Development of antisense therapy for the treatment of Spinal Muscular Atrophy and Investigating the role of Aquaporins in skeletal muscle function
Creators: Aslesh, Tejal
Publisher: name: University of Alberta Library
Year: 2023

<p>Spinal muscular atrophy (SMA), the most frequent genetic cause of infant mortality, is caused by deletion of the survival of motor neuron 1 (SMN1) gene. Humans have a paralogous gene SMN2 that produces the same SMN protein; however, only ~10% of functional SMN protein is produced as ~90% of SMN2 transcripts lack exon 7 due to splicing of this essential exon. Synthetic DNA-like molecules called antisense oligonucleotides (AOs) are promising agents for the treatment of SMA. Spinraza, the first approved drug for SMA, is an AO that binds to an intronic splicing silencer region in SMN2 transcripts and restores the production of full-length SMN2 (FL-SMN2). SMA is a multi-organ disorder affecting the heart, liver, muscle, and spleen; however, Spinraza is injected intrathecally to avoid renal toxicity and therefore can treat only motor neurons. Therefore, a compound providing effective yet safe delivery of AOs to the central nervous system (CNS) and body-wide organs is needed to prevent SMA-related morbidity and death. Here, we demonstrate that a novel peptide-conjugated antisense morpholino oligomer DG9-PMO is safe and improves the efficacy of the AO treatment in a mouse model of SMA.<br>
    DG9 was conjugated to a safe AO called phosphorodiamidate morpholino oligomer (PMO) targeting SMN2. We subcutaneously injected SMA mice with DG9-PMO on postnatal day 0 (PD0). DG9-PMO-treated mice had significantly increased FL-SMN2 expression by reaching the CNS as well as the peripheral tissues. DG9-PMO treatment significantly prolonged survival, improved breathing and motor function, and ameliorated muscle pathology. No apparent toxicity was observed from serum and urine analyses. DG9-PMO is a promising therapeutic option to treat SMA, overcoming the necessity for invasive injections with a single peripheral administration and treating body-wide tissues without apparent toxicity.<br>
    In the second part of the thesis, we focus on the role of aquaporins (AQPs) in skeletal muscle function. AQPs are integral membrane proteins that facilitate the movement of water, urea, and glycerol across biological membranes. Out of the 13 identified AQPs, AQP4 and AQP1 are expressed in the skeletal muscle that regulate homeostasis and the osmotic gradient. Patients suffering from Duchenne muscular dystrophy (DMD) showed reduced levels of AQP4 in the muscle. These biopsies also revealed a concomitant rise in Aqp1 expression in the endothelial cells of blood capillaries. However, ex vivo contractile properties of the fast twitch muscles in Aqp4 knockout (KO) mice revealed normal muscle force generation and fatigue index. We compared the effects of AQP4 and AQP1 ablation on open-field activity before and after a moderate exercise in the KO mice. We provide evidence that Aqp1 KO mice exhibited significantly reduced activity both before and after exercise.  We also created a double mutant model lacking both Aqp4 and Aqp1 (double KO, DKO). The DKO and Aqp4 KO appear phenotypically normal and exhibited higher activity than Aqp1 KO mice. <br>
    To determine whether ablation of AQP4, AQP1 or both affects muscle regeneration, we introduced acute injury to the tibialis anterior (TA) muscle. The histology of the muscle assessed at different time points revealed a similar distribution of regenerating and degenerating myofibers across all mice. Thus, the deficiency of AQPs in the muscle does not unmask an underlying myopathy. Future studies to determine the distribution of water in the muscle will help investigate edema in the DKO mice, as these mice had larger myofibers in baseline studies. Finally, to determine the relevance of AQP1 overexpression and AQP4 reduction in DMD pathology, we are creating an Aqp1 KO model on an mdx background. This model will be useful to explore the effects of AQP4 reduction on the dystrophic musculature, without the compensatory role of AQP1. <br>
    Overall, we provide evidence for a non-invasive therapy for SMA using a cell-penetrating peptide (CPP) platform. We also provide insight into the relevance of AQP4 and AQP1 on muscle pathology and activity. Together, our findings will serve as a platform for understanding the implications of CPP technology in SMA that can be extended to other neuronal disorders that are difficult to target using AOs due to the impermeable blood-brain barrier (BBB), as well as the relevance of AQPs in DMD, and other neuromuscular disorders (NMDs) such as limb-girdle muscular dystrophy type 2B (LGMDR2), Fukuyama congenital muscular dystrophy (FCMD) and sarcoglycanopathies. </p>