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

Abstract

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.

Physical and functional convergence of the autism risk genesScn2aandAnk2in neocortical pyramidal cell dendrites

“…This suggests that restoration of Ank2 function would have similar benefits, at least in dendritic regions where it actively scaffolds Na V 1.2. ANK genes are too large for traditional gene therapy approaches; however, several other approaches are maturing for gene regulation in neurodevelopmental disorders, with marked progress for a number of genetic conditions (Colasante et al, 2020; Derbis et al, 2021; Han et al, 2020; Tamura et al, 2022; Ure et al, 2016; Weuring et al, 2021; Wolter et al, 2020). In addition, a better understanding of the unique roles of different ankyrin proteoforms in scaffolding and function of various binding partners (Gidon and Segev, 2012; Ujfalussy et al, 2015; Wilson et al, 2012; Zhang et al, 2013) may provide insight into methods that allow ankyrins to better compensate for one another in dendritic compartments.…”

“…Excitatory synapses appear similar to those found in immature neurons, with relatively small spine heads and low AMPA:NMDA receptor ratios, suggesting that synapses may be maintained in an immature, pre-critical period state (Spratt et al, 2019). Indeed, restoration of near WT levels of Scn2a , either via Cre-induced genetic rescue or CRISPR activator-based upregulation of the residual, functional allele in Scn2a heterozygotes, restores dendritic excitability, synapse morphology, and synapse function to WT levels (Tamura et al, 2022). This suggests that restoration of Ank2 function would have similar benefits, at least in dendritic regions where it actively scaffolds Na V 1.2.…”

Proximity Analysis of Native Proteomes Reveals Interactomes Predictive of Phenotypic Modifiers of Autism and Related Neurodevelopmental Conditions

“…Thus, their downregulation in Scn2a +/R102Q are likely contributing factors to VGSC channelopathy. These results also explain why Scn2a-CRISPRa treatment alone is insufficient to restore the deficits, especially since the treatment does not differentiate the functional allele from the point-mutant allele 67 . It appears that supplying additional SCN1B and FGF12 provides the molecular environment needed to restore functional VGSC complexes and reinstate the level of spontaneous neural activity.…”

“…S5A). This strategy has recently been used to phenotypically rescue Scn2a haploinsufficiency 67 but has not yet been tested in neurons harboring patient-derived missense mutations. This treatment transcriptionally activates Scn2a expression; however, it is expected to amplify both the WT and mutant allele.…”

rAAV-CRISPRa therapy corrects Rai1 haploinsufficiency and rescues selective disease features in Smith-Magenis syndrome mice

“…However, the development of Rai1 gene transfer therapy is limited by the size of Rai1 coding sequence, which exceeds rAAV’s payload capacity. The advent of clustered regularly interspaced short palindromic repeats activation (CRISPRa) system and the discovery of the smaller Staphylococcus aureus CRISPR-associated protein 9 (saCas9) hold great promise for rAAV-mediated, gene size-independent therapy in treating SMS and other disorders associated with genetic forms of obesity, epilepsy, blindness, and muscular dystrophy ( 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ). A common strategy to express Cas9 and single-guide RNAs (sgRNAs) is using dual rAAV systems that could result in low co-infection efficiency ( 28 ).…”