In Vitro Modeling as a Tool for Testing Therapeutics for Spinal Muscular Atrophy and IGHMBP2-Related Disorders

SIMPLE SUMMARY: Spinal Muscular Atrophy (SMA) is a genetic disease that can cause infant mortality. It is typically caused by mutations in the SMN1 gene. On the other hand, mutations in the IGHMBP2 gene can lead to a range of diseases, including the rare form of SMA known as SMARD1, as well as Charcot–Marie–Tooth 2S (CMT2S). In this study, we developed a patient-derived in vitro model system to generate induced neurons and explore disease pathogenesis and test the response to gene therapies. The generated induced neurons from SMA and SMARD1/CMT2S patient cell lines were then treated with the clinical gene therapies AAV9.SMN (Zolgensma) for SMA and AAV9.IGHMBP2 for IGHMBP2-related disorders. We found that the SMA neurons had morphological defects that partially responded to treatment with AAV9.SMN, while the SMARD1/CMT2S neurons showed similar improvement after the restoration of IGHMBP2. The model also helped us identify whether an unclassified mutation was disease-causing in a patient with suspected SMARD1/CMT2S. These findings could help improve our understanding of SMA and SMARD1/CMT2S, as well as aid in the development of new treatments for these diseases. ABSTRACT: Spinal Muscular Atrophy (SMA) is the leading genetic cause of infant mortality. The most common form of SMA is caused by mutations in the SMN1 gene, located on 5q (SMA). On the other hand, mutations in IGHMBP2 lead to a large disease spectrum with no clear genotype–phenotype correlation, which includes Spinal Muscular Atrophy with Muscular Distress type 1 (SMARD1), an extremely rare form of SMA, and Charcot–Marie–Tooth 2S (CMT2S). We optimized a patient-derived in vitro model system that allows us to expand research on disease pathogenesis and gene function, as well as test the response to the AAV gene therapies we have translated to the clinic. We generated and characterized induced neurons (iN) from SMA and SMARD1/CMT2S patient cell lines. After establishing the lines, we treated the generated neurons with AAV9-mediated gene therapy (AAV9.SMN (Zolgensma) for SMA and AAV9.IGHMBP2 for IGHMBP2 disorders (NCT05152823)) to evaluate the response to treatment. The iNs of both diseases show a characteristic short neurite length and defects in neuronal conversion, which have been reported in the literature before with iPSC modeling. SMA iNs respond to treatment with AAV9.SMN in vitro, showing a partial rescue of the morphology phenotype. For SMARD1/CMT2S iNs, we were able to observe an improvement in the neurite length of neurons after the restoration of IGHMBP2 in all disease cell lines, albeit to a variable extent, with some lines showing better responses to treatment than others. Moreover, this protocol allowed us to classify a variant of uncertain significance on IGHMBP2 on a suspected SMARD1/CMT2S patient. This study will further the understanding of SMA, and SMARD1/CMT2S disease in particular, in the context of variable patient mutations, and might further the development of new treatments, which are urgently needed.