UC San Diego

The past decade of research has yielded much success in the identification of risk genes for Autism Spectrum Disorder (ASD), with many studies implicating loss-of-function (LoF) mutations within these genes. Despite these successes, no significant clinical advances have been made so far in the development of therapeutics for ASD. Given the role of LoF mutations in ASD etiology, many of the therapeutics in development are designed to rescue the haploinsufficient effect of genes at the transcriptional, translational, and protein levels. The first half of this thesis will begin by reviewing the various therapeutic techniques being developed from each level of the central dogma with examples including: CRISPR activation (CRISPRa) and gene delivery at the genetic level, antisense oligonucleotides (ASOs) at the mRNA level, and small-molecule drugs at the protein level, followed by a review of current delivery methods for the aforementioned therapeutics. The second half of this thesis will detail our own lab’s experimental results using mRNA-level therapeutics to target natural antisense transcripts (NATs) that are complimentary to mRNA transcripts for ASD-associated, haploinsufficient genes with the following goals: 1. Delineating relationships between NATs and their respective sense genes and 2. Increasing the expression of the sense gene through degradation of the respective NAT. Specifically, the two methods we have utilized are the previously mentioned ASOs, as well as a new system known as CAS13d—a method of mRNA knockdown through a CRISPR/CAS construct. Given the bidirectional effect of NATs on the translation of genes, many of our experiments elucidated the relationships that NATs had on their respective sense genes. Additionally, although some ASOs effectively degraded the NAT mRNA as well as increased the expression of the sense gene, the CAS13d system showed no such success. Thus, further experiments are needed to optimize the CAS13d system in this therapeutic context.