Objective: Our objective was to target a specific cryptic splice site variant affecting immunoglobulin mu‐binding protein 2 (IGHMBP2) protein levels with a novel antisense oligonucleotide (ASO) designed to avoid nonsense‐mediated decay (NMD) in an individual Charcot-Marie-Tooth disease Type 2S (CMT2S) patient and characterize their neuromuscular phenotype by analyzing the physiology of disease motor neurons (MNs) and their neuromuscular junction (NMJ) differences.

Background: Variants in IGHMBP2 may result in abnormal RNA processing leading to alpha‐motor neuron degeneration, causing CMT2S.

A patient was reported with pathogenic variants within IGHMBP2. Whole genome sequencing (WGS) revealed a paternally inherited cryptic splice site variant (non‐coding variant (c.1235+894 C>A)) deep in intron 8. The resulting transcript undergoes NMD.

Method: WGS confirmed the variant and a 19mer ASO was designed targeting deep in intron 8. Fibroblasts underwent ASO treatment (1µM) and 48-hour incubation. With Hesperos Inc., MNs and skeletal myofibers were generated through expansion and differentiation of patient’s fibroblasts. CMT2S-MN physiology was characterized through patch-clamp electrophysiology and NMJ defects were examined. Derived MNs were incubated with ASO and treatment effects were quantified.

Results: IGHMBP2 increased (~50-70%) in treated samples. qPCR confirmed increased ratio of restored WT transcript to cryptic exon-containing transcript (~1.8-fold). RNAseq confirmed increased IGHMBP2 expression of 1.3log₂-fold post treatment. Data support this ASO as a potential treatment for restoring IGHMBP2, with limited off-target effects in silico. Patch-clamp electrophysiology revealed hyperexcitability and spontaneous firing of CMT2S-MNs and reduced resting membrane potential, capacitance, and cell body area compared to WT-MNs. CMT2S-NMJ analysis revealed higher fatigue index (FI) than WT-NMJ.

We showed rescue of NMJ functioning post ASO treatment, captured by FI decrease and reduction in decay and sporadic responses, likely to lead to clinical motor control restoration. Potential ASO toxicity was evaluated in a 3-month rat study. The no-adverse-event-level was the highest dose.

Conclusion: N-of-1 precision medicine approach is instrumental in designing treatments for this diverse genetic disorder. This case exemplifies how WGS-based clinical diagnoses and research capabilities allow for the design of potential personalized ASO-based treatments.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/novel-aso-targeting-ighmbp2-cryptic-splice-variant-efficacy-and-safety/