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.