A Novel Gene Therapy Approach to Prevent Alpha-synuclein Misfolding in Multiple System Atrophy

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Multiple system atrophy (MSA) is a prion-like movement disorder caused by misfolding and self- templating of the protein α-synuclein (α-syn), which spreads throughout the central nervous system to cause progressive degeneration. Similar to many other prion and prion-like neurodegenerative diseases, there are currently no therapeutics available that alter the course of disease for MSA patients. To interfere with α-syn self-templating, several groups have proposed various strategies for knocking down α-syn expression to reduce the amount of protein available as substrate. Unfortunately, these strategies may interfere with normal α-syn function in the brain, leading to loss-of-function deficits for MSA patients. Alternatively, MSA cannot propagate in transgenic (Tg) cells or mice expressing α-syn with the E46K mutation, raising the possibility of using gene therapy to generate conversion-incompetent α-syn to disrupt self-templating. However, to date, this approach has not been tested as a therapeutic intervention for MSA. The objective of the proposed work is to establish proof-of-concept that introducing a single residue change in the α-syn primary sequence can disrupt templated misfolding. We hypothesize that generating conversion-incompetent α-syn using CRISPR prime editing will reduce or prevent MSA propagation. Our approach will capitalize on our recent discovery that non- pathogenic α-syn mutations at residue K80 inhibit MSA propagation in vitro. In Aim 1, we will use CRISPR prime editing to insert our novel K80 mutations into Tg cells and mice expressing wild-type human α-syn prior to challenging the models with MSA patient samples. We have shown that MSA induces α-syn aggregation in unedited cells and mice expressing wild-type protein. We anticipate that successful gene editing will block transmission to these model systems. Cryo-electron microscopy has been used to resolve the structures of α- syn fibrils in MSA patient samples. This work has shown that misfolded α-syn adopts a Greek key conformation that is stabilized by a salt bridge between residues E46 and K80. In Aim 2, we will determine if our non-pathogenic K80 mutations exert their protective effectives by preventing salt bridge formation. We will also quantify the effect of these mutations on lipid binding and protein fibrillization. These orthogonal studies will determine if the K80 mutations are a viable clinical candidate for an MSA gene therapy. This work is innovative because it represents a paradigm-shift in how we approach gene therapies. Rather than focusing on correcting a disease-causing point mutation, we will establish proof-of-concept that gene therapy can be used to interfere with the self-templating disease mechanism underlying prion and prion-like neurodegenerative disorders. This work is significant because it has the potential to serve as a novel treatment strategy for patients with both sporadic and familial prion-like diseases. Through investigating the ability of conversion- incompetent α-syn to prevent MSA propagation, this work has the potential to transform the way we approach therapeutic development for neurodegenerative disease patients.