Development of splice modulating oligomers targeting AMPA receptor alternative splicing as therapeutics for neurological diseases
Year: 2011
Type: dissertation
Abstract: Amyotrophic lateral sclerosis (ALS) and neonatal epilepsies are particular patient populations for which sufficient drug therapies are currently unavailable. Neuronal hyperexcitability and excitotoxicity through enhanced AMPA receptor (AMPA-R) function is linked to the pathogenesis of many neurodegenerative diseases, including epilepsy and ALS. As the major excitatory neurotransmitter receptors in the CNS, AMPA-Rs are obvious drug targets that have not been sufficiently exploited. AMPA-R subunits (GluAs1-4) have high and low gain alternative splice isoforms, termed flip and flop, respectively. When GluA-flip levels are elevated, calcium influx is high, and neurons are hyperexcitable and susceptible to excitotoxicity. This dissertation describes the development and characterization of novel AMPA-R targeted splice modulating oligonuclotides (SMOs) as drug candidates for the treatment of ALS and intractable seizures. By developing novel in silico molecular engineering methods, we predicted SMOs that would specifically and potently decrease flip isoform levels of individual and groups of GluA isoforms and validated their efficiency using a rapid in vivo screening process. The SMOs of highest clinical interest, termed GR1 and GR3, specifically and robustly reduced expression of GluA1-flip (by 99%) and GIuA3-flip (by 97%), respectively. GR1 was evaluated in a murine model of neonatal seizures and through electrophysiological characterization of SMO effects in hippocampal slices. When delivered by Intracerebroventricular (ICV) bolus directly into the CNS, GR1 reduced seizure activity, and reversed post-seizure AMPA-R potentiation that precedes epileptogenesis, without negative effects on cognition. Additionally, a single ICV injection of GR1 robustly suppressed GluA1-flip levels for at least 60 days, demonstrating long lasting effects. GR3 SMO was evaluated in dual (G93A) ALS mouse models to confirm efficacy and compare SMO delivery methods. GR3 treatment produced a significant increase in longevity for both models using continuous ICV infusion or periodic lumbar bolus injection. GR1 also significantly increased lifespan after lumbar bolus injections. Lumbar injections appeared to improve compound delivery, such that GR3- and GR1-treated ALS mice showed significant improvements on several functional motor measures. This work demonstrates a novel drug design method, and validates the efficacy of the GR1 and GR3 SMOs as possible therapeutics for the treatment of seizures and ALS.
DOI: 10.17918/00007605
Institution: Drexel University
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4400184041