Trojanowski, N. F. | Search bioRxiv

bioRxiv2026

PV interneuron-targeted CRISPRa rescue of SCN1A haploinsufficiency in Dravet syndrome

Dravet syndrome is a severe epileptic encephalopathy caused by SCN1A haploinsufficiency, which leads to reduced NaV1.1 expression in parvalbumin (PV)-expressing interneurons and disrupted excitatory-inhibitory balance in the brain. We developed an AAV-based CRISPR activation system (AAV9-E2-dCas9-VP64) to selectively upregulate SCN1A from its endogenous locus in PV interneurons. An in vitro saturating guide RNA (gRNA) screen across the human SCN1A promoter identified a lead guide with robust and highly specific engagement of the SCN1A locus. This lead gRNA was validated in human Dravet syndrome model GABAergic neurons, where dose-dependent and specific SCN1A upregulation was observed. Intracerebroventricular (ICV) administration in a mouse model of Dravet syndrome produced dose-dependent improvement in survival as well as reduced susceptibility to hyperthermia-induced seizures and increased NaV1.1 protein expression, with maintained PV interneuron selectivity and minimal off-target expression. In a study in juvenile cynomolgus macaques, MRI-guided ICV administration of the vector was well tolerated, achieved broad cortical biodistribution, and maintained strong detargeting of peripheral tissues, with substantially lower peripheral dCas9 expression relative to the brain. These results support PV interneuron-selective SCN1A gene modulation via CRISPR activation as a promising therapeutic strategy for Dravet syndrome. AAV9-E2-dCas9-VP64 (RT101) is currently in preclinical development and is being advanced toward evaluation in the clinic.

neuroscience↗

bioRxiv2019

Activity labeling in vivo using CaMPARI2 reveals electrophysiological differences between neurons with high and low firing rate set points

Individual excitatory neurons in visual cortex (V1) display remarkably stable mean firing rates over many days, even though these rates can differ by several orders of magnitude between neurons. When perturbed, each neurons firing rate is slowly regulated back to its pre-perturbation level, demonstrating that neurons maintain their mean firing rate around an individual firing rate set point (FRSP). To better understand the mechanisms that neurons within a single cell type use to maintain different FRSPs in vivo, we implemented a novel method of activity labeling that uses CaMPARI2, a fluorescent protein that undergoes Ca2+- and UV-dependent green-to-red photoconversion, to permanently label neurons in freely behaving mice based on their firing rates. We found that immediate early gene (IEG) expression was correlated with CaMPARI2 red/green ratio following an activity stimulation paradigm, and that neurons with greater photoconversion in vivo tended to have a higher firing rate ex vivo. In layer 4 (L4) pyramidal neurons in mouse monocular V1, which comprise a single transcriptional cell type, we found that high activity neurons had a left-shifted F-I curve, lower rheobase current, and decreased spike adaptation index relative to low activity neurons, demonstrating increased intrinsic excitability. Surprisingly, we found no difference in total excitatory or inhibitory synaptic current or in E/I ratio between high and low activity neurons. Thus, within a single cell type differences in intrinsic excitability and spike frequency adaptation can contribute to divergent activity set points. These results reveal that E/I ratio plays only a minor role in determining the firing rate set point of L4 pyramidal neurons, while intrinsic excitability is an important factor.

neuroscience↗