Biology subjects

Bridges, R. J.

Publications and source records attributed to Bridges, R. J..

4 recordsLinked to original sources

bioRxiv2024

Systematic deletion of symmetrical CFTR exons reveals new therapeutic targets for exon skipping antisense oligonucleotides

There is a major need for therapeutics that treat diseases caused by pathogenic gene variants that disrupt protein open-reading frames. Splice-switching antisense oligonucleotides (ASOs) offer a potential solution by inducing the skipping of exons containing these variants, removing them from the mRNA and correcting the open-reading frame. Cystic fibrosis (CF), caused by disruption of the CF transmembrane regulator (CFTR) gene, is one such disease that has many chain-terminating variants, which are untreatable with standard protein-targeted modulator therapies. Using CFTR as a model, we demonstrate the utility of ASOs in engineering protein isoforms through exon skipping to rescue protein function disrupted by truncating variants. We functionally screened all CFTR isoforms generated by the deletion of symmetrical exons, which can be skipped without disrupting the open-reading frame. We identified exons that can be removed and produce CFTR isoforms that remain functionally responsive to modulators. We screened for ASOs that induce skipping of these exons and show that they recover CFTR function in airway cells derived from individuals with terminating CFTR variants. This study demonstrates that systematic functional analysis of in-frame exon-deleted protein isoforms can identify targets for ASO-based splice-switching therapies, a concept that can be broadly applied to any multi-exon protein-coding gene.

molecular biology↗

bioRxiv2023

In Vitro Modulator Responsiveness of 655 CFTR Variants Found in People With CF

BackgroundIn 2017, the US Food and Drug Administration initiated expansion of drug labels for the treatment of cystic fibrosis (CF) to include CF transmembrane conductance regulator (CFTR) gene variants based on in vitro functional studies. This study aims to identify CFTR variants that result in increased chloride (Cl-) transport function by the CFTR protein after treatment with the CFTR-modulator combination elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA). These data may benefit people with CF (pwCF) who are not currently eligible for modulator therapies. MethodsPlasmid DNA encoding 655 CFTR variants and wild-type (WT) CFTR were transfected into Fisher Rat Thyroid cells that do not natively express CFTR. After 24 hours of incubation with control or TEZ and ELX, and acute addition of IVA, CFTR function was assessed using the transepithelial current clamp conductance assay. Each variants baseline activity, responsiveness to IVA alone, and responsiveness to the TEZ/ELX/IVA combination were measured in three different laboratories. Western blots were conducted to evaluate CFTR protein maturation and complement the functional data. Results and Conclusions253 variants not currently approved for CFTR modulator therapy showed low baseline activity (<10% of normal CFTR Cl- transport activity). For 152 of these variants, treatment with ELX/TEZ/IVA improved the Cl- transport activity by [≥]10% of normal CFTR function, which is suggestive of clinical benefit. ELX/TEZ/IVA increased CFTR function by [≥]10 percentage points for an additional 140 unapproved variants with [≥]10% but <50% of normal CFTR function at baseline. These findings significantly expand the number of rare CFTR variants for which ELX/TEZ/IVA treatment should result in clinical benefit.

pharmacology and toxicology↗

bioRxiv2021

Open reading frame correction using antisense oligonucleotides for the treatment of cystic fibrosis caused by CFTR-W1282X

CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR mRNA expression as a result of nonsense mediated mRNA decay (NMD), as well as production of a non-functional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations, due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs) designed to induce skipping of exons in order to restore the mRNA open reading frame have shown therapeutic promise pre-clinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the 5th most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from homozygote CFTR-W1282X patients. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein. Significance StatementFrameshift and nonsense mutations pose a major problem for disease therapeutic development. Eliminating these mutations from the mRNA by inducing exon skipping is a relatively unexplored treatment approach, though it has shown promise for some diseases. Here, we show that eliminating a common stop mutation associated with cystic fibrosis by inducing skipping of the exon it is located in, results in a restoration of the open reading frame and recovers CFTR protein function in a manner expected to be therapeutic in CF patients who dont currently have effective treatment options. These results are an important advancement for the cystic fibrosis community but also have implications for other diseases where terminating mutations are responsible for dysfunction.

cell biology↗

bioRxiv2020

Antisense oligonucleotide-mediated correction of CFTR splicing improves chloride secretion in cystic fibrosis patient-derived bronchial epithelial cells

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in more than 15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10kbC>T), which creates a new 5 splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.

molecular biology↗