Objective:

Our study aims to develop a genetic treatment strategy using antisense oligonucleotide treatment targeting the causal missense variant in Charcot-Marie-Tooth type 2E in an iPSC-derived motor neuron model system. 

Background:

Charcot-Marie-Tooth disease type 2E is caused by a missense mutation (p.N98S) in  NEFL  encoding neurofilament light chain (NFL), an integral component of the axonal cytoskeleton. Previous studies have established intracellular toxic NFL-positive aggregates in motor neurons and increased supernatant NFL as molecular phenotypic biomarkers. Recently, peripherin (PRPH), an intermediate filament specific to the peripheral nervous system, has been reported as increased in patient serum and CSF in other neuromuscular disorders. Using these established molecular phenotypes and axonal injury biomarkers, our study aims to develop an antisense oligonucleotide (ASO) mediated genetic treatment strategy to mitigate NFL aggregate formation by targeting allele-specific knockdown of the mutant NEFL transcripts.

Design/Methods:

ASOs were designed with 2'-O-methyl modifications flanking 4 base pairs on each end and phosphorothioate modifications between 16 base pairs. Patient and control derived spinal spheroids (SpS), projecting axonal neurites were grown from iPSC-differentiated motor neurons. Using a CX5 high content screening platform, SpS were analyzed for NFL-positive aggregate formation and clearance post-ASO treatment while supernatant was collected to determine NFL and PRPH concentration post-ASO treatment. Using iPSC-derived 2D motor neurons, RT-PCR was performed to assess transcript knockdown efficiency.

Results:

Axonal injury biomarkers, supernatant NFL and PRPH, were significantly decreased in SpS treated over the course of 21 days. NFL-positive aggregate formations were also attenuated with a decrease in detectable deposits. ASO allele-specificity was assessed using allele-specific RT-PCR, showing significant decrease in mutant transcripts. 

Conclusions:

Our results suggest a viable genetic therapeutic strategy by directly targeting the causal missense variant for CMT2E. This work in patient-derived iPSC-differentiated motor neurons is a crucial step in assessing ASO efficacy and viability for further clinical development.