Na v igating Phenotypic Characterization and CRISPRa Restoration for SCN2A Haploinsufficiency Disorders

Abstract

Sex-Specific Behavioral Features of Juvenile and Adult Haploinsufficient Scn2a^+/− Female Mice, Model of Autism Spectrum Disorder

Marcantonio W, Simonti M, Lena I, Mantegazza M. Genes Brain Behav. 2025;24(5):e70034.

Genetic variants of the SCN2A gene, encoding the Na_V1.2 sodium channel, cause a spectrum of neurodevelopmental and epileptic disorders, and are among those that show the strongest association with Autism Spectrum Disorder (ASD). ASD has a male-bias prevalence, but several studies have proposed that female prevalence may be underestimated due to different symptomatic expression compared with males. However, it is unclear whether this is related to actual different pathological features or to greater masking abilities in females. Studies on Scn2a^+/− mice, a model of SCN2A haploinsufficiency and ASD, have shown an age-dependent ASD-like phenotype attenuated at adulthood in males. However, little is known about the behavioral features of Scn2a^+/− female mice. We performed a battery of behavioral tests that are relevant for assessing ASD-like features, investigating juvenile and adult Scn2a^+/− female mice. Our results demonstrate that female Scn2a^+/− mice exhibit an overall milder phenotype than males, showing increased risk-taking in juveniles, hyper-reactivity to cold stimuli, and mild memory impairments in adults, abnormally increased sociability, and altered decision-making related behaviors in both juveniles and adults. Thus, this aligns with the male-biased prevalence of ASD and supports the existence of sex-specific phenotypic differences, potentially arising from distinct underlying pathophysiological mechanisms. Both sexes should be investigated in studies of mouse models of ASD.

CRISPR Activation for SCN2A-Related Neurodevelopmental Disorders

Tamura S, Nelson AD, Spratt PW, Hamada EC, Zhou X, Kyoung H, Li Z, Arnould C, Barskyi V, Krupkin B, Young K. Nature, 2025;646, 983–991.

Most neurodevelopmental disorders with single gene diagnoses act via haploinsufficiency, in which only one of the two gene copies is functional. SCN2A haploinsufficiency is one of the most frequent causes of neurodevelopmental disorder, often presenting with autism spectrum disorder, intellectual disability, and, in a subset of children, refractory epilepsy. Here, using SCN2A haploinsufficiency as a proof-of-concept, we show that upregulation of the existing functional gene copy through CRISPR activation (CRISPRa) can rescue neurological-associated phenotypes in Scn2a haploinsufficient mice. We first show that restoring Scn2a expression in adolescent heterozygous Scn2a conditional knock-in mice rescues electrophysiological deficits associated with Scn2a haploinsufficiency (Scn2a^+/−). Next, using an adeno-associated virus CRISPRa-based treatment in adolescent mice, we show that we can correct intrinsic and synaptic deficits in neocortical pyramidal cells, a major cell type that contributes to neurodevelopmental disorders and seizure aetiology in SCN2A haploinsufficiency. Furthermore, we find that systemic delivery of CRISPRa protects Scn2a^+/− mice against chemoconvulsant-induced seizures. Finally, we also show that adeno-associated virus CRISPRa treatment rescues excitability in SCN2A haploinsufficient human stem-cell-derived neurons. Our results showcase the potential of this therapeutic approach to rescue SCN2A haploinsufficiency and demonstrates that rescue even at adolescent stages can ameliorate neurodevelopmental phenotypes.

Commentary

SCN2A, which encodes the alpha subunit of the Na_v1.2 sodium channel, plays a critical role in regulating neuronal excitability. Mutations in SCN2A are associated with a broad spectrum of clinical presentation, encompassing mild seizures to severe epileptic encephalopathy, autism spectrum disorder (ASD), intellectual disability (ID), and ataxia. Some genotype–phenotype correlations have been identified. ¹ For example, gain-of-function (GOF) mutations that increase Na_v1.2 channel activity have been associated with benign familial-neonatal seizures, neonatal seizures and late episodic ataxia, and developmental and epileptic encephalopathy (DEE). In contrast, loss-of-function mutations that decrease channel activity or expression (i.e., protein truncations and frameshift) have been associated with late-onset DEE (after 3 months of age), ASD, and ID with or without epilepsy. There are also variants in SCN2A that exhibit a combination of gain- and loss-of-function effects on channel activity.

Given the wide range of clinical presentations in patients with SCN2A mutations, numerous mouse models have been developed to gain insight into the underlying mechanisms of SCN2A-associated disorders. From mouse models, we have learned that Na_v1.2 localization is developmentally regulated. In the first week of life, Na_v1.2 is found at the axon initial segment, but it is soon replaced by Na_v1.6 and redistributed to the dendrites. ² Furthermore, immature and mature glutamatergic cortical neurons from heterozygous knockout Scn2a^+/− mice exhibit impaired action potential generation, while complete deletion of Scn2a from mature glutamatergic cortical neurons using a conditional Scn2a knockout mouse (postnatal day, P28-44) also leads to hyperexcitability.^3,4 These observations highlight the unique contribution of Na_v1.2 to neuronal excitability during development and adulthood. Some mouse models of Scn2a dysfunction recapitulate the seizure phenotypes observed in patients, including generalized tonic–clonic seizures and absence seizures. Since mice that model Scn2a GOF mutations die prematurely from spontaneous seizures, behavior has not been thoroughly examined in those models. In contrast, heterozygous knockout mice (Scn2a^+/−) have been shown to exhibit ASD-like phenotypes, absence seizures, learning and memory impairment, and reduced anxiety-like behavior.^5–7

Most behavior studies in Scn2a^+/− mice have utilized male mice, which is not surprising given the male-bias in ASD prevalence. In a recent study, Marcantonio et al., ⁸ address an important gap in ASD modeling, which is the underrepresentation of female subjects in preclinical studies. The authors performed a battery of ASD-related behavioral assessments in juvenile (P21-44) and adult (>P70) female Scn2a^+/− mice. ⁸ Compared to the authors’ previous analysis of male Scn2a^+/− mice, ⁶ female Scn2a^+/− mice exhibited a milder ASD-like phenotype, displaying normal locomotor behavior, no anxiety-like behavior, normal recognition memory, no stereotypies, increased sociability, a slight reduction in working memory, and altered decision-making. ⁸ Possibly due to the milder phenotype in female Scn2a^+/− mice, there was no difference in behavior in the female mutants at the different ages tested, unlike the authors’ prior analysis of male Scn2a^+/− mice that revealed an age-related attenuation of ASD-like behaviors. ⁶ Interestingly, a previous characterization of a gene-trap mouse with ∼75% reduction in Scn2a expression revealed increased sensitivity to hot and cold temperatures. ⁹ In the current study, Marcantonio and colleagues ⁸ observed increased sensitivity only to cold temperatures in juvenile and adult male and adult female Scn2a^+/− mice. Together, the observations from this study reveal sex-specific phenotypic differences in Scn2a^+/− mice, consistent with patterns reported clinically in ASD. They emphasize the importance of including both sexes in ASD research to gain insight into biological mechanisms that may differ between males and females, and these findings introduce an important sex-specific consideration when evaluating potential treatments.

The ideal treatment strategy for SCN2A haploinsufficiency would be to restore gene expression, which in turn might ameliorate or reverse clinical symptoms. However, given the size of the open reading frame for human SCN2A (>6 kb), this prevents the utilization of traditional adeno-associated viruses (AAVs), which are limited by their packaging capabilities (∼4.7 kb). Tamura and colleagues ¹⁰ developed a CRISPR activation (CRISPRa) approach to target the Scn2a promoter to upregulate gene expression in Scn2a haploinsufficient mice. ¹⁰ As a proof-of-principle, the authors first generated a conditional knock-in mouse where exons 3–5 of Scn2a were flipped and flanked by LoxP sites (Scn2a^+/KI). The authors injected rAAV-EF1α-Cre-mCherry into the medial prefrontal cortex (mPFC) of adolescent Scn2a^+/KI mice, and in the presence of Cre recombinase, neuronal and synaptic function were comparable to wild-type (WT) mice, ¹⁰ demonstrating that reactivation of Scn2a during adolescence can restore Scn2a-mediated intrinsic and synaptic function. Next, Tamura et al., ¹⁰ unilaterally injected their optimized CRISPRa construct (Scn2a-rAAV-CRISPRa) into the mPFC of constitutive Scn2a^+/− mice at P30. ¹⁰ With this approach, the authors were able to increase Scn2a mRNA expression by 1.5-fold and restore Scn2a-mediated action potential depolarization, dendritic excitability, and excitatory synapse function to WT levels. Using a more clinically-relevant delivery strategy, intravenous administration of Scn2a-rAAV-CRISPRa was also able to normalize Scn2a-mediated electrophysiological defects.

Since some patients with SCN2A haploinsufficiency exhibit seizures that are often refractory to treatment, ¹ Tamura et al., ¹⁰ also investigated whether increasing Scn2a expression could alter seizure susceptibility in Scn2a^+/− mice. Scn2a-rAAV-CRISPRa was delivered via retro-orbital administration to Scn2a^+/− mice at P30, and susceptibility to the chemiconvulsant 4-aminopyridine (4-AP, 8 mg/kg) was assessed 42–65 days later. Notably, ictal spike frequency was comparable in mice treated with Scn2a-rAAV-CRISPRa and WT mice. Since patients with gain-of-function SCN2A mutations are associated with severe epileptic encephalopathy, ¹ one potential concern for the CRISPRa approach is that overexpression of Scn2a beyond normal physiological levels could result in neuronal hyperexcitability. Importantly, while the injection of Scn2a-rAAV-CRISPRa into the mPFC of WT mice resulted in a 1.75-fold increase in Scn2a mRNA expression, action potential properties, and ictal spike frequency following 4-AP administration were comparable to untreated WT mice. ¹⁰ Altogether, Tamura and colleagues ¹⁰ demonstrated that their CRISPRa approach was able to increase Scn2a mRNA levels and rescue electrophysiological defects in a mouse model and human neuronal cell model of Scn2a haploinsufficiency without adverse effects on neuronal excitability.

Together, these two recent studies highlight a powerful convergence between phenotypic understanding and molecular rescue of Scn2a haploinsufficiency disorders. Marcantonio and colleagues ⁸ identified sex- and age-specific behavioral outcomes in Scn2a^+/− mice that could serve as downstream endpoints for evaluating CRISPRa-based interventions. Complementing this, Tamura et al., ¹⁰ demonstrated that restoring Scn2a mRNA expression in adolescence is sufficient to normalize neuronal excitability and synaptic function, which could be impairments that contribute to the observed behavioral deficits Scn2a^+/− mice. Although SCN2A is often discussed in the context of epilepsy, it is one of the most important genes associated with autism. ¹¹ Future studies exploring whether CRISPRa can rescue behavioral deficits in Scn2a^+/− mice will be particularly informative. Given the age- and sex-dependent effects on behavior in Scn2a^+/− mice documented by Marcantonio and colleagues ⁸ treatment responses may also vary by sex, and the developmental timing of CRISPRa administration could be especially important. Another key consideration is the developmental shift between the neonatal (5N) and adult (5A) isoforms of SCN2A, as the 5A isoform was shown to increase neuronal excitability and seizure susceptibility in mice.^12,13 Thus, it is possible that CRISPRa-induced upregulation of Scn2a during 5N- or 5A-dominant stages could have different functional consequences. By integrating molecular upregulation of SCN2A with how SCN2A haploinsufficiency manifests across development and sex, these studies offer a foundation for advancing targeted therapies not only for SCN2A-related disorders but for a broad range of neurological disorders.