Florey Department of Neuroscience and Mental Health - Theses

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Subject: Epilepsy × Author: Blackburn, Todd ×

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    Mass Spectrometry As A Tool For Drug Development In SCN2A Developmental and Epileptic Encephalopathies
    Blackburn, Todd ( 2023-11)
    Mutations within the SCN2A are recognized as a prominent cause of autism spectrum disorder and a spectrum of developmental and epileptic encephalopathies (DEEs). As more patients are affected by mutations in SCN2A, it drives the need for precision medicines and to better understand the biology and pathogenesis of the disorder. The SCN2A gene encodes the voltage-gated sodium channel, Nav1.2. Antisense oligonucleotides (ASOs) are a class of drugs being developed to treat SCN2A disorders by knocking down SCN2A mRNA and therefore protein levels. In this study, targeted mass spectrometry methods are utilised to measure Nav1.2 protein levels directly and untargeted, or “discovery”, proteomic methods are used to measure the entire proteome in brain tissue collected from various SCN2A mouse models and mice treated with an experimental ASO therapy. Three SCN2A knock-in missense mutation mouse models are included in the study, each representing a phenotypic group within the SCN2A disease population. These results all support that the ASO has strong target engagement on the protein expression similar to mRNA level. The mutant mouse models are R1882Q representing the early seizure onset phenotype, R853Q representing the late seizure onset phenotype, and S1758R representing the autism with no seizure phenotype. When measuring Nav1.2 in R1882Q mouse whole-brain treated with an ED80 dose (80% knockdown Scn2a mRNA) of an Scn2a-targeting ASO, Nav1.2 was reduced 72% compared to R1882Q mice treated with a scrambled-control ASO. WT mice treated with an ED50 dose of SCN2A-targeting ASO at P30 (post-natal day 30) with brain tissue collected over a 5-week period showed consistent knockdown of Nav1.2 protein of approximately 50% 2- and 3-weeks post-injection in cerebellum, hippocampus, and cerebellum. In the mutant models of Scn2a encephalopathy, Nav1.2 expression remained unchanged in R1882Q and R853Q mutant mice compared to WT littermates while Nav1.2 expression was reduced ~50% in S1758R mutant mice compared to WT littermates, suggesting haploinsufficiency may be a major driver of the autism phenotype. Global proteomic analysis revealed several potential off-target and/or toxicity biomarkers of ASO treatment. These biomarkers were primarily associated with neuroinflammation, including neurofilament heavy (Nefh) and programmed cell death 5 (Pdcd5). Global proteomic analysis in the 3 mutant models showed unique proteomic profiles in each, with minimal overlap, suggesting the very different phenotypes also lead to differences in protein expression and dysregulation. However, dysregulated proteins across the 3 models were involved in several shared pathways, including those responsible for regulation of synaptic signalling and mitochondrial function and metabolism. The exploration of novel epileptic mouse models and mice treated with experimental antisense oligonucleotides through proteomic analysis has unveiled promising prospects for potential new biomarkers. This integrated approach has provided invaluable insights. It is anticipated that certain biomarkers identified may undergo further validation and potentially be employed in clinical trials for emerging SCN2A drugs. These biomarkers could serve to monitor disease progression and assess the effectiveness of innovative treatments, building upon prior research efforts.