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Dirkx, N.

Publications and source records attributed to Dirkx, N..

2 recordsLinked to original sources

KCNQ2 Loss-of-Function variants disrupt neuronal maturation via early hyperexcitability followed by maladaptive network remodeling

Loss-of-function (LOF) variants in the potassium channel subunit KCNQ2 cause a spectrum of neonatal epilepsies from self-limiting familial neonatal epilepsy (SeLFNE) to severe developmental and epileptic encephalopathy (DEE). To dissect the developmental consequences of KCNQ2 LOF, we conducted a longitudinal and multimodal comparative analysis in a human neuronal model generated from patients with KCNQ2-DEE and KCNQ2-SeLFNE. KCNQ2-LOF induced a biphasic network dysfunction, with early Kv7-driven hyperexcitability rescued by acute Retigabine (RTG) treatment, followed by maladaptive remodeling in the opposite direction. Transcriptomic analysis mirrored this biphasic dynamic trajectory, revealing an initial upregulation followed by a subsequent downregulation of synaptic genes. Structural analysis showed a steeper decline in presynaptic density alongside a distal shift in the axon initial segment (AIS) throughout maturation, and impaired AIS plasticity at later stages. Overall, KCNQ2-LOF disrupts human neuronal maturation through dynamic, biphasic changes in function, gene expression and structure, offering insights into disease mechanisms and therapeutic options.

neuroscience↗

Efficient and accurate prime editing strategy to correct genetic alterations in hiPSC using single EF-1alpha driven all-in-one plasmids

Prime editing (PE) is currently the most effective and versatile technology to make targeted alterations in the genome. Several improvements to the PE machinery have recently been made, and have been tested in a range of model systems, including immortalized cell lines, stem-cells and animal models. While nick RNA (ncRNA)-dependent PE systems like PE3 and PE5 are currently considered to be the most effective, they come with undesired indels or SNVs at the edit locus. Here, we aimed to improve ncRNA-independent systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids, driven by a tissue-broad EF-1alpha promoter, that is especially suitable for human iPSC models, and linked to a GFP tag for fluorescent based sorting. These novel pAIO systems effectively corrected mutations observed in patients suffering from epileptic encephalopathy, including a truncating SCN1A R612* variant in HEK293T cells and a gain-of-function KCNQ2 R201C variant in patient-derived hiPSC, with edit efficiency up to 50%. We also show that introducing additional silent PAM-removing mutations can negatively influence edit efficiency. Finally, we observed an absence of genome-wide PE off-target effects at pegRNA homology sites. Taken together, our study shows an improved efficacy and accuracy of EF-1alpha driven ncRNA-independent pAIO PE plasmids in hiPSC.

molecular biology↗