Antisense Oligonucleotide Therapeutics for Cystic Fibrosis: Recent Developments and Perspectives

Abstract

Antisense oligonucleotide (ASO) technology has become an attractive therapeutic modality for various diseases, including Mendelian disorders. ASOs can modulate the expression of a target gene by promoting mRNA degradation or changing pre-mRNA splicing, nonsense-mediated mRNA decay, or translation. Advances in medicinal chemistry and a deeper understanding of post-transcriptional mechanisms have led to the approval of several ASO drugs for diseases that had long lacked therapeutic options. For instance, an ASO drug called nusinersen became the first approved drug for spinal muscular atrophy, improving survival and the overall disease course. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Although Trikafta and other CFTR-modulation therapies benefit most CF patients, there is a significant unmet therapeutic need for a subset of CF patients. In this review, we introduce ASO therapies and their mechanisms of action, describe the opportunities and challenges for ASO therapeutics for CF, and discuss the current state and prospects of ASO therapies for CF.

Keywords: RNA therapeutics; antisense oligonucleotide; cystic fibrosis; cystic fibrosis transmembrane conductance regulator; nonsensemediated mRNA decay; splicing.

Fig. 1. Overview of ASO properties and mechanisms.

(A) ASO chemical modifications. ASOs can be modified at the phosphate inter-nucleotide linkage, and at the 2’ position in the sugar. Replacement of phosphate linkages with PS linkages improves nuclease resistance and improves binding to plasma proteins. Modification of the 2’ position of the sugar (e.g., 2’-OMe, 2’-MOE, LNA, and cEt) enhances nuclease stability and increases affinity for target RNAs. PMO modification replaces the sugar with the morpholino group and the phosphate linkages with neutral phosphorodiamidate linkages. Various molecules can be conjugated to the 3’ or 5’ termini of ASOs (Winkler, 2013). The most popular and successful conjugate is the N-acetylgalactosamine (GalNAc) moiety, which binds to the asialoglycoprotein receptor and enhances delivery of ASOs to hepatocytes. (B) General mechanisms of gene-expression modulation by ASO. Gapmer ASOs downregulate gene expression by inducing RNase H-mediated degradation of the target RNA. Steric-blocking ASOs are uniformly modified ASOs that sterically hinder the binding of proteins or RNPs that regulate post-transcriptional RNA processing (e.g., splicing, NMD, or polyadenylation) or translation. ASO, antisense oligonucleotide; PMO, phosphorodiamidate morpholino oligomers; NMD, nonsense-mediated mRNA decay; uORF, upstream open reading frame; pORF, primary open reading frame; PAS, polyadenylation signal.

Fig. 2. Classification of CFTR mutations.

CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum; PTC, premature termination codon; N/A, not applicable. Mutation classification based on De Boeck and Amaral (2016).

Fig. 3. Mechanisms of ASOs for CF.

Overview of the available mechanisms for class I mutations (top panel) and other mutations (bottom panel). Gapmer ASOs can trigger RNase H-mediated downregulation of NMD-factor mRNAs (①) in the cytoplasm and nucleus. Uniformly modified ASOs that block the binding of EJCs downstream of a PTC can specifically stabilize nonsense-mutant CFTR mRNA (②), which is translated to make truncated CFTR protein or, in the presence of RTC, to make full-length CFTR. Uniformly modified ASOs targeting the splice sites or ESEs (exonic splicing enhancers) of an in-frame exon containing a PTC can induce exon skipping, and the resulting mRNA can be translated to produce partially functional CFTR (③). Uniformly modified ASOs can correct aberrant splicing mutations (④). Fully modified ASOs can also insert missing bases in the mutant CFTR mRNA through an unknown RNA-repair mechanism (⑤). Enhance translation in the pORF of CFTR by suppressing translation in the uORF by binding to the uORF start codon or TSE in the 5’ UTR (⑥). Gapmer ASOs can trigger RNase H-mediated downregulation of CF modifier genes, such as SCNN1A, to improve mucociliary clearance (⑦). ASO, antisense oligonucleotide; CF, cystic fibrosis; NMD, nonsense-mediated mRNA decay; EJCs, exon-junction complex(es); PTC, premature termination codon; CFTR, cystic fibrosis transmembrane conductance regulator; RTC, read-through compound; TSE, translational-suppression elements; uORF, upstream open reading frame; pORF, primary open reading frame.