Food and Drug Administration (FDA) approval and clinical use of antisense oligonucleotide (ASO) therapies have demonstrated their potential for treating monogenic disorders. An ASO is a synthetic single-stranded oligonucleotide that binds to a complementary RNA sequence. To date, ASO therapies function either as inhibitors of gene expression (e.g., mipomersen for reducing apolipoprotein B cholesterol levels) or as splice-modulators that block the binding of proteins to splice enhancers or junctions (e.g., nusinersen for the treatment of spinal muscular atrophy).

More than 100 autism spectrum disorder (ASD) susceptibility genes have been identified so far¹. A substantial fraction of risk variants (25 percent) are protein-truncating de novo mutations, suggesting that genetic therapies designed to restore the function of such genes are a promising strategy for the development of new treatments for ASD. However, current ASO technologies are not applicable to most protein-truncating variants observed in ASD cases.

For haploinsufficiency, a broadly applicable therapeutic strategy would be one that restores gene expression to biallelic levels by boosting the overall expression of the gene. To this end, ASOs have been developed that can disinhibit messenger RNA (mRNA) expression via multiple mechanisms. In particular, Jonathan Sebat’s lab has narrowed its focus to two types of ASOs and techniques: (1) AntagoNATs, which are ASOs that target natural antisense transcripts (NATs) that negatively regulate mRNAs and (2) targeted augmentation of nuclear gene output (TANGO), which involves the use of splice-modulating ASOs to increase mRNA abundance by targeting naturally occurring alternative-splice isoforms that are degraded by nonsense-mediated decay (AS-NMD).

While ASOs have been developed that can raise the expression of target genes, ASO development has been unsuccessful for the majority of targets because they lack the desired function as a repressor of mRNA expression. Thus, a critical step toward ASO development for haploinsufficiency is the identification of targets that have demonstrable inhibitory effects on the expression of ASD genes.

In this project, Sebat and his team propose to accelerate the development of antisense therapies for ASD genes by applying novel platforms for the identification of ASO targets. Aim 1 of this study will develop a high-throughput single-cell CRISPR screen for AntagoNAT targets. Aim 2 will seek to identify TANGO targets by transcriptome-wide analysis of AS-NMD in neural cells derived from induced pluripotent stem cells. Aim 3 will develop lead ASO molecules for a subset of targets identified in this study. Sebat’s platform-based approach, where ASO targets could be identified rapidly from whole-transcriptome screens, could significantly accelerate the lead-generation stage of ASO development.