Manufacturing and testing of a polymer formulation for long-time release delivery of antisense oligonucleotides in hiPSC-derived motoneurons

Antisense oligonucleotides (ASOs) are an emerging therapeutic approach for genetically induced neurological disorders such as spinal muscular atrophy (SMA). For SMA, the Food and Drug administration (FDA)-approved ASO Nusinersen (Spinraza®) restores exon 7 inclusion in survival motor neuron (SMN) 2, leading to improved motoneuron survival. However, current administration to patients requires repeated intrathecal (IT) injections via lumbar puncture, an invasive procedure with considerable side effects. There is therefore an urgent need for more efficient and sustained drug-delivery strategies capable of overcoming these limitations. In this study, we developed and tested a system for the controlled, long-term release of Nusinersen ASO. Using motoneurons derived from human induced pluripotent stem cells (hiPSCs) from SMA and control patients, two formulations were evaluated: (I) pure ethylene vinyl acetate (EVA, 40% vinyl acetate) and (II) a composite of EVA (40% vinyl acetate) and silica (50:50 w/w). Both formulations were found to be biocompatible in the co-cultures in vitro. Moreover, the efficient uptake of ASOs and the optimization of ASO dose and time-response in motoneurons was achieved. Additionally, fluorescently labelled ASOs confirmed sustained release from the implants. Importantly, sustained polymer-released ASO significantly increased exon 7 inclusion in SMN2 transcripts and motoneuron viability over time, demonstrating therapeutic efficacy and preserved biological activity. These findings demonstrate the initial feasibility for a minimally invasive, long-term ASO delivery system that could overcome the limitation for repeated lumbar punctures in SMA patients and potentially be adapted as ASO delivery systems for other neurodegenerative diseases.