Exome sequencing studies for autism spectrum disorder (ASD) have identified variants in SCN2A as being among the most common risk factors for ASD¹. SCN2A encodes the alpha subunit of the voltage-gated sodium channel Na_V1.2, which plays a role in neuronal excitability, particularly during early development. Using Scn2a heterozygous mice, the laboratory of Kevin Bender at the University of California, San Francisco have unpublished findings that suggest that Scn2a haploinsufficiency leads to deficits in neuronal excitability during early development, as well as deficits in synaptic function that persist into adulthood.

In this proposal, Nadav Ahituv plans to use these mice as a tool to test a CRISPR activation (CRISPRa) therapeutic for this gene, upregulating the existing Scn2a functional copy in these mice and analyzing the phenotypic consequences of this upregulation. Preliminary results from Ahituv’s laboratory suggest that CRISPRa can be used as a therapeutic tool to rescue deficits in Scn2a haploinsufficient mice in vivo.

The specific aims of this proposal are:

1. Optimize adeno-associated virus (AAV) CRISPRa conditions in mice.

Ahituv’s laboratory will generate AAV vectors that target Scn2a in mice and optimize the titers, single guide RNA (sgRNA) targets and injection conditions (both location and developmental age) to achieve activation levels in Scn2a heterozygous mice similar to that of wild-type mice.

2. Assess the phenotypic consequences of Scn2a CRISPRa.

Using the optimized CRISPRa conditions, Ahituv’s laboratory will rescue Scn2a levels in Scn2a heterozygous mice to wild-type levels and determine whether neuronal excitability and synaptic function is restored.

This work will provide insights into the possible therapeutic potential of CRISPRa gene therapy for the treatment of ASDs that are a result of SCN2A genetic mutations. In addition, this mouse model system will allow future testing, via targeted CRISPRa injections into Scn2a heterozygous mice, of the neural and temporal specificity through which SCN2A haploinsufficiency can lead to ASD.