Autism spectrum disorder (ASD) is a highly heterogeneous and highly heritable condition with complex genetic contributions, and yet 20 percent of genetic risk is imparted by de novo mutations of major effect. Most of the currently known high-confidence ASD risk genes harbor de novo mutations that are either known to or predicted to lead to truncated mRNA and protein products, strongly indicating likely haploinsufficiency¹. Given that these mutations affect only one allele, a potential therapeutic avenue would be activation of expression from the non-mutant allele to restore gene expression to normal or near-normal levels.

Daniel Geschwind and his colleagues propose to develop such an approach by leveraging advances in gene editing technology, using CRISPR-mediated activation (CRISPRa) to target enhancer regions of high-confidence ASD genes, combined with stem cell–based three-dimensional cortical spheroids in vitro², which have been shown to faithfully recapitulate many features of in vivo cortex development. To facilitate this approach, over the last five years, Geschwind and his team have built genome-wide maps of active enhancer-gene combinations from the human brain based on Hi-C, ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing) and eQTL (expression quantitative trait loci)^3,4. Using these maps, the team has developed a comprehensive list of evolutionarily conserved enhancer-gene pairs for 13 high-confidence ASD genes predicted to be haploinsufficient.

In preliminary experiments, Geschwind’s team has been able to provide proof-of-principle data showing that CRISPRa increased expression levels for most of these 13 ASD risk genes when paired with enhancer-targeting guide RNA. They next plan to use stem cell–derived cortical spheroids to test this approach during neuronal differentiation and determine if spheroids carrying ASD risk mutations can be successfully rescued using CRISPRa. Once functional guides are identified, they can be tested in vivo in preclinical studies with the eventual goal of moving towards therapy in humans.

Taken together, this proposal will enhance understanding of the effects of haploinsufficiency in ASD genes, the mechanisms whereby they impact neuronal development, and provide a proof-of-principle for gene activation as a therapeutic intervention.

1. Ruzzo E.K. et al. Cell 178, 850-866 (2019) PubMed
2. Yoon S.J. et al. Nat. Methods 16, 75-78 (2019) PubMed
3. de la Torre-Ubieta L. et al. Cell 172, 289-304 (2018) PubMed
4. Walker R.L. et al. Cell 179, 750-771 (2019) PubMed