Development of an UNC13A antisense oligonucleotide treatment for ALS and FTD Project Summary Background: In 97% of ALS cases, and roughly half of FTD cases, TAR DNA-binding protein 43 (TDP-43) is lost from the nucleus to the cytoplasm, where it forms into aggregates. A major function of TDP-43 in the nucleus is to repress cryptic exon (CE) inclusion during RNA splicing. Recent studies have shown that a CE is included in UNC13A mRNA when TDP-43 is depleted from the nucleus of neurons resulting in a loss of UNC13A protein. UNC13A single nucleotide polymorphisms (SNPs) are among the strongest hits associated with ALS and FTD in human GWAS studies. The CE is located in the same intronic region as the primary risk SNP. The risk SNP exacerbates the UNC13A CE inclusion - ALS / FTD patients with both risk alleles have more CE inclusion than patients with one risk allele, who have more CE inclusion than patients with the non-risk alleles. Similarly, risk SNP carriers have a dose-dependent reduction in survival. This additive risk strongly suggests that targeting UNC13A to suppress the CE inclusion could have a substantial therapeutic benefit. Since nearly all ALS patients have TDP-43 pathology, such a treatment would benefit them and not be limited to SNP carriers. UNC13A plays a critical role in synaptic transmission and is essential for synaptic vesicle release at most excitatory synapses and neuromuscular junctions. Among all the genes known to be dysregulated by the loss of TDP-43 from the nucleus, only UNC13A has such strong genetic validation. Altered STMN2 expression in the context of TDP-43 depletion is a hallmark of ALS / FTD, yet no link between ALS risk and STMN2 variants has been established. Thus, UNC13A targeting treatments should be prioritized for rapid advancement into clinical proof of concept studies. AcuraStem’s mission is to identify targets that rescue multiple forms of ALS / FTD. Thus we have established patient-specific ALS / FTD disease models of both genetically defined and sporadic diseases. We show in these models that cortical neurons derived from an ALS patient with the risk SNP have more UNC13A CE inclusion than neurons derived from ALS patients with non-risk alleles. When we depleted TDP-43 in patient-derived neurons using short interfering RNAs (siRNA) it induced a robust inclusion of CEs and reduced normal UNC13A mRNA and protein levels across many patient lines. Antisense oligonucleotides (ASOs) are an attractive approach for genetic targets in the CNS like UNC13A with several successes, including nusinersen for spinal muscular atrophy (SMA) and tofersen, soon to be approved for SOD1-ALS. We used our proprietary ASO design platform to synthesize >125 ASOs and identified several novel ASO sequences that potently blocked CE inclusion and restored UNC13A mRNA and protein levels. AcuraStem is uniquely positioned with potent UNC13A ASO candidates that can be advanced towards the clinic for ALS and FTD. Objectives & Impact: T...