Rnai Therapy Mitigates Spontaneous Seizures in a Mouse Model of KCNQ3 Neurodevelopmental Disorder
Abstract number :
3.133
Submission category :
2. Translational Research / 2D. Models
Year :
2022
Submission ID :
2204914
Source :
www.aesnet.org
Presentation date :
12/5/2022 12:00:00 PM
Published date :
Nov 22, 2022, 05:27 AM
Authors :
Divyalakshmi Soundararajan, MS – Columbia University Irving Medical Center; Osasumwen Aimiuwu, PhD – Columbia University Irving Medical Center; Megha Sah, PhD – Columbia University Irving Medical Center; Anastasios Tzingounis, PhD – Professor, University of Connecticut; Scott Harper, PhD – Professor, Nationwide Children’s Hospital; Wayne Frankel, PhD – Professor, Institute for Genomic Medicine, Columbia University Irving Medical Center; Tristan Sands, MD, PhD – Assistant Professor, Neurology, Columbia University Irving Medical Center
Rationale: Recurrent de novo missense mutations affecting one arginine codon (R230) in the KCNQ3 gene are responsible for an electroclinical syndrome that accounts for as many as 1 in 1,000 individuals with intellectual disability and autism spectrum disorder. KCNQ3 encodes a voltage-gated potassium channel activated at subthreshold potentials and concentrated at the axon initial segment, where it opposes depolarizing currents that drive neurons to fire action potentials. R230 variants cause gain-of-function (GoF) effects and impair neuronal firing in vitro. The development of effective disease modifying therapy for KCNQ3 GoF neurodevelopmental disorder (NDD) represents a critical unmet need. To address this need, we developed knockin mouse models carrying two different R230 GoF variants and examined key phenotypes, which include spontaneous generalized seizures, suitable for testing efficacy of therapy._x000D_
Methods: Novel knock-in mice lines in C57BL/6J harboring a mutation in Kcnq3 orthologous to human disease variants, R230H and R230C, were developed respectively by the Frankel and Tzingounis labs. Heterozygous mutant animals were crossed to FVB/NJ to generate F1 hybrid heterozygotes and wildtype littermates. Approximately 9E+10 AAV9 viral particles that encode Kcnq3 miRNA (or a similar amount of control) were delivered to postnatal day 1 pups by intracerebroventricular injection. Mice were anaesthetized and injected at approximately 2/5 the distance from the lambda suture to each eye. Injections were executed free hand using a point style 4, 33G needle and a 10 mL or 25 mL Hamilton syringe. Subsequently, RNA and protein levels were assessed and EEG was recorded. For EEG recordings, silver subdural electrodes were implanted on mice aged 6 to 10 weeks with three recording electrodes positioned over the cerebral cortex and one reference electrode over the cerebellum. SWD analysis was performed blinded to genotype.
Results: The Harper laboratory devised miRNA shuttle that targets human or mouse KCNQ3 mRNA, evaluating in vitro efficacy by using a dual luciferase assay before cloning into a self-complementary AAV9 vector and producing virus for in vivo use. Neonatal delivery of scAAV9.miKcnq3, but not control virus led to reduction in Kcnq3 mRNA and Kv7.3 protein in Kcnq3 mutant heterozygotes and wildtype littermates. EEG recordings demonstrated frequent spontaneous spike-wave discharges (SWD) in untreated heterozygous animals. A significant reduction in SWD incidence was observed in mutant mice treated with miKcnq3, including complete elimination of SWD in most animals treated. Control virus injected mutants were not significantly different from the uninjected group. Preliminary data with injections at lower titers support a dose response.
Conclusions: We demonstrate use of a novel RNAi therapy to suppress spontaneous seizures in a new mouse model of KCNQ3 GoF NDD. This non-allele-specific approach to reducing KCNQ3 expression may be a viable therapeutic strategy across KCNQ3 gain-of-function mutations (R230C/H/S and R227Q).
Funding: Sackler Brain and Spine Institute at NewYork-Presbyterian Research Grant
Translational Research