Structure-based antisense therapeutics targeting dystrophin exons 44 and 45 for Duchenne Muscular Dystrophy

Project Summary Duchenne muscular dystrophy (DMD) is a lethal pediatric neuromuscular disorder that affects 1/5000 boys globally. This rare disease is caused by mutations in the gene encoding the dystrophin protein, often by disrupting the reading frame. Antisense oligonucleotide (ASO) therapeutics target the dystrophin pre-

mRNA, induce exon skipping to restore the reading frame, and partially rescue dystrophin expression. Four ASO drugs have been approved by the U.S. Food and Drug Administration. However, their clinical efficacies are dismally low due to significant barriers in activity and delivery. To address these problems,

we propose two innovative strategies for developing highly effective ASOs targeting dystrophin exons 44 and 45, which together could treat about 14% of DMD patients. Both strategies take advantage of the fact that RNA tends to fold into structures. In the first strategy, we design ASOs with tertiary interactions

that dramatically expand the ASO-exon molecular interface, in addition to conventional Watson-Crick base pairing. The tertiary contacts enable the ASO to recognize both the sequence and the structure of the target exon, potentially driving higher affinity, specificity, and exon skipping activity. For exon 44 (Aim

1) we design the ASO sequence and backbone chemistry to generate tertiary contacts with a short hairpin in the pre-mRNA, which enhances binding affinity. The crystal structure of the ASO-exon complex reveals opportunities for creating additional interactions via chemically modified bases. In subsequent rounds of

design and testing, we will synthesize modified oligos, characterize them structurally and biochemically, and measure exon-skipping activity in DMD patient-derived muscle cell culture. In the second strategy, we develop a bifunctional cell-penetrating peptide (CPP) that recognizes a unique hairpin adjacent to the

binding site of existing ASO drug Casimersen in exon 45 (Aim 2). Conjugation of the CPP to Casimersen should enhance both delivery and target RNA recognition. In preliminary studies, we solved a high- resolution crystal structure of the exon 45 hairpin and discovered non-canonical base pairs that create

unusual structural features in the major groove. We will computationally design CPP sequences to recognize the novel major groove conformation. We will co-crystallize candidate CPPs with the exon 45 hairpin and quantify their affinity and specificity. For promising leads, we will produce CPP-ASO conjugates and measure cell penetrance and exon 45 skipping activity in patient cell lines. We anticipate

testing the best ASOs developed in this study in animal models in the future, with potential for clinical trials. The structure-based strategies can drive development of more effective ASO drugs for skipping other dystrophin exons, leading to increased access to precision therapies for this debilitating childhood

disease.