Prime Editing as a Potential Therapeutic Intervention for Generalized Epilepsy with Febrile Seizures Plus (GEFS+): From Gene Editing to Functional Recovery

This thesis investigates the in vivo therapeutic potential of prime editing by applying it to correct a disease-causing mutation in the SCN1A gene associated with Generalized Epilepsy with Febrile Seizures Plus (GEFS+), a severe neurodevelopmental disorder characterized by hyperthermia-induced seizures and premature mortality. Using an intein-split prime editor delivered via neuron-specific adeno-associated viral vectors (AAV-PHP.eB), we targeted the recurrent Scn1a K1259T missense mutation in neonatal mice. We optimized pegRNA design and delivery via intracerebroventricular injection at postnatal day 1, achieving editing rates of over 36% in bulk cortical DNA, with even higher efficiencies in neurons as confirmed by RNA sequencing. This approach led to robust editing in relevant neuronal populations and significantly reduced seizure frequency and delayed seizure onset in heterozygous animals, establishing in vivo proof-of-concept for prime editing in the mammalian brain. However, the limited rescue observed in homozygous mice highlighted remaining challenges in achieving efficient and widespread editing across critical brain regions such as the cortex and hippocampus. These findings emphasize the need for improved spatial targeting and cell-type specificity when treating network-based neurological diseases. Notably, functional improvements in heterozygous animals correlated with higher editing levels in certain regions and the restoration of inhibitory neurotransmission. Accordingly, electrophysiological recordings revealed enhanced synaptic inhibition in prime-edited animals, indicating compensatory plasticity that may contribute to the therapeutic effect. Importantly, the editing strategy demonstrated high specificity and safety, with minimal off-target events and no detectable genomic toxicity. In summary, this thesis establishes prime editing as a precise and durable approach for correcting pathogenic mutations in the brain. While current delivery and efficiency limitations remain, this study provides a foundational step toward the development of genetic therapies for intractable monogenic epilepsies and other neurodevelopmental disorders. Full-text embargoed until: 2026-12-05

Author dc:creator

• Pietrafesa, Francesca

• 610 Medicine & health
• 570 Life sciences; biology

Statement dc:rights

• info:eu-repo/semantics/closedAccess

Language dc:language

eng