A Multimodal and Spatially Resolved Dissection of Antisense Oligonucleotide Therapy of FUS-Related ALS Disease Progression

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons in the spinal cord, universally leading to progressively severe paralysis and, ultimately, respiratory failure. While the majority of cases are sporadic, individuals with a family history of ALS exhibit heritability. Roughly 5% of these familial cases are due to mutations in the FUS gene. Among these cases are patients that develop juvenile onset ALS, a rare manifestation in the general ALS population. Juvenile FUS-ALS is characterized by symptoms that appear before 25 years of age and are often accompanied by aggressive and rapid disease progression. There is currently no effective treatment for ALS; however, a promising antisense oligonucleotide (ASO) therapy targeting FUS-ALS, ION363, is being allowed compassionate use in clinical trials. The objective for ION363 is to deplete toxic mutant FUS protein from the spinal cord with the intention of stopping disease spread and allowing cells to recover as much as possible from existing pathology, thereby extending patient life expectancy and improving quality of life. Unfortunately, despite having achieving the desired molecular result (mutant FUS depletion from the patient spinal cord), ION363 treatment has not significantly improved the clinical outcome in patients enrolled in the trial.

Our proposed research project focuses on first identifying the molecular underpinnings associated with the efficacy and potential limitations of ION363 treatment and subsequently identifying opportunities for enhancing treatment efficacy. There is a potential disconnect between the established readout for successful treatment, FUS protein depletion, and the desired outcome, which is reestablishment of normal cellular function. In order to connect and fill the gap between these differences, we propose to study them in a common underlying space: gene expression, as measured by mRNA transcript abundance. ION363 is an ASO, and exerts its effects by binding to FUS pre-mRNA transcripts. Instead of being processed into mature mRNA and translated into protein, these ASO-bound transcripts are degraded. Therefore, loss of mature FUS mRNA is an equivalent readout to loss of FUS protein expression. Furthermore, each cell type in the spinal cord has a distinct gene expression profile in accordance with its biological role. Gene expression profiles of cells can be assessed through measuring the mRNA abundance for each gene in the cell. By determining the gene expression profiles of individual cells in the spinal cord from healthy individuals, it is possible to define what healthy 'control' cells looks like in gene expression space. During ALS disease, as cells become sick and die, their gene expression profiles change in accordance with how they are impacted by the disease. Consequently, by measuring mRNA abundance in cells from an ALS patient spinal cord, it is possible to define what 'diseased' cells look like in gene expression space. Finally, by measuring mRNA abundance in cells from ION363-treated ALS patient spinal cords, it is possible to define what 'treated' cells look like in gene expression space. Notably, because of the intrinsic differences between different cells in the spinal cord, it is important to take these measurements in individual cells. By analyzing and comparing the gene expression profiles of control, diseased, and treated cells, it becomes possible to ask how ALS dysregulates gene expression, how ION363 treatment affects diseased cells, and to what extent did treatment return diseased cells to a control state. These data also make it possible to probe off-target effects ASO treatment has, which could potentially disrupt cells returning to a healthy state. Given that the FUS protein is involved in RNA processing, removing it from cells completely will have an impact on gene expression that is distinct from any restorative effects of ablating toxic mutant FUS.

A better understanding of the impact ION363 treatment has on cells within the human spinal cord will make it possible to enhance this ASO therapy. A possible optimization strategy could include use of a combination of ASOs that target not just FUS, but also genes that had been dysregulated in disease and not restored upon treatment. Another possible strategy, if off-target effects are severe, would be to modify the ASO so that it has a reduced impact on FUS mRNA degradation. This project, which is projected to take two years to complete, will provide an important resource for the ALS community and will provide valuable insight toward optimizing a promising clinical intervention that has not yet reached its full potential.