Development of a UNC13A Cryptic Exon Skipping Antisense Oligonucleotide as a Treatment for ALS


Translational Research

Poster Number: M224


Zhihua Feng, PhD, AcuraStem, Stacee Mendonca, BS, AcuraStem, Joscany Perez, BS, AcuraStem, Emily Lee, BS, AcuraStem, Sarah Hong, BS, AcuraStem, Sarvatha Mukundan, MS, AcuraStem, Chien-Ping Ko, PhD, AcuraStem, Wen-Hsuan Chang, PhD, AcuraStem, Sam Alworth, MS, MBA, AcuraStem

Background: TDP-43 is a RNA-binding protein normally located in the nucleus, but in ALS patients it is depleted in the nucleus and forms into toxic aggregates in the cytoplasm of neurons. A variant in the UNC13A gene is one of the strongest risk factors for ALS. When TDP-43 is lost from the nucleus, a cryptic exon with a premature termination signal is introduced into the UNC13A messenger RNA, which leads to the loss of UNC13A protein. The UNC13A risk variant is located very close to the TDP-43 binding site and exacerbates the cryptic exon inclusion. Importantly, the risk variant is predictive of reduced survival in ALS and FTD patients in a dose-dependent manner. This link between patient outcomes and UNC13A pathology strongly suggests that UNC13A dysregulation could be central to disease progression in TDP-43 proteinopathies.

Objectives: We sought to develop and identify a novel development candidate antisense oligonucleotide (ASO) treatment that could potently and safely suppress the inclusion of the cryptic exon and restore the full-length UNC13A transcript by 1) using our iNeuroRx® disease-modeling platform to model and establish disease phenotypes for lead optimization, and 2) using cross-species models for target validation and pharmacodynamic modeling.

Methods: To assess the potential of candidate ASOs in mitigating cryptic exon inclusion and restoring neuronal function, we have created a novel in vivo target engagement model to evaluate human-specific sequences in a murine model alongside in vitro assays designed to measure synaptic function. RT-qPCR and western blot assessed varying concentrations of ASO to establish in vitro and in vivo potency.

Results: Several ASO candidates potently suppressed UNC13A cryptic exon inclusion in vitro and in vivo, and these candidates also rescued functional deficits in patient-derived neurons from multiple forms of familial and sporadic ALS / FTD. These candidates are now being advanced into toxicity studies.