Develop an allele-specific antisense oligonucleotide (ASO) that selectively targets the mutant SNCA A53T allele while preserving expression of the wild-type (WT) allele.

Delineated by Dr. Polymeropoulos et al. (1997), the SNCA p.A53T mutation is a significant risk factor for early-onset Parkinson’s disease (PD). Current ASO approaches broadly target SNCA regardless of mutation status, risking α-synuclein depletion. We propose an allele-specific strategy that achieves genotype-selective downregulation, targeting the aberrant A53T allele without compromising WT function.

An ASO that selectively targets the SNCA A53T allele was designed in silico to have a higher affinity for the mutant allele. HEL cells were treated via gymnotic uptake for 72-hours, followed by qPCR and RNA-seq.

hiPSCs from p.A53T carriers were differentiated into a mixed neuronal model displaying PD-relevant phenotypes. Neurons were treated with A53T-specific ASOs (0.2-1 µM) for two weeks, and neuronal morphology, viability, and synaptic connectivity were quantified.  

A53T-specific ASOs reduced SNCA expression by ~40%, consistent across qPCR and RNA-seq, with limited off-target effects in silico. In the hiPSC-derived model, treatment resulted in a notable effect on neuronal outgrowth and viability,  restoring neuronal networks and diminishing levels of phosphorylated α-synuclein. Network metrics analysis demonstrated significantly increased neurite branching with treatment comparable to healthy control levels (p<0.0001). Additionally, measurements of synaptic connectivity show significantly increased connectivity of neurons following treatment (p<0.0001).

It has been demonstrated that using CRISPR-Cas9 to delete the A53T mutation improves PD-related conditions. Achieving a ratio in favor of mutant allele downregulation is advantageous, as it spares WT function while targeting the aberrant gain-of-function allele.

We present a novel ASO that selectively downregulates A53T SNCA, with promising restoration of synaptic function and neuronal health. Given the high prevalence of PD, a treatment that works by directly targeting an underlying molecular cause holds potential to significantly improve disease manifestation and quality of life.