Axonal degeneration has been recognized as a key early contributor to the clinical presentation and pathogenesis of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Activation of the calcium-dependent protease calpain-2 is proposed as a critical effector of axonal degeneration.
To develop antisense oligonucleotides (ASOs) targeting the gene encoding calpain-2 (CAPN2), quantitatively assess their capacity to inhibit CAPN2 expression, and evaluate the neuroprotective effects of the lead ASO candidate, AMX0114, in in vitro ALS models.
ASOs targeting CAPN2 were evaluated in human induced pluripotent stem cell (iPSC)–derived glutamatergic neurons (ioGlutamatergic Neurons; bit.bio). CAPN2 RNA levels were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). Varying concentrations of AMX0114 were then evaluated in an iPSC–derived human spinal motor neuron cell line (iCell Motor Neurons; FUJIFILM Cellular Dynamics, Inc.). An ASO targeting a long noncoding RNA was included on each plate as a control owing to lack of known positive control against CAPN2. Knockdown was analyzed with primer-probe sets to confirm ASO delivery and assess plate variability. The effects of pretreatment with AMX0114 on neurite degeneration and excretion of axonal damage biomarker, neurofilament light chain (NfL), were evaluated in neurotoxically triggered human motor neurons.
Six ASOs reduced CAPN2 RNA levels by ≥30% in the human iPSC–derived glutamatergic neurons without evident cytotoxicity; AMX0114 reduced RNA levels by ≥74%. In the human motor neuron cell line, AMX0114 reduced CAPN2 RNA levels by ≥99%; the potency (half maximal effective concentration, EC50) was ~40 nM. Treatment with ASO controls had no effect on CAPN2 RNA levels. Pretreatment with AMX0114 significantly reduced NfL excretion and rescued neurite degeneration following exposure to neurotoxic triggers.
ASOs targeting CAPN2 were developed and showed concentration-dependent CAPN2 knockdown in 2 cell types. Studies in additional models of neurodegenerative diseases will further assess the functional efficacy of AMX0114.