ASO Blockade of the miR-145 Binding Site on CFTR Enhances F508del Correction in Primary Airway Epithelial Cells and Patient-Derived Nasal Organoids
Year: 2021
Type: conference-abstract
Abstract: Rationale: miRNA-145 (miR-145) regulates CFTR gene expression by binding to the 3'-untranslated region (3'-UTR) to destabilize the mRNA transcript. We utilize a highly selective antisense oligonucleotide (ASO)-based approach that sterically blocks miR-145 binding to CFTR 3'UTR (target site blockade, TSB). This study investigates utility of ASO TSB to enhance elexacaftor/ivacaftor/tezacaftor (E/T/I) correction of F508del in primary airway epithelia and tests the individualized response to miR-145 inhibition in human nasal epithelial (HNE) organoids derived from CF patients. Hypothesis: ASO TSB of the miR-145 binding site increases E/T/I correction of F508del CFTR in primary airway epithelia and HNE-derived nasal organoids. Methods: HNE cells were conditionally reprogrammed, grown in a 3D culture system, and differentiated into CFTR-responsive organoids. HNE organoids and human primary polarized airway epithelial cells (AECs) were transfected with a miR-145 antagonist (25M), ASO (1uM), or appropriate controls. For F508del cells, elexacaftor and tezacaftor (3uM) were added for 24 hours following transfection and ivacaftor was introduced at the time of functional readout. qPCR quantified miRNA and CFTR expression. Modified Ussing chambers assayed CFTR function. Lionheart FX bright-field microscopy quantified forskolin-induced organoid swelling. Results: Consistent with our previous miR-145 data in AECs, the TGF-/miR-145/CFTR mechanistic axis is present in patient-derived HNE organoids. In non-CF HNE, TGF- stimulation quadrupled miR-145 and halved CFTR expression whereas ASO administration nullified TGF- inhibition and doubled CFTR mRNA (p<0.01). In F508del HNE organoids, ASO administration increased the CFTR mRNA substrate by nearly 50% (p<0.01) compared to controls. In functional studies, ASO treatment (1uM, 3x/week) blocked TGF- inhibition of CFTR and significantly increased organoid swelling (p<0.01). In F508del HNE organoids, both ASO and miR-145 antagonism improved E/T/I correction of F508del by 50% (p<0.01). Parallel data in F508del primary AECs similarly revealed that ASO TSB increases E/T/I functional correction by > 50% (p<0.05) (Figure RNA-Seq data in gene-edited F508del 16HBE14o -cells quantified the selectivity of the ASO approach, with alterations above the priori threshold (FC>2, q<0.05) for only 3 protein-encoding genes out of 25,000 candidates. Conclusions: ASO TSB of the miR-145 seed region on CFTR is a novel strategy to inhibit miR-145 degradation of CFTR selectively, while limiting genome-scale unintended sequelae that could occur with full miR-145 antagonism. ASO TSB boosts E/T/I correction of F508del by 50% in both AECs and HNEs, suggesting that nasal derived organoids may provide a personalized substrate to test for ASO benefit in patients suboptimally responsive to E/T/I correction.
Authors: F. Lutful Kabir, Z. Liu, J.D. Anderson, S. Sasaki, L. Huang, S. Guo, J.S. Guimbellot, S.M. Rowe, W.T. Harris
Language: English
Subfield: Pulmonary and Respiratory Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: gold
OpenAlex ID: W4230580708