(650) Ribosomal Silencing Confers CFTR Nonsense Suppression and is Synergistic with Highly Effective Modulators

Background: Approximately 10% of people with CF remain without an effective therapeutic intervention, as they harbor CFTR variants unresponsive to clinically approved modulators. Refractory variants prominently encompass “Class I” defects in protein synthesis, including premature termination codons (PTCs) such as W1282X. PTCs confer both an aberrant transcript and truncated protein, which are subject to nonsense-mediated decay (NMD) and proteasomal degradation, respectively. To address unmet medical needs, our work focuses on elucidating mechanistic and therapeutic impact of genetic modifiers that influence PTC biogenesis.

Methods: We previously employed an innovative approach, genome-wide yeast phenomics, to ascertain gene-gene interaction networks responsible for read-through of W1282X and other CF-causing PTCs. We designed a phenomic model of nonsense suppression using the CFTR homologue, Yeast Oligomycin Resistance 1 (YOR1). Mutations corresponding to CFTR PTCs were introduced into YOR1, and “hits” that emerged from subsequent yeast deletion library screens included evolutionarily conserved translation initiation factors, tRNA processing enzymes, and ribosomal proteins. Translational relevance of molecular targets was assessed in Fischer rat thyroid (FRT) cells and CF bronchial epithelia (CFBE). FRT were transduced with W1282X-CFTR cDNA encoding intron-flanked exon mini-genes (to generate transcripts sensitive to NMD) and a horseradish peroxidase tag in the fourth extracellular loop (to detect cell surface localization). Patch-clamp was employed to quantify CFTR channel open probability. CFBE stably expressing W1282X cDNA were grown at air-liquid interface and assessed by short-circuit current measurement.

Results: Preliminary data show that siRNA knockdown of several candidate genes, most notably ribosomal protein L8 (RPL8/uL2), enhanced W1282X-CFTR plasma membrane density ~3-fold. We also demonstrated synergistic correction of W1282X cell surface trafficking (~12-fold increase) in response to combined treatment with RPL8 silencing and elexacaftor/tezacaftor/ivacaftor. In addition, RPL8 suppression augmented W1282X transepithelial ion transport (~29% of wild-type CFTR) and open probability (~7-fold enhancement), indicating improved channel activity and/or gating rate. Ongoing studies will test effects of RPL8 depletion alone and together with highly effective modulators during rescue of W1282X functional expression. Assays will include evaluation of CFTR mRNA utilization (ribo-seq), protein maturation (western blot using CFTR C-terminal antibodies), and short-circuit current analysis performed in FRT, CFBE, or primary human airway epithelia.

Conclusions: RPL8 interfaces with both 18S and 25S rRNA near the E-site of the peptidyltransferase center. These interactions confer structural rearrangements in functional centers of the 60S and 40S subunits, thereby facilitating alterations to translational fidelity. Ribo-seq experiments underway will determine whether RPL8 depletion influences PTC read-through, NMD, and/or other aspects of mRNA metabolism. Together, findings indicate specific ribosomal proteins such as RPL8 should be considered as therapeutic targets for overcoming CFTR PTCs. Our work also illustrates the utility of yeast phenomics as a method for deducing principles of PTC rescue, which could be extendable to other hereditary conditions caused by premature termination.

Acknowledgements: This work was supported by NIH (HL151965 to KEO; HL136414 to EJS/JLH) and CFF (OLIVER22A0-KB to KEO).

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