Biology subjects

Spratt, P. W.

Publications and source records attributed to Spratt, P. W..

4 recordsLinked to original sources

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↗

bioRxiv2022

CRISPR activation rescues abnormalities in SCN2A haploinsufficiency-associated autism spectrum disorder

The majority of autism spectrum disorder (ASD) risk genes are associated with ASD due to haploinsufficiency, where only one gene copy is functional. Here, using SCN2A haploinsufficiency, a major risk factor for ASD, we show that increasing the expression of the existing functional SCN2A allele with CRISPR activation (CRISPRa) can provide a viable therapeutic approach. We first demonstrate therapeutic potential by showing that restoring Scn2a expression in adolescent heterozygous Scn2a conditional knock-in mice rescues electrophysiological deficits associated with Scn2a haploinsufficiency. Next, using an rAAV-CRISPRa based treatment, we restore electrophysiological deficits in both Scn2a heterozygous mice and human stem-cell-derived neurons. Our results provide a novel therapeutic approach for numerous ASD-associated genes and demonstrate that rescue of Scn2a haploinsufficiency, even at adolescent stages, can ameliorate neurodevelopmental phenotypes.

neuroscience↗

bioRxiv2021

Paradoxical hyperexcitability from NaV1.2 sodium channel loss in neocortical pyramidal cells

Loss-of-function variants in the gene SCN2A, which encodes the sodium channel NaV1.2, are strongly associated with autism spectrum disorder and intellectual disability. An estimated 20-30% of children with these variants are co-morbid for epilepsy, with altered neuronal activity originating in neocortex, a region where NaV1.2 channels are expressed predominantly in excitatory pyramidal cells. This is paradoxical, as sodium channel loss in excitatory cells would be expected to dampen neocortical activity rather than promote seizure. Here, we examined pyramidal neurons lacking NaV1.2 channels and found that they were intrinsically hyperexcitable, firing high-frequency bursts of action potentials (APs) despite decrements in AP size and speed. Compartmental modeling and dynamic clamp recordings revealed that NaV1.2 loss prevented potassium channels from properly repolarizing neurons between APs, increasing overall excitability by allowing neurons to reach threshold for subsequent APs more rapidly. This cell-intrinsic mechanism may therefore account for why SCN2A loss-of-function can paradoxically promote seizure.

neuroscience↗

bioRxiv2020

Functional microstructure of CaV-mediated calcium signaling in the axon initial segment

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (CaV). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we utilize conventional 2-photon laser scanning and diffraction-limited, high-speed spot 2-photon imaging to resolve action potential-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of CaV2 and CaV3 channels that differentially localized to discrete regions. CaV3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-dependent stores, whereas CaV2 localized to non-hotspot regions. Thus, different pools of CaVs appear to play distinct roles in AIS function.

neuroscience↗