Feinstein, E. | Search bioRxiv

bioRxiv2026

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

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.

molecular biology↗

bioRxiv2020

RTP801 Is A Key Modulator For Motor Learning

RTP801/REDD1 is a stress-regulated protein whose upregulation is necessary and sufficient to trigger neuronal death in in vitro and in vivo models of Parkinsons and Huntingtons diseases and is up regulated in compromised neurons in human postmortem brains of both neurodegenerative disorders. Indeed, in both Parkinsons and Huntingtons disease mouse models, RTP801 knockdown alleviates motor-learning deficits. Here, we investigated the physiological role of RTP801 in neuronal plasticity. RTP801 is found in rat, mouse and human synapses. The absence of RTP801 enhanced excitatory synaptic transmission in both neuronal cultures and brain slices from RTP801 knock-out (KO) mice. Indeed, RTP801 KO mice showed improved motor learning, which correlated with lower spine density but increased basal filopodia and mushroom spines in the motor cortex layer V. This paralleled with higher levels of synaptosomal GluA1 and TrkB receptors in homogenates derived from KO mice motor cortex, proteins that are associated with synaptic strengthening. Altogether, these results indicate that RTP801 has an important role modulating neuronal plasticity in motor learning.

neuroscience↗