Florey Department of Neuroscience and Mental Health - Theses
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ItemMass Spectrometry As A Tool For Drug Development In SCN2A Developmental and Epileptic Encephalopathies( 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.
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ItemAntisense oligonucleotide precision therapy in KCNT1 - severe epilepsy( 2019)In recent years, the advancement of sequencing technology used in conjunction with thorough clinical evaluation has enabled clinicians to attribute genetic factors to the etiology of epilepsy. A significant number of mutations in genes encoding ion channels have been identified as the cause of the developmental and epileptic encephalopathies (DEE), a group of severe epilepsy syndromes of childhood and infancy characterized by the presence of abundant epileptiform activity, refractory seizures, intellectual disability, developmental regression, movement disorders, and increased mortality. The gene KCNT1 encodes the sodium activated potassium channel subunit KNa1.1. De novo mutations in this gene have been identified in patients with both severe and milder forms of epilepsy. The most commonly associated phenotypes are epilepsy of infancy with migrating focal seizures (EIMFS) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). EIMFS is situated on the severe end of the spectrum and manifests during the first six months of life with frequent multifocal seizures in addition to developmental plateau or regression. Patients with KCNT1 associated epilepsy typically respond poorly to conventional anti-seizure medications, resulting in an unfavorable prognosis and compromising their quality of life. Biophysical studies in heterologous expression systems have shown that KCNT1 pathogenic variants found in epilepsy result in strongly increased potassium currents. This overall gain of function (GoF) is currently accepted as the primary mechanism of disease in KCNT1 associated epilepsy. To directly address this pathologic mechanism, antisense oligonucleotide (ASO) mediated knockdown was used to test the idea that reducing Kcnt1 expression would be therapeutic in a mouse model of KCNT1 associated epilepsy. This approach was supported by the tolerance for KCNT1 loss of function (LoF) observed in the general population and the mild phenotype found in Kcnt1 knockout mice. Using the CRISPR Cas9 system, an orthologous pathogenic variant found in KCNT1 EIMFS was inserted in Kcnt1 to develop a rodent model. Mice, homozygous for the mutation display frequent spontaneous seizures, abundant interictal activity in the electrocorticogram (ECoG), behavioral abnormalities and early death. Thus, recapitulating key aspects of KCNT1 severe epilepsy and offering a valuable tool for therapeutic screening. Then, an ASO was designed to specifically hybridize with Kcnt1. An additional nontargeting sequence was used as a control. After a single intracerebroventricular bolus injection, homozygous mice showed a marked knockdown of Kcnt1 mRNA and in comparison to control treated animals, displayed almost complete abolition of seizures, prolonged survival, and improved cognition. Exaggerated pharmacology was explored in wild type mice treated with a dose of ASO that produced more than 90 percent knockdown. Here, behavioral measures revealed some anxiety like traits in ASO treated mice, but knockdown was otherwise tolerated. Additional seizure susceptibility in LoF was tested in Kcnt1 knock out mice, which indicated that LoF was not related to a reduction in seizure threshold. To conclude, the preclinical evidence presented in this thesis supports ASO based gene silencing as a therapeutic approach in KCNT1 GoF epilepsies. This body of work provides proof of concept for such approach and encourages the translation of ASO based therapies for genetic epilepsies, in particular for those with an underlying GoF pathomechanism.