Hayley S. McLoughlin, Ph.D. - US grants

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD), is the most common dominantly inherited ataxia in the world and caused by an expansion of a polyglutamine-coding CAG repeat in the ATXN3 gene. There currently is no effective treatment for this relentlessly progressive and fatal disease. Because expression of the mutant protein is an early and necessary step in disease pathogenesis, strategies to reduce expression of the disease gene itself are high on the list of potential therapies. Our previous therapeutic studies suggested the need for broad CNS delivery of gene silencing reagents. Chemically modified ASOs can be delivered broadly to the CNS and are known to be highly stable in vivo. Prior studies employing ASOs have documented well-tolerated, long-term knockdown in mouse and non-human primate models of neurodegenerative diseases including SMA, HD and ALS. Moreover, ASO therapy in spinal muscular atrophy (SMA) was recently FDA- and EMA-approved following multiple highly successful human clinical trials. We recently established ASO therapy proof-of-concept in a SCA3 mouse model, concluding that the strategy to reduce levels of the nonessential ATXN3 disease protein in patients would likely be well tolerated. This U01 discovery proposal extends from the proof-of-concept studies to characterize a final human candidate lead ASO compound for IND-enabling studies. Aim 1 will confirm the viability of 4-6 lead ATXN3 ASOs through in vitro screens for inflammatory and off-target effects and in vivo tolerability in rodent and non-human primates that will support IND-enabling studies. Aim 2 will assess the in vivo efficacy of the lead ATXN3 ASOs to suppress mutant ATXN3 expression and ameliorate behavioral deficits, alter disease pathology and molecular signatures in a SCA3 mouse model expressing the full-length human mutant ATXN3 transcript. The results will culminate in the selection of a single lead compound for future IND-enabling studies. Concurrent with these studies, a necessary next step for therapeutic success is the optimization of SCA3 disease biomarkers that may directly assess therapeutic target engagement and treatment response. Cerebrospinal fluid (CSF) is an ideal biological sample in which to look for therapeutic biomarkers as it is readily accessible and can be sampled repeatedly throughout disease progression and therapeutic trials. Significant advances in immunoassay technology now make it possible to quantify low abundance proteins using an ultrasensitive single molecule counting (SMC) immunoassay detection system. In Aim 3, we will optimize a SMC immunoassay to detect ATXN3 protein levels from SCA3 patient CSF samples. Developing this novel ATXN3 SMC immunoassay will enable detection of in vivo therapeutic target engagement and efficacy during ASO treatment in SCA3 patients.

ABSTRACT Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD), is one of nine polyglutamine expansion diseases and the most common dominantly inherited ataxia in the world. SCA3 is caused by an expansion of a polyglutamine-coding CAG repeat in the ATXN3 gene. Currently, there is no effective treatment for this relentlessly progressive and fatal disease. With the goal of preventive therapy for SCA3, several ongoing studies seek to reduce levels of the disease protein, ATXN3. Our published work with antisense oligonucleotides (ASOs) targeting human ATXN3 provides strong preclinical evidence of gene silencing efficacy in a SCA3 mouse model of disease. As we progress gene silencing and other therapeutics toward the clinic, a necessary hurdle for success is to define robust biomarkers of SCA3 disease that may directly/indirectly assess disease progression and treatment response. To address this gap in the field, we have designed a longitudinal trial that will repeat dose SCA3 mice with a gene silencing treatment beginning at an early symptomatic age and assess noninvasive high field magnetic resonance spectroscopy (MRS) and blood plasma biomarkers repeatedly over one year. These biomarkers will be correlated with disease progression as defined by brain pathology and motor assessment and assess how well the biomarkers reflect gene silencing efficacy in brain tissue. Based on our data with SCA3 gene silencing therapy and MRS in the SCA3 mouse model that expresses the full-length human ATXN3 disease gene, we expect to uncover reliable biomarkers of disease progression that are readily exacerbated by gene silencing therapy and are predicted to translate well to future human trials.

Spinocerebellar Ataxia type 3 (SCA3), also known as Machado-Joseph disease, is one of at least nine diseases caused by CAG repeat expansions that encode abnormally long polyglutamine tracts in the disease proteins. Despite advances in disease understanding, much remains unknown about how the CAG expansion in the SCA3 disease gene, ATXN3, causes brain dysfunction and cell death. We recently discovered selective oligodendrocyte vulnerability across SCA3 mouse disease brain regions and identified early and robust changes that implicate oligodendrocytes in disease pathogenesis. Our long-term objective is to understand pathogenic mechanisms in SCA3 and related polyglutamine diseases so that therapies targeting the most promising molecular and cellular targets can be developed for these fatal and currently untreatable disorders. Toward that objective, the team of investigators will leverage their diverse research expertise and a wide range of model systems including primary cell cultures, mouse models, and human disease tissue. Building on recent discoveries in mouse models and human disease tissue, we will investigate nonneuronal contributions to disease pathogenesis, with an emphasis on cells of the oligodendroglial lineage. In Aim 1, we will determine how widespread oligodendrocyte dysfunction is in SCA3 disease. In Aim 2, we will generate novel conditional SCA3 mouse models to establish if the mutant protein elicits cell-autonomous oligodendrocyte dysfunction and define oligodendrocyte dysfunction contributions to disease pathogenesis. In Aim 3, we will elucidate the molecular mechanisms that underlie oligodendrocyte dysfunction in SCA3 disease. Results of these studies will help guide therapeutic development in SCA3 and related polyglutamine diseases.