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