Enhancing Therapeutic Efficacy in Spinal Muscular Atrophy: DG9-PMO Conjugates Amplify Cellular Uptake, Nuclear Localization, and Long-term Benefits in a Murine Model

Introduction: Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by progressive muscle weakness and atrophy. It is also one of the leading causes of infant death. This disease is caused by mutations in the survival of the motor neuron 1 (SMN1) gene. A paralogous gene, SMN2, encodes the vital SMN protein but generates only minimal levels due to a sequence variant leading to the exclusion of exon 7 from approximately 90% of mature transcripts. Antisense oligonucleotide (ASO)–based therapies have emerged as a promising approach to treat SMA by targeting the SMN2 gene. Although the FDA-approved ASO, Nusinersen, has demonstrated efficacy in SMA treatment, its application is marred by invasive intrathecal injections, substantial cost, and limited impact on the broader multi-organ manifestations of SMA. Notably, SMA is now recognized as a multi-organ condition involving the heart, liver, thymus, and spleen, rather than just a motor neuron disease. To address these limitations, we recently developed DG9-a novel moiety modified from a human T-cell peptide, and showed that DG9-PMO exhibits a remarkable capacity to augment the cellular uptake of ASOs, eliminating the need for intrathecal injections. Here, we examined the long-term efficacy of DG9-PMO with multiple injections, as well as its capacity to enhance endosomal escape and nuclear targeting. Methods: A severe SMA mouse model was utilized to evaluate the therapeutic effects of DG9-PMO conjugates. We injected 40 mg/kg DG9-PMO into a severe SMA mouse model subcutaneously on postnatal day 0, day 2, day 28, and day 56 to determine the in vivo efficacy. Cellular uptake and endosomal escape of DG9-PMO were assessed by immunocytochemistry and live imaging using confocal microscopy. The therapeutic efficacy was evaluated by survival rates, weights, and functional tests to assess the motor function. Results: Our results indicate that multiple injections of DG9-PMO increase the survival rate and weight in the SMA mouse model without requiring intrathecal injections. Functional assessment data demonstrated increased motor ability in the DG9-PMO-treated mice, indicating remarkable amelioration of motor deficits, and improved locomotor activity, muscle strength, and coordination. Notably, DG9-PMO prevents necrosis better than MOE (molecule equivalent to nusinersen) as indicated by the mice’s tail lengths. DG9 was also found to greatly improve intracellular uptake, endosomal escape, and nuclear localization of PMO, as evidenced by increased fluorescent signals. Conclusion: In this study, DG9-PMO conjugates displayed improved cellular uptake, nuclear localization, and therapeutic efficacy in a severe SMA mouse model. These results highlight the potential of DG9-PMO conjugates as a novel SMA therapeutic approach, providing hope for better patient outcomes.