Objective: Our objective was to develop an antisense oligonucleotide (ASO) allele-specific strategy to target the mutant A53T SNCA allele, reducing aberrant α-synuclein production, without downregulating the wild-type (WT) SNCA allele, for the treatment of Parkinson’s disease (PD).

Background: First delineated by Dr. Polymeropoulos et al. in 1997, the Ala53Thr (A53T) missense mutation in SNCA has been identified as one of the most significant risk factors in early-onset PD. We propose an allele-specific ASO strategy to target the mutant A53T allele. This is unique compared to ASOs under development, as these target general knockdown of SNCA. A treatment that directly targets the underlying molecular cause of PD holds significant potential to improve patients’ disease manifestation and quality of life.

Method: We designed an 18-mer gapmer phosphorothioate methoxyethyl ASO that selectively targets the A53T allele. This ASO was designed to have a higher affinity for the mutant allele, predicted in silico, as opposed to the same sequence and length from WT. HEL cells, chosen for their abundant SNCA expression, were incubated for 72-hours with ASO (1µM) using gymnotic uptake.

Results: ASO treatment resulted in a 40% reduction of SNCA expression (qPCR) and was consistent with RNAseq data. Additional gapmers and siRNA were used as positive controls. Limited off-target effects of this ASO were demonstrated in silico.

Furthermore, fibroblasts from a patient with confirmed A53T mutation status were obtained and iPSCs were generated. Currently, molecular characterization in a human iPSC-derived PD model, containing dopaminergic, GABAergic, and glutamergic neurons, is planned to assess the effects of A53T-specific ASO silencing in rescuing PD phenotype.

Conclusion: Achieving a ratio in favor of mutant allele downregulation is desirable as it does not knockdown the WT allele while targeting the aberrant gain-of-function allele, a phenomenon that is not possible with other proposed lines of treatment. We present a novel ASO that achieves this goal in vitro. We plan to utilize the iPSC model of a mixed neuronal population to examine the efficacy of our ASO in reducing numerous parameters, including the presence of Lewy neurites, Lewy bodies, extracellular Lewy body-like formations, varicosities, bulbous endings, and degenerating neurons.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/first-in-class-aso-targeting-a53t-allele-preclinical-efficacy/