(675) Personalized medicine approaches directed at rare CFTR trafficking mutations contribute to elucidation of correction mechanisms

Background: Among the more than 2,100 variants in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, more than 1,000 are very rare, being present in less than 5 individuals worldwide. As the respective functional defect is still uncharacterized, establishment of a CF diagnosis/prognosis is difficult. Moreover, several of these variants may respond to the available modulator drugs.

Our aim here was to assess the molecular/functional CFTR defect associated with rare mutations both in patient-derived materials and cellular models, and to determine their responsiveness to highly effective CFTR modulator therapies (HEMTs). Furthermore, we aimed at analyzing if mutation location would somehow influence/predict the response to the correctors.

Methods: We produced novel CFBE-based cell lines stably expressing fifteen different CFTR variants (P5L, R75G, H139R, I148T, D192G, G194R, H199Y, V201M, P205S, W361R, A550T, L558S, L571S, S945L and M1137R) and used them to assess CFTR expression and maturation by Western blot (WB) after treatment with correctors (VX-809, VX-661, VX-445 and the double corrector combination VX-661+VX-445). CFTR function was assessed by Ussing chamber. Additionally, we performed the forskolin-induced swelling (FIS) assay in intestinal organoids from individuals bearing these variants.

Results: All the mutations analyzed led to total lack or severe reduction (I148T, G194R and S945) of CFTR processing i.e, impairing the fully glycosylated mature form (band C), and thus confirming them as class II.

When assessing the response to correctors, all mutations evidenced some level of response (at least to some of the treatments), except for those localized in NBD1 (A550T, L558S, and L571S) that could not be corrected by none of correctors or combinations tested. Interestingly, considering the localization of the responders in CFTR structure, several of them are located at a particular position at TMD1, that apparently correlates with easy correction.

Functional assessment in intestinal organoids obtained from individuals with CF showed that organoids with P5L/F508del and P205S/Y1092X genotypes were rescued by VX-661/VX-770 although a more robust rescue was observed with the VX-445/VX-661/VX-770 combination. In contrast, in S945L/F508del and M1137R/F508del organoids CFTR function was only rescued by VX-445/VX-661/VX-770 – which confirms the different response observed to correctors in cell lines expressing each mutation individually.

Conclusions: Our study confirms the relevance of assessing the response of rare mutations to HEMTs (in a personalized medicine approach, that is best achieved by combining data from cellular and patient-derived models) and suggest the existence of a location-dependent pattern of response to modulators that may contribute to further clarify the mechanism of action of correctors.

Acknowledgements: Work supported by UIDB/04046/2020 and UIDP/04046/2020 centre grants from FCT, Portugal (to BioISI); DysMut2 grant (Ref. FARINH19I0) from CFF (USA); and "HIT-CF" (H2020-SC1-2017-755021) from EU. SR is recipient of a BioSys PhD programme fellowship (SFRH/BD/142857/2018) from FCT (Portugal).

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