To rescue neuromuscular defects caused by immunoglobulin mu-binding protein 2 (IGHMBP2) variants by targeting a Charcot-Marie-Tooth disease Type 2S (CMT2S) patient-specific splice site variant with a novel antisense oligonucleotide (ASO).

CMT2S is a rare Charcot-Marie-Tooth disease subtype caused by IGHMBP2 variants resulting in alpha‐motor neuron degeneration. A patient was reported with a paternally inherited cryptic splice site non‐coding IGHMBP2 variant (c.1235+894 C>A) deep in intron 8, which leads to nonsense‐mediated decay resulting in reduced IGHMBP2. We developed an ASO that targets this splice site to restore canonical splicing, rescuing levels of IGHMBP2.

We designed a 19-mer ASO targeting this variant. CMT2S-MNs were differentiated from the CMT2S patient-derived iPSC line and integrated into a dual-chamber neuromuscular junction (NMJ) system. NMJs underwent ASO treatment and were assessed. To further evaluate axonal defects, CMT2S-MNs were integrated on microelectrode arrays with wild-type (WT) Schwann cells. Conduction velocity (CV) effects were assessed before and after ASO treatment.  

CMT2S-NMJ analyses revealed a higher fatigue index (FI) than WT-NMJs and showed high levels of decay and chaotic tetanus, as compared to WT-NMJs. We demonstrate rescue of NMJ functioning following ASO treatment, captured by an FI decrease and reduction in decay and sporadic tetanus responses.

While CMT2S-MNs had a significantly higher CV average on Day 14, they exhibited consistently fewer conductions at all testing time points. CMT2S-MNs experienced a significant dose-dependent rescue of CV by ASO treatment, compared to WT control MNs or vehicle treatment.

We show rescue of NMJ functioning post-ASO treatment, captured by FI decrease and reduction in decay and sporadic responses. Furthermore, ASO treatment resulted in a dose-dependent increase in CV and number of conductions. This provides translational evidence that this ASO may restore clinical motor control and improve fatigue. We are further analyzing this patient-specific model to continue phenotyping CMT2S caused by IGHMBP2 variants.