Al Saneh, A. | Search bioRxiv

bioRxiv2026

Programmable Repair of Disease-Causing UGA Stop Codons in Mammalian Brain

Protein truncating variants caused by UGA stop codons are the most prevalent class of rare variant mutations in neurodevelopmental diseases. Suppressor transfer RNA (sup-tRNA) have therapeutic potential for premature termination codon (PTC) repair, but have thus far underperformed by traditional AAV delivery platforms and progress has been hampered by the lack of methods to non-invasively assess in vivo activity in mammalian brain. To fill this material gap, we utilize transcranial in vivo bioluminescence imaging data from a luciferase-UGA mouse model to enable payload optimization. These data demonstrate that U6 promotor and AAV2/9 capsids have the lowest in vivo activity, whereas self-complementary AAV2/9 with the tRNA in a minimal 100bp genomic context provide broad and efficacious PTC rescue. Further, payload tRNA multiplexing and use of tRNA introns enable efficacy of low viral titers and sustained rescue. tRNA sequencing of scAAV delivered ArgUGA sup-tRNA in brain demonstrate no effects on endogenous tRNA levels, their acylation or processing, and these features are also maintained in scAAV delivered ArgUGA sup-tRNA. Collectively, this work defines a scalable strategy for precision UGA stop codon suppression, supporting development of durable genetic rescue therapies for neurodevelopmental disorders in the mammalian brain. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/724978v3_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@d0fefeorg.highwire.dtl.DTLVardef@1150446org.highwire.dtl.DTLVardef@cbb839org.highwire.dtl.DTLVardef@abddfc_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Position-dependent effects of SCN2A premature stop codons on neuronal excitability and behavior

SCN2A encodes the voltage-gated sodium channel NaV1.2, a key determinant of spike initiation and propagation in glutamatergic neurons. Premature termination codons are often assumed to produce uniform haploinsufficiency via nonsense-mediated decay, yet whether distinct SCN2A premature stop codons yield equivalent molecular, cellular, and behavioral outcomes remains unknown. We generated two mouse lines carrying patient mutations--Scn2aY84X/+ (p.Tyr84UAA; early coding sequence) and Scn2aR1627X/+(p.Arg1627UGA; terminal coding exon)--on a C57BL/6J background. Allele-specific expression was quantified by targeted next-generation sequencing of whole-brain reverse transcribed cDNA. NaV1.2 protein was measured in half-brain lysates by automated western blot and ex vivo whole-cell recordings were obtained from layer 5b pyramidal-tract neurons in medial prefrontal cortex. A panel of behavioral assays assessed locomotion/exploration, motor learning, anxiety-like behavior, sociability, sensorimotor gating, and seizure susceptibility. Allele-specific RNA handling diverged by position: mRNA carrying Y84X engaged partial nonsense-mediated decay, whereas R1627X transcripts were at allelic balance. Despite this difference in RNA fate, NaV1.2 protein was comparably reduced in both lines. Electrophysiologically, both premature termination codon mutations slowed the action-potential upstroke, with a larger decrement in Scn2aY84X/+than in Scn2aR1627X/+. Spike threshold was depolarized only in Scn2aY84X/+, whereas Scn2aR1627X/+ remained similar to wild type. Frequency-current relations showed reduced firing at near-rheobase inputs in both mutants, with responses approaching wild type at stronger currents. Behaviorally, locomotion, sociability, and sensorimotor gating were preserved. Both lines exhibited increased grooming--consistent with restrictive, repetitive behavior; Scn2aY84X/+alone showed greater exploration in the elevated-risk context and a male-predominant deficit in rotarod learning. In maximal electroshock testing, mortality was lower in both lines without differences in seizure threshold or severity. Our results show that distinct SCN2A premature termination codons are not equivalent to one another, nor to a uniform haploinsufficient state. An early, nonsense-mediated decay-competent premature stop codon (Y84X) and a terminal-exon one (R1627X) produce partially overlapping yet allele-specific effects on neuronal excitability and behavior. These findings establish premature termination codon position as a determinant of phenotype, supporting allele-tailored mechanistic studies and therapeutic strategies.

neuroscience↗