Abstracts

This is a Late Breaking abstract

Rationale: Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a rare autosomal dominant form of progressive myoclonic epilepsy (PME) caused by intraneural inclusions of mutant neuroserpin. A severe form of FENIB is caused by a missense mutation in the SERPINI1 gene c.1175 G >A (p. Gly392Glu). Symptoms typically manifest around age 10 in the form of progressive decline in global cognitive and social functions associated with absence seizures that are followed by myoclonic and tonic-clonic seizures. EEGs reveal generalized slowing and 2-3 Hz generalized spike-wave discharges. We are combining gene-editing and high resolution, single neuron patch-clamp electrophysiology in iPSC-derived neurons to assess whether the SERPINI1 G >A mutation affects intrinsic excitability or network level synaptic excitability. CRISPR-mediated gene editing can generate patient-specific mutations as well as permanently correct single nucleotide transition mutations including those responsible for FENIB and other PMEs. To establish proof of concept for therapeutic genome editing, we corrected this very mutation using an optimized adenine base editing system.

Methods: We developed a personalized CRISPR gene editing approach to precisely knock-in the SERPINI1 c.1175 G >A mutation in human iPSCs to model FENIB. Single clones with the patient-specific mutation were treated with adenine base editor (ABE) to assess mutation correction efficiency. Wildtype, heterozygous mutant, homozygous mutant, and corrected isogenic iPSCs were differentiated into neurons through Neurogenin-2 (NGN-2) overexpression. Current- and voltage-clamp electrophysiology was then used to assess intrinsic and passive membrane properties of iPSC-derived neurons. We conducted pair-wise comparisons between groups to examine expression of FENIB pathology in vitro and determine whether ABE-mediated correction can rescue the phenotype.

Results: Differentiation of wildtype, mutant, and corrected iPSCs through NGN-2 overexpression resulted in electrically active neurons which express neuron markers validated by patch-clamp recordings and immunocytochemistry, respectively. Preliminary data suggests an increase in the frequency of spontaneous post-synaptic excitatory activity in homozygous neurons compared to wildtype, heterozygous and corrected iPSC-derived neurons.

Conclusions: In summary, we established an “Epilepsy-in-a-Dish” model of FENIB to examine changes in neuronal and synaptic activity and corrected the patient-specific FENIB mutation to demonstrate the potential of personalized gene editing. Efforts on electrophysiological characterization of intrinsic excitability, passive membrane properties, and synaptic activity are ongoing to further examine in vitro manifestation of pathology and validate preliminary data. Personalized genome editing can model and correct monogenic epilepsies caused by mutations in critical genes in vitro and eventually, in vivo.

Funding: Kids Connect Charitable Fund; UL1 TR001445 from the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH) Clinical and Translational Science Award (CTSA) program; FACES (Finding a Cure for Epilepsy and Seizures), NYU Langone Health