(611) A Mutation-Independent Role of TMEM16A for Airway Cystic Fibrosis Therapy.

Background: With the announcement of Trikafta^TM now approved for the treatment of the main cystic fibrosis (CF) mutation F508del, the majority of the 90% of the CF population will have access to this life-saving treatment. Effective responses to this drug for individuals harboring at least one F508del allele remain to be determined. For a small percentage of patients under the age approved for this treatment and for those harboring rare mutations (like W1282X) for which current CF transmembrane conductance regulator (CFTR) modulators are not expected to work, there needs to be other therapeutic options. Here we are evaluating a mutation-independent approach by targeting an alternative chloride channel TMEM16A (ANO1) to compensate for defective CFTR in CF patient-derived cells as a potential therapeutic option for all CF. Leveraging our iPSC-derived lung models we will determine the effects of up-regulating the TMEM16A gene or activating TMEM16A function in restoring chloride transport properties in CF airway epithelial cells.

Methods: The human wild-type ES cell line (CA1) and CF hiPSC (harboring W1282X mutations) were utilized to generate the TMEM16A-mCherry iPSC reporter line by a CRISPR-based editing platform. The expression of TMEM16A and CFTR was analyzed by single-cell RNA sequencing, qRT-PCR and immunohistochemistry (IHC). The TMEM16A membrane potential was measured by FLiPR with TMEM16A activator (Eact) and inhibitor (Ani9). An inducible TMEM16A cell line was generated using a piggyBac CRISPR-activation system in the 16HBE14o- (wild-type CFTR).

Results: First, we generated lung epithelial cells in CA1 using our differentiation protocol and analyzed TMEM16A and CFTR expression by single-cell RNA sequencing. We also performed IHC on human adult lung, CF lung and trachea tissue to identify colocalization of CFTR and TMEM16A on the same epithelial cells. We confirmed the CFTR and TMEM16A co-expression in our CA1-derived lung epithelial cells and co-localization in adult lung and trachea tissue. In CF hiPSC-derived lung epithelial cells, higher TMEM16A function was observed compared to cells derived from wild-type ES as measured by an apical chloride conductance assay (FLiPR) when treated with TMEM16A potentiator Eact (1mM). Specificity of TMEM16A activity was confirmed by the specific inhibitor Ani9 (10mM). High TMEM16A function correlated with higher expression of TMEM16A in the CF mutant cells. CFTR expression was found to be 3-fold higher than TMEM16A in normal cells conversely, a 5-fold higher expression of TMEM16A versus CFTR was found in CF mutant cells. To determine whether upregulating TMEM16A expression will restore chloride transport activity in CF cells, we have generated and will test an inducible CRISPR activation system increase TMEM16A expression in CF iPSC-derived cells.

Conclusions: Altogether, our TMEM16A-mCherry iPSC reporter line and TMEM16A activation/inhibition by small molecules and the piggyBac system will help to understand the role of TMEM16A during lung epithelial development and determine whether targeting alternative ion channels may be an efficacious strategy to treat CF.

Acknowledgements: We received the Dualase CRISPR-Cas9 protein from our collaborator Dr. Brent Stead (Specific Biologics Inc). This work was supported by the Canada First Research Excellence Fund Medicine by Design New Ideas Award. Dr. Lee holds a Cystic Fibrosis Canada post-doctoral research fellowship.

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