Reply to Finotti et al.: Enhancing the Expression of CFTR Using Antisense Molecules against MicroRNA miR-145-5p.

To the Editor:

Fabbri and colleagues’ investigations (1) complement well the work of our group (2) and others (3–5) to emphasize the importance of the TGF-β (transforming growth factor-β)/miR-145 (microRNA 145) axis in regulating CFTR (cystic fibrosis transmembrane conductance regulator) expression and function in airway epithelia. In brief, miR-145 mediates TGF-β suppression of CFTR through miR-145 binding to the 3′-untranslated region (3′-UTR) of CFTR mRNA. TGF-β/miR-145 stimulation nullifies the benefit of currently available CFTR modulators, and miR-145 antagonism augments F508del correction. The authors outline well the therapeutic potential of miR-145 antagonism for each CFTR mutational category (e.g., class I–VI defects).

In their letter to the editor, Finotti and colleagues elucidate two important considerations in the development of oligotherapeutics: target specificity and preclinical model selection. With regard to antagonist selection, the question arises as to whether to block the microRNA (miRNA) itself (full antagonism) or the binding site on the transcript of interest (target site blockade). Pursuing the latter approach, we use an antisense oligonucleotide to block miR-145 binding to the CFTR 3′-UTR sequence and limit off-target disruption of TGF-β signaling. Finotti and colleagues describe the possible benefit of full miR-145 antagonism to suppress additional consequences of increased TGF-β stimulation. Their letter highlights the potential antiinflammatory benefit of miR-155 downregulation. As miR-145 and miR-155 are involved in lung fibrosis, full antagonism may also ameliorate pathogenic airway remodeling in cystic fibrosis (CF). Next-generation sequencing of Calu-3 cells suggests that 80% of miRNAs are unaffected by miR-145 antagonism. The acceptable threshold of off-target miRNA perturbation remains to be determined, but it likely will be balanced by therapeutic necessity and the consequences of dysregulated signaling.

A second consideration in the development of oligotherapeutics is the adequacy of the preclinical modeling system. Like Finotti and colleagues, we often use Calu-3 to test biochemical and functional readouts of CFTR manipulation as an initial step. However, Calu-3 cells are an adenocarcinoma cell line with an altered miRNA profile compared with primary cells. For example, miR-145 expression in Calu-3 cells is >5-fold lower than in non-CF cells and >10-fold lower than in CF primary airway epithelial cells grown to terminal differentiation. The consequences of miR-145 blockade may be less impactful in a low-expressing cell line such as Calu-3 than in a higher-expressing primary epithelial cell line. The recently discovered ionocyte (6) may have an even greater discordance with the Calu-3 cell line. A second consideration involves common limitations of gene-edited lines such as F508del CFBE cells in which stably transfected cDNA lacks the 3′-UTR binding domain. This consideration extends to most transgenic animal models. For miRNA manipulation in animals with CF, it may be preferable to use the recently developed full-length human CFTR model rather than standard CF mice. A third consideration for preclinical work is the variability of miRNA-binding sites across species. Although the miR-145 sequence itself is faithfully replicated, the 3′-UTR binding domain on human CFTR is not preserved in rodents. A preclinical evaluation of miR-145–directed intervention should address these in vitro and in vivo considerations.

Finotti and colleagues' report represents an important preliminary step along the preclinical roadmap toward miR-145 intervention. An immediate next priority will be to confirm the utility of miR-145 inhibition for bolstering next-generation CFTR correctors. Our recently published results tested lumacaftor/ivacaftor (Orkambi), and unpublished results with tezacaftor/ivacaftor (Symdeko) performed similarly. Once the benefit of miR-145 is established with upcoming triple-combination strategies, the safety profile in primary CF and non-CF airway epithelial cells should be assessed, as Fabbri and colleagues have already done in Calu-3 cells (1). In vivo safety measures focusing on TGF-β signaling pathways should then be tested in animal models with full-length human sequence CFTR. Through this rigorous assessment of miRNA efficacy, selectivity, and safety, the value of miR-145 oligotherapeutics can be realized.