Enhancing In Vitro SMN Protein Expression and Cell Viability through Xeno-Nucleic Acid-Based ASOs in Spinal Muscular Atrophy

Abstract

SUMMARYSpinal Muscular Atrophy (SMA) stands as a devastating ailment arising from the dearth of functional SMN (Survival Motor Neuron) protein due to genetic anomalies within the SMN1 gene. This condition is marked by the consequential attrition of motor neurons, precipitating a progressive decline in muscular strength and culminating in the disruption of neuromuscular junctions. Existing therapeutic approaches encompassing Zolgensma, Nursinersen, and Evrysdi employ innovative genetic therapeutic strategies involving transgene delivery, Antisense Oligonucleotide (ASO) technology, and modulation of pre-mRNA processing to enhance functional SMN protein expression. However, the ASO therapeutics remain suboptimal in establishing a sustained panacea for SMA, as they inadequately maintain consistent levels of functional SMN protein expression. In this study, we present a discerning inquiry into focusing on XNA-DNA-ASO products that exhibit enhanced safety and stability compared to conventional DNA/RNA-ASO sequences. Through precise targeting of the ISSN-1 region within SMN2 gene’s intron 7, our approach seeks to amplify SMN protein expression. Employing Xeno Nucleic Acid (XNA) bases, known for their augmented hydrophobicity and stability, our strategy surmounts previous limitations associated with chemical modifications, showcasing heightened endonuclease resistance. Comparative analyses with conventional DNA/RNA-ASO products substantiate the superiority of XNA-DNA-ASO sequences, underscoring elevated SMN protein expression and reduced toxicity. In a comprehensive evaluation, our gene therapy paradigm is scrutinized within a type 1 SMA fibroblast cell line. Utilizing diverse analytical methodologies, encompassing Annexin V-PI analysis for cytotoxicity, MTT assays for mitochondrial activity, and flow cytometry for SMN protein expression profile, we gauge therapeutic impact and potential toxicity. In conclusion, our investigation not only spotlights the promise of XNA-DNA-ASO sequences but also holds implications for refining SMA treatment strategies, converging on minimized dosages, lowered toxicity, and heightened therapeutic efficacy, thus shaping the landscape of gene therapy for SMA.