M.; Tsouris, A.; Diendorfer, A.; Andrieux, G.; Boerries, M.; Hackl, M.; Cornu, T. I.; Zurr, D.; Cathomen, T.; Feinstein, E.; Mussolino, C.

doi:10.64898/2026.01.28.702195

bioRxiv · 10.64898/2026.01.28.702195

Castling, a novel therapeutic concept for rewiring pathological gene-expression networks, enabled by the TRIPLE technology

Antony, D.·Roman Azcona, M. S.·Kalinski, H.·Pultar, M.·Adamsky, S.·Nachum, S. A.·Shalom, E.·Rhiel, M.·Tsouris, A.·Diendorfer, A.·Andrieux, G.·Boerries, M.·Hackl, M.·Cornu, T. I.·Zurr, D.·Cathomen, T.·Feinstein, E.·Mussolino, C.

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Abstract

Pathological conditions often arise from dysregulation of complex gene networks, with microRNAs (miRNAs) acting as central modulators. Disease progression is frequently characterized by upregulation of "disease-promoting" miRNA, suppressing beneficial pathways, and concomitant downregulation of "protective/therapeutic" miRNAs, normally restraining pathological programs. Since individual miRNAs coordinately regulate multiple genes, their manipulation represents powerful therapeutic intervention, yet synthetic or ectopically overexpressed miRNA mimics or inhibitors may perturb physiological miRNA processing and/or cause off-target effects. We hypothesized that pathological gene regulatory imbalances could instead be corrected by rewiring endogenous miRNA regulation. Specifically, by placing downregulated "protective/therapeutic" miRNAs under the control of promoters activated in pathology, and driving overexpression of "disease-promoting" miRNAs, thereby disabling the pathogenic program while inducing the therapeutic one in a single editing event. We termed this concept castling, after the chess move. For effective implementation of castling, we developed TRIPLE (Targeted Replacement Induced by Persistent Locus Editing), a novel genome-editing procedure enhancing homology-directed repair through sequential cleavage. As proof of concept, we castled miRNAs inversely regulated during onset of CAR T cell dysfunction in a model of chronic antigen stimulation. Castled CAR T cells exhibited a delayed dysfunction enabled by up- and downregulation of relevant gene subsets.

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Antony, D., Roman Azcona, M. S., Kalinski, H., Pultar, M., Adamsky, S., Nachum, S. A., Shalom, E., Rhiel, M., Tsouris, A., Diendorfer, A., Andrieux, G., Boerries, M., Hackl, M., Cornu, T. I., Zurr, D., Cathomen, T., Feinstein, E., Mussolino, C.. 2026-01-30. Castling, a novel therapeutic concept for rewiring pathological gene-expression networks, enabled by the TRIPLE technology. https://doi.org/10.64898/2026.01.28.702195

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