Tejal Aslesh

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by DMD gene mutations, leading to the loss of functional dystrophin. While antisense oligonucleotide (ASO)-mediated exon skipping offers therapeutic potential, its efficacy in cardiac muscle remains limited. Here, we investigate DG9, a cell-penetrating peptide derived from human polyhomeotic 1 homolog (Hph-1) transcription factor, as an enhancer of phosphorodiamidate morpholino oligomer (PMO)-based therapy targeting exon 44. In a humanized DMD mouse model (hDMDdel45;mdx), DG9-PMO significantly increases exon skipping, restores dystrophin expression, and improves muscle function, particularly in the heart. Mechanistically, DG9-PMO enhances intracellular uptake through multiple endocytic pathways and achieves superior nuclear localization. Compared to the benchmark R6G peptide, DG9-PMO exhibits greater efficacy in cardiac tissue with no detectable toxicity. These findings highlight DG9-PMO as a promising next-generation exon-skipping therapy with potential clinical relevance for improving both skeletal and cardiac outcomes in DMD patients.

Antisense oligonucleotide–mediated (AO-mediated) therapy is a promising strategy to treat several neurological diseases, including spinal muscular atrophy (SMA). However, limited delivery to the CNS with AOs administered intravenously or subcutaneously is a major challenge. Here, we demonstrate a single subcutaneous administration of cell-penetrating peptide DG9 conjugated to an AO called phosphorodiamidate morpholino oligomer (PMO) reached the CNS and significantly prolonged the median survival compared with unconjugated PMO and R6G-PMO in a severe SMA mouse model. Treated mice exhibited substantially higher expression of full-length survival of motor neuron 2 in both the CNS and systemic tissues compared with nontreated and unmodified AO–treated mice. The treatment ameliorated the atrophic musculature and improved breathing function accompanied by improved muscle strength and innervation at the neuromuscular junction with no signs of apparent toxicity. We also demonstrated DG9-conjugated PMO localized in nuclei in the spinal cord and brain after subcutaneous injections. Our data identify DG9 peptide conjugation as a powerful way to improve the efficacy of AO-mediated splice modulation. Finally, DG9-PMO is a promising therapeutic option to treat SMA and other neurological diseases, overcoming the necessity for intrathecal injections and treating body-wide tissues without apparent toxicity.

Water transport across the biological membranes is mediated by aquaporins (AQPs). AQP4 and AQP1 are the predominantly expressed AQPs in the skeletal muscle. Since the discovery of AQP4, several studies have highlighted reduced AQP4 levels in Duchenne muscular dystrophy (DMD) patients and mouse models, and other neuromuscular disorders (NMDs) such as sarcoglycanopathies and dysferlinopathies. AQP4 loss is attributed to the destabilizing dystrophin-associated protein complex (DAPC) in DMD leading to compromised water permeability in the skeletal muscle fibers. However, AQP4 knockout (KO) mice appear phenotypically normal. AQP4 ablation does not impair physical activity in mice but limits them from achieving the performance demonstrated by wild-type mice. AQP1 levels were found to be upregulated in DMD models and are thought to compensate for AQP4 loss. Several groups investigated the expression of other AQPs in the skeletal muscle; however, these findings remain controversial. In this review, we summarize the role of AQP4 with respect to skeletal muscle function and findings in NMDs as well as the implications from a clinical perspective

Significance: Duchenne muscular dystrophy (DMD) is a fatal disorder of progressive body-wide muscle weakness, considered the most common muscular dystrophy worldwide. Most patients have out-of-frame deletions in the DMD gene, leading to dystrophin absence in muscle. There is no cure for DMD, but exon skipping is emerging as a potential therapy that uses antisense oligonucleotides to convert out-of-frame to in-frame mutations, enabling the production of truncated, partially functional dystrophin. Currently approved exon skipping therapies, however, have limited applicability and efficacy. Here, we developed a more economical approach to skip DMD exons 45 to 55 (a strategy that could treat nearly half of all DMD patients) and identified DG9 peptide conjugation as a powerful way to improve exon skipping efficiencies in vivo.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder and one of the most common genetic causes of infant death. It is characterized by progressive weakness of the muscles, loss of ambulation, and death from respiratory complications. SMA is caused by the homozygous deletion or mutations in the survival of the motor neuron 1 (SMN1) gene. Humans, however, have a nearly identical copy of SMN1 known as the SMN2 gene. The severity of the disease correlates inversely with the number of SMN2 copies present. SMN2 cannot completely compensate for the loss of SMN1 in SMA patients because it can produce only a fraction of functional SMN protein. SMN protein is ubiquitously expressed in the body and has a variety of roles ranging from assembling the spliceosomal machinery, autophagy, RNA metabolism, signal transduction, cellular homeostasis, DNA repair, and recombination. Motor neurons in the anterior horn of the spinal cord are extremely susceptible to the loss of SMN protein, with the reason still being unclear. Due to the ability of the SMN2 gene to produce small amounts of functional SMN, two FDA-approved treatment strategies, including an antisense oligonucleotide (AON) nusinersen and small-molecule risdiplam, target SMN2 to produce more functional SMN. On the other hand, Onasemnogene abeparvovec (brand name Zolgensma) is an FDA-approved adeno-associated vector 9-mediated gene replacement therapy that can deliver a copy of the human SMN1. In this review, we summarize the SMA etiology, the role of SMN, and discuss the challenges of the therapies that are approved for SMA treatment.

Exon skipping using antisense oligonucleotides (ASOs) has recently proven to be a powerful tool for mRNA splicing modulation. Several exon-skipping ASOs have been approved to treat genetic diseases worldwide. However, a significant challenge is the difficulty in selecting an optimal sequence for exon skipping. The efficacy of ASOs is often unpredictable, because of the numerous factors involved in exon skipping. To address this gap, we have developed a computational method using machine-learning algorithms that factors in many parameters as well as experimental data to design highly effective ASOs for exon skipping. eSkip-Finder (https://eskip-finder.org) is the first web-based resource for helping researchers identify effective exon skipping ASOs. eSkip-Finder features two sections: (i) a predictor of the exon skipping efficacy of novel ASOs and (ii) a database of exon skipping ASOs. The predictor facilitates rapid analysis of a given set of exon/intron sequences and ASO lengths to identify effective ASOs for exon skipping based on a machine learning model trained by experimental data. We confirmed that predictions correlated well with in vitro skipping efficacy of sequences that were not included in the training data. The database enables users to search for ASOs using queries such as gene name, species, and exon number.

Introduction: Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder that affects approximately one in 3500–5000 male births. Patients experience muscle degeneration, loss of ambulation, and eventual death from cardiac or respiratory failure in early adulthood due to a lack of functional dystrophin protein, which is required to maintain the integrity of muscle cell membranes. Out-of-frame mutations in the DMD gene generally lead to no dystrophin protein expression and a more severe phenotype (DMD). Conversely, in-frame mutations are often associated with milder Becker muscular dystrophy (BMD) with a truncated dystrophin expression. Areas covered: Genome editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system can induce permanent corrections of the DMD gene, thus becoming an increasingly popular potential therapeutic method. In this review, we outline recent developments in CRISPR/Cas9 genome editing for the correction of DMD, both in vitro and in vivo, as well as novel delivery methods. Expert opinion: Despite recent advances, many limitations to CRISPR/Cas9 therapy are still prevalent such as off-target editing and immunogenicity. Specifically, for DMD, intervention time and efficient delivery to cardiac and skeletal muscles also present inherent challenges. Research needs to focus on the therapeutic safety and efficacy of this approach.

The field of neuromuscular and neurodegenerative diseases has been revolutionized by the advent of genetics and molecular biology to evaluate the pathogenicity, thereby providing considerable insight to develop suitable therapies. With the successful translation of antisense oligonucleotides (AOs) from in vitro into animal models and clinical practice, modifications are being continuously made to the AOs to improve the pharmacokinetics and pharmacodynamics. In order to activate RNase H-mediated cleavage of the target mRNA, as well as to increase the binding affinity and specificity, gapmer AOs are designed to have a phosphorothioate (PS) backbone flanked with the modified AOs on both sides. Antisense-mediated knockdown of mutated huntingtin is a promising therapeutic approach for Huntington’s disease (HD), a devastating disorder affecting the motor and cognitive abilities. This chapter focuses on the modified gapmer AOs for the treatment of HD. Key words Huntingtin (HTT) Trinucleotide repeat disorders 2′-O-methoxyethyl (MOE) Gapmer HTT_RX (Alternative Names: HTT ASO; IONIS-HTTRx; ISIS-443139; ISIS-HTT Rx; RG 6042; RO7234292) WVE-120101/WVE-120102 Nusinersen (brand name Spinraza) Eteplirsen (brand name Exondys 51) Zinc-finger proteins (ZFPs) Short interfering RNA (siRNA)

Although technological advances in molecular genetics over the last few decades have greatly expedited the identification of mutations in many genetic diseases, the translation of the genetic mechanisms into a clinical setting has been quite challenging, with a minimum number of effective treatments available. The advancements in antisense therapy have revolutionized the field of neuromuscular disorders as well as lipid-mediated diseases. With the approval of splice-switching antisense oligonucleotide (AO) therapy for nusinersen and eteplirsen for the treatment of spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD), several modified AOs are now being evaluated in clinical trials for the treatment of a number of disorders. In order to activate RNase H-mediated cleavage of the target mRNA, as well as to increase the binding affinity and specificity, gapmer AOs are designed that have a PS backbone flanked with the modified AOs on both sides. Mipomersen (trade name Kynamro), a 2′-O-methoxyethyl (MOE) gapmer, was approved by the Food and Drug Administration (FDA) for the treatment of homozygous familial hypercholesterolemia (HoFH) in 2013. Volanesorsen, another 20-mer MOE gapmer has shown to be successful in lowering the levels of triglycerides (TGs) in several lipid disorders and has received conditional approval in the European Union for the treatment of Familial chylomicronemia syndrome (FCS) in May 2019 following successful results from phase II/III clinical trials. This chapter focuses on the clinical applications of gapmer AOs for genetic dyslipidemia and lipodystrophy. Key words 2′-O-methoxyethyl (MOE) Low-density lipoprotein (LDL) Apolipoprotein B-100 (ApoB-100) Homozygous familial hypercholesterolemia Hypertriglyceridemia Familial chylomicronemia Familial partial lipodystrophy Nusinersen (brand name Spinraza) Eteplirsen (brand name Exondys 51) Cardiovascular diseases (CVD)

Spinal muscular atrophy (SMA) is the most common genetic cause of infantile death caused by mutations in the SMN1 gene. Nusinersen (Spinraza), an antisense therapy-based drug with the 2′-methoxyethoxy (2’MOE) chemistry approved by the FDA in 2016, brought antisense drugs into the spotlight. Antisense-mediated exon inclusion targeting SMN2 leads to SMN protein expression. Although effective, 2′MOE has weaknesses such as the inability to cross the blood–brain barrier and the high cost of treatment. To investigate new chemistries of antisense oligonucleotides (ASOs), SMA mouse models can serve as an important source. Here we describe methods to test the efficacy of ASOs, such as phosphorodiamidate morpholino oligomers (PMOs), in a severe SMA mouse model.