Uncovering 3′-UTR regulatory elements with therapeutic potential for haploinsufficiency of VGSCs
Graduation Year
2025
Document Type
Master's Thesis
Degree
Master of Science
Program
Biological Science
Partner Organization
BioMarin Therapeutics
Program Director
Patti Culros, MD, MPH
First Reader
Gina Caldas
Second Reader
Jan Lui
Abstract
Voltage-gated sodium channels (VGSCs) are crucial to the initiation and propagation of action potentials (APs) in electrically excitable cells, particularly within the central and peripheral nervous systems. Given their role in transmitting electrical signals throughout the brain, proper VGSC expression is essential for neuronal communication and plasticity. Genetic screening has linked de novo loss-of-function (LoF) mutations in VGSCs of heterozygous patients to severe neurodevelopmental disorders (NDDs), including developmental encephalopathy (DE), intellectual disability (ID), autism spectrum disorder (ASD), and infantile seizures. These LoF mutations often result in a haploinsufficiency, where one allele fails to produce a functional protein, leading to approximately 50% of normal protein levels. Studies in animal models of VGSC haploinsufficiency have shown that increasing wild-type protein can rescue neuronal phenotypes, suggesting that reversibility of NDD phenotypes in LoF patients may be achievable through upregulation of protein expression in neurons. Therefore, boosting protein expression of VGSCs by enhancing RNA transcription or RNA stability is a viable therapeutic strategy for patients with VGSC haploinsufficiency. Here, I investigate multiple predicted candidate regulatory regions within a human VGSC transcript by evaluating their impact on RNA stability when targeted with sequence-complementary oligonucleotides. Using human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons and an engineered GFP-3′-UTR reporter system, this study demonstrates that blocking elements near multiple polyadenylation sites and predicted miRNA sites within the target 3′-UTR results in approximately 1.4-fold increase in target RNA and protein levels. Similarly, this study also demonstrates that oligo-mediated blocking of alternative splicing events that result in non-productive RNA, leads to a 1.2-fold increase in productive RNA. Together, these studies uncover destabilizing cis-elements within a VGSC human transcript and demonstrate that oligo-mediated steric blocking of such elements, increases mRNA stability, offering a potential strategy to restore low protein levels in the context of VGSC haploinsufficiency.