Dissertation
Investigating NaV1.2 Channelopathies: characterization and therapeutic approaches for neurological diseases
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2026
Abstract
SCN2A encodes the voltage-gated sodium channel NaV1.2, a central regulator of action potential firing in glutamatergic neurons and one of the highest-confidence monogenic risk genes for autism spectrum disorder (ASD). Premature termination codons (PTCs) in SCN2A are broadly classified as loss-of-function alleles and are presumed to produce uniform haploinsufficiency through nonsense-mediated mRNA decay (NMD). Nevertheless, how PTC position influences molecular outcome and neuronal phenotype remains a critical knowledge gap in the field. To address this, we generated two patient-derived knock-in mouse lines, Scn2aY84X/+ and Scn2aR1627X/+. We characterized each mouse model at the transcript, protein, and electrophysiological levels. Allele-specific mRNA quantification revealed that Y84X engaged partial NMD, whereas R1627X transcripts remained at allelic balance. Despite this divergence in transcript fate, NaV1.2 protein was comparably reduced in both lines to approximately 57% of wild-type levels. Mass spectrometry confirmed the absence of translational readthrough at either PTC. At the cellular level, both variants slowed somatic action-potential upstroke velocity in medial prefrontal cortex layer 5b pyramidal-tract neurons, with a larger decrement in Scn2aY84X/+. Spike threshold was depolarized only in Y84X-expressing neurons, and axon initial segment–associated depolarization kinetics were reduced in Scn2aY84X/+ but preserved in Scn2aR1627X/+. These findings establish PTC position as a determinant of neuronal phenotype and demonstrate that distinct nonsense variants within the same gene are not necessarily mechanistically equivalent to one another, nor to a uniform haploinsufficient state. We also developed a platform for anticodon-edited suppressor transfer RNAs (sup-tRNAs) as a therapeutic strategy for UGA premature stop codons. Using a Luciferase-UGA reporter mouse and transcranial bioluminescence imaging, we optimized sup-tRNA payload design for in vivo delivery to mammalian brain. Our data demonstrate that self-complementary AAV9 packaging of tRNA in a minimal genomic context provides broad and efficacious PTC rescue, and that tRNA multiplexing and use of intronic sequences enable potency at low viral titers and sustained rescue over time. tRNA sequencing of scAAV-delivered ArgUGA sup-tRNA confirmed no perturbation of endogenous tRNA levels, aminoacylation, or processing and demonstrated that the sup-tRNA was efficiently processed, charged, and functional at relatively low abundance. Collectively, our data demonstrate that SCN2A premature termination codons are allele-specific molecular variants that produce position-dependent effects on transcript handling, protein output, and neuronal physiology. This work challenges the prevailing assumption that protein-truncating variants within the same gene converge on a single molecular outcome and can be modeled or treated interchangeably. Our suppressor tRNA platform provides a scalable strategy for precision nonsense codon suppression in the mammalian brain, and the convergence of allele-specific disease modeling with transcript-targeted rescue establishes a preclinical framework for developing durable genetic therapies for nonsense variants in neurodevelopmental disorders.
Details
- Title: Subtitle
- Investigating NaV1.2 Channelopathies: characterization and therapeutic approaches for neurological diseases
- Creators
- Ahmad Al Saneh
- Contributors
- Christopher A Ahern (Advisor)Robert C Piper (Committee Member)Stephanie C Gantz (Committee Member)Aislinn J Williams (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Molecular Physiology and Biophysics
- Date degree season
- Spring 2026
- Publisher
- University of Iowa
- Number of pages
- xii, 141 pages
- Copyright
- Copyright 2026 Ahmad Al Saneh
- Language
- English
- Date submitted
- 04/28/2026
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (page 118-141).
- Public Abstract (ETD)
Autism spectrum disorder (ASD) is a condition that affects how individuals communicate and interact with the world. While ASD has complex origins, some cases are caused by changes in a single gene. One of the most strongly linked genes to ASD is SCN2A, which provides the instructions for making a protein called NaV1.2. This protein acts as a tiny gate in brain cells that controls the flow of sodium, which is essential for brain cells to send electrical signals to one another. When SCN2A is disrupted, brain cells cannot fire properly, which is thought to underlie the features seen in affected individuals. A common type of disruption in SCN2A involves mutations that introduce an early "stop sign" in the gene's instructions. These stop signs cause the cell to stop making NaV1.2 before it is complete. It has been widely assumed that all such stop signs produce the same result, which is half the normal amount of NaV1.2 protein regardless of where in the gene they occur. The goal of my thesis work is to test whether the location of these early stop signs matters and to develop a therapeutic strategy that can correct them. To do this, we created two mouse models, each carrying a different patient-derived stop sign in SCN2A: one near the beginning of the gene and one near the end. We found that although both mutations reduced NaV1.2 protein to similar levels, they did so through different mechanisms. The early stop sign triggered a cellular quality-control process that partially destroyed the faulty genetic message, while the late stop sign did not. More importantly, brain cells from these two models behaved differently. Cells with the early stop sign had greater difficulty initiating electrical signals than cells with the late stop sign, demonstrating that the location of the stop sign determines the specific consequences for brain cell function. In the second part of this work, we developed a potential treatment for one class of these premature stop signs. We engineered a biological tool called a suppressor transfer RNA (sup-tRNA) that can read through a specific type of stop sign (UGA) and allow the cell to continue building the full-length protein. Using a specialized virus to deliver the sup-tRNA to the brains of mice, we showed that this approach can restore protein production broadly across the brain and that the effect is long-lasting. Importantly, the delivered sup-tRNA was properly processed and functional in brain cells at low levels without disrupting the cell's normal machinery. Together, this work shows that not all stop-sign mutations are created equal. This means that treatments must be designed with the same level of specificity. Our sup-tRNA platform offers a promising path toward correcting these mutations directly, and the framework we have established can be applied to SCN2A and other genes involved in neurodevelopmental disorders.
- Academic Unit
- Molecular Physiology and Biophysics
- Record Identifier
- 9985177373402771
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