To target a Charcot-Marie-Tooth disease Type 2S (CMT2S) patient-specific cryptic splice site variant within immunoglobulin mu-binding protein 2 (IGHMBP2) with a novel antisense oligonucleotide (ASO), rescuing IGHMBP2 protein levels.

CMT2S is a rare autosomal recessive Charcot-Marie-Tooth disease subtype. IGHMBP2 is an upstream regulator of the THO complex that affects cellular mRNA homeostasis by regulating mRNA production and nuclear export. Variants within IGHMBP2 may result in abnormal RNA processing leading to alpha-motor neuron degeneration, causing CMT2S.

A patient was reported with IGHMBP2 variants. Whole genome sequencing (WGS) revealed a paternally inherited cryptic splice site, non-coding variant (c.1235+894 C>A), in intron 8. The resulting transcript undergoes nonsense-mediated decay (NMD), resulting in IGHMBP2 haploinsufficiency.

We confirmed the variant with WGS and the existence of NMD in the patient’s fibroblasts. Several ASOs were designed with a phosphorothioate methoxyethyl backbone and prioritized based on in silico binding affinity. A 19mer ASO was designed, targeting intron 8 (c.1235+894 C>A) around sequence CACTTCCAC(A)GGGGGAAGA. Fibroblasts underwent ASO treatment (1µM) and 48-hour incubation. Flow cytometry and fluorescein-labeled ASO (GFP+99.8%) confirmed cellular entry.

Upon ASO treatment, we observed a significant IGHMBP2 protein level increase (~50-70%) in ASO-treated samples compared to untreated samples (WB antibody Sigma SAB2106426). qPCR confirmed an increased ratio of restored wild-type transcript to cryptic exon-containing transcript (~1.8-fold). RNAseq data further confirms an increase in IGHMBP2 expression of 1.3log₂-fold (p value<0.002). Preclinical data support this ASO as a potential treatment for restoring IGHMBP2 protein levels, with limited off-target effects in silico.

CMT2S cases caused by IGHMBP2 variants are increasingly reported. A precision medicine approach may prove instrumental to the design of treatments for this highly diverse genetic disorder. This case exemplifies the shifting boundary between rapid WGS-based clinical diagnoses and research capabilities allowing for the design of personalized ASO-based treatments.