Project Summary / Abstract Successful development of gene-based therapeutics, including gene delivery and RNA interference (RNAi)-based therapies, requires establishing the mechanism by which gene mutations contribute to pathogenesis. For instance, therapeutics for Loss of Function (LoF) mutations will be focused on increasing gene expression, possibly through viral gene delivery. The development of gene-based therapies for dominant disorders based on Gain of Function (GoF) or Dominant Negative (DN) mechanisms is more complex. For GoF mutations, researchers have mainly focused on using RNAi to knockdown gene expression that targets both the mutant and wild-type allele. However, in many cases, the reduction of the single wild-type allele may have deleterious consequences. For DN diseases, knockdown of both alleles will unlikely be successful as the ratio of mutant:wild-type protein is not expected to change. One method to overcome these limitations is to develop RNAi that are specific to the actual mutation, however, single base pair discrimination is technically very challenging and would require a different therapeutic for each mutation. Based on these premises, there is a dire need to develop mutant-specific RNAi for the therapeutic treatment of diseases resulting from dominant mutations with a GoF or DN pathogenic mechanism. We have recently developed a novel method for mutant specific RNAi which overcomes the limitations described. Here, we will focus on advancing this technology through the comprehensive development of therapeutics for ALS/FTD. The objective of this application is to identify and develop potential therapeutics for the allele-specific knockdown of mutant alleles in the TARDBP and KIF5A genes which are causative for ALS/FTD. To test our hypothesis, we have designed two specific aims: (1) To Design and Evaluate Allele- Specific ASOs for TARDBP/KIF5A. ASOs spanning target regions will be tested for mutant-specific knockdown within isogenic iPSC-derived cortical neurons (iCNs) harboring either TARDBP or KIF5A mutations. Further modifications will be incorporated as needed to improve allelic discrimination, potency, and possible toxic effects. (2) To Evaluate the Rescue of Allele-Specific ASOs in Mutant TARDBP/KIF5A iCNs. IPSc-derived neurons harboring mutant TARDBP/KIF5A display alterations in gene expression, alternative/cryptic splicing, nucleocytoplasmic transport, and survival. Here, we will evaluate the rescue of these defects by mutant specific ASOs to isogenic iCNs harboring mutations in TARDBP/KIF5A. Impact: This study will provide a comprehensive development of allele specific- ASOs (AS-ASOs) for mutations in TARDBP/KIF5A. Undoubtedly, AS-ASOs will have broad applications for therapeutic development across numerous genes contributing to ALS and numerous other human diseases.