Specific Aim 1 – To investigate electrophysiological properties of iPSC-derived AHC neurons. Summary of proposed experimental objectives: A. Complete electrophysiological studies of D801N and the splice site mutation c.2542+2T>C. B. Investigate the electrophysiological properties of iPSC-neurons with mutations E815K and R756H or R756L (depending on our success generating these iPSC lines). C. Optimize a neuron co-culture system and investigate the electrophysiological properties of neurons with the G947R mutation. D. Implement methods to generate dopaminergic neurons from iPSC carrying the G947R mutation. E. Use genomic engineering with patient derived iPSC lines harboring specific ATP1A3 mutations to disrupt the mutant copy and then compare functional deficits between neurons generated from the two isogenic cell lines. F. Complete validation of new anti-ATP1A3 monoclonal antibodies Specific Aim 2 – To investigate effects of compounds on iPSC-derived AHC neurons. Summary of proposed experimental objectives: A. Determine the effect of chlormadinone on cortical excitatory neurons derived from AHC patients with the ATP1A3 mutation D801N. B. Re-optimize the virtual compound screen using data from the originally identified compounds. Re-run the screen and identify compounds to test experimentally using the thallium-uptake assay. C. Determine the effects of select compounds identified by the second stage virtual screen on cortical excitatory neurons derived from AHC patients with the ATP1A3 mutation G947R or D801N. Specific Aim 3 – To evaluate the success of therapeutic gene editing in iPSC-derived AHC neurons. Summary of proposed experimental objectives: A. Design and test the ability of adenine base editing to correct the G947R mutation in patient-derived iPSC lines. B. Quantify the efficiency of G947R base editing. C. Generate neurons from the corrected G947R iPSC line and test electrophysiologically. D. Determine if base editing can be applied directly in neurons.
|Effective start/end date||7/1/18 → 3/31/22|
- Alternating Hemiplegia of Childhood Foundation (Award Letter 7/23/18)
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