DG9-Conjugated Morpholino Rescues Phenotype in Spinal Muscular Atrophy Mice

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease characterized by decreased expression of the survival of motor neuron 1 (SMN1) gene. Antisense oligonucleotide (ASO)-based therapies have been developed recently as a means of increasing the production of functional SMN protein by targeting the SMN2 gene. Nusinersen, an FDA-approved ASO, has shown success in SMA treatment. However, it faces several challenges that limit its therapeutic potential. One of the main challenges is the administration route, as nusinersen requires intrathecal injection, which involves direct delivery into the cerebrospinal fluid (CSF). This invasive method can be burdensome for patients and may pose accessibility and practicality issues. Another challenge is the limited ability of nusinersen to efficiently penetrate the blood-brain barrier (BBB). The BBB restricts the passage of molecules to bodywide tissues. Cost and affordability are significant challenges associated with nusinersen. The therapy is expensive, which can limit its accessibility for all patients who could benefit from it. Overcoming these challenges is crucial to optimize the therapeutic benefits of ASOs and broaden their availability to a larger population of SMA patients. To address these challenges, phosphorodiamidate morpholino oligomer (PMO)-based ASOs have emerged as a potential solution. However, PMO's inability to cross the BBB remains a significant hurdle. Recent advancements include the development of DG9 conjugated to PMO, which has demonstrated CNS distribution following subcutaneous injection. DG9-PMO treatment shows promise in increasing survival rates, improving motor coordination, mitigating muscle pathology, and enhancing neuromuscular junction characteristics, compared to the molecule equivalent to nusinersen and a benchmark cell-penetrating peptide R6G-conjugated PMO. Importantly, it also leads to a significant increase in full-length SMN2 expression in both CNS and systemic tissues. This chapter describes the methodologies employed to evaluate the efficacy and safety of DG9-PMO in a severe SMA mouse model. These methodologies include systemic ASO injection, quantitative RT-PCR, western blotting, histology, ELISA-based quantification of DG9-PMO, functional tests, and toxicology analysis. By addressing challenges related to administration route, BBB penetration, and cost, overcoming these obstacles is essential to optimize the therapeutic benefits of ASO and broaden its accessibility to a larger population of SMA patients. The advancements in DG9-PMO offer promising prospects for the development of ASO-based therapeutics.