Therapeutic potential of base editing strategies to convert CAG to CAA in Huntington's disease Huntington's disease (HD) defies development of effective treatments despite its long-known genetic cause and numerous mechanisms implicated in model systems, reflecting limited clinical utility of model-based investigations. By contrast, observations in HD patients may reveal therapeutics that actually works in people. All cases of HD are due to an expanded CAG repeat in huntingtin gene. However, age at clinical manifestation varies widely, and unexplained variance in age at onset by the mutation size shows heritability, indicating HD is modified by genes. Therefore, we performed genome-wide association study, and discovered that repeat instability-related DNA repair genes modify HD onset. Importantly, we revealed that duplicated and loss of CAG-CAA interruption in the huntingtin CAG repeat robustly delay and hasten HD onset age, respectively. Together, our data indicate that the rate of HD is largely determined by the size of uninterrupted CAG repeat and modified by repeat instability, providing insights into driver of the disease and therapeutic strategies. Capitalizing on these clinically relevant observations in humans, we conceived novel therapeutic Base Editing (BE) strategies to convert CAG to CAA aiming at delaying clinical manifestation by decreasing the size of uninterrupted CAG repeat and potentially further suppressing repeat expansion. Our novel therapeutic BE strategies have a number of advantages over other gene targeting approaches. Observations in patients suggest that CAG-to-CAA conversion produces very strong therapeutic benefit (i.e., delaying onset more than 10 years). In addition, our BE strategies, targeting the root cause of the disease, do not alter huntingtin protein since both CAG and CAA encode glutamine. Therefore, same single treatment strategies can be applied to all HD patients to produce allele-specific benefits. Here, we propose to determine therapeutic potential of selected BE strategies to convert CAG to CAA using relevant cell and animal models of HD. Briefly, we will 1) evaluate conversion efficiencies and allele specificity of BE strategies with high efficiencies, 2) test whether CAG-to- CAA conversion affects HTT expression levels, neuronal differentiation, and other molecular phenotypes, 3) determine impacts of CAG-to-CAA conversion on CAG repeat instability, and 4) evaluate off-target effects, and further optimize to reveal the BE strategy with the highest feasibility and therapeutic potential. This research will 1) produce a complete evaluation chart for combinations of different base editors and conversion strategies, 2) generate knowledge regarding allele specificity, off-targeting, and molecular consequences, 3) provides considerations for subsequent optimization, and 4) produce expected outcomes when BE strategies are applied to HD patients. Our research testing novel and innovative therapeutic routes for HD therefore will significantly contribute to the development of effective therapeutics for HD and other CAG expansion disorders.

Public Health Relevance

Our large scale genetic investigation of Huntington's disease (HD) patients revealed that the timing of onset is significantly delayed in subjects carrying duplicated CAA-CAG interruption in HTT gene, due to decreased length of uninterrupted CAG repeat and potentially reduced somatic repeat expansion. In order to advance this promising human-validated therapeutic target, we propose to determine potential of base editing strategies to convert CAG at various locations in the huntingtin CAG repeat to CAA. Our human-based therapeutic base editing strategies for CAG-to-CAA conversion, which do not change huntingtin protein sequence, can be applied to all HD patients and are expected to produce very strong effects, representing a significant and clinically relevant advancement toward development of effective and safe treatments for HD.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Molecular Neurogenetics Study Section (MNG)
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Miller, Daniel L
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Massachusetts General Hospital
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