Development of gene and drug based therapies to target rare mutations causing severe cystic fibrosis.

Cystic fibrosis (CF) is the most common life-threatening autosomal recessive disorder, which affects over 105,000 people worldwide. Progressive lung disease is the main cause of morbidity and mortality in people with CF (PwCF), although many organs are affected in CF. Symptoms arise because of an imbalance in ion and water homeostasis in the secretory epithelia of these organs. This is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene resulting in loss of function of the CFTR chloride and bicarbonate channel. To date, over 401 CF-causing mutations have been described, which result in different defects on the protein level. Mutations are therefore grouped into seven classes (Ia-Ib-II-III-IV-V-VI), where class Ia/b represents the most severe defects, i.e. no protein, and class VI represents mutations that produce a normally functioning CFTR with an increased turn-over at the plasma membrane (PM).  Many mutations however, present with a combination of multiple protein defects, all of which need to be tackled to reverse the phenotype.

CFTR modulators have been developed that either improve the folding and trafficking of CFTR to the PM (correctors) or increase the open probability of the channel (potentiators). Today, two highly effective CFTR modulator therapies are on the market: Kalydeco™ (potentiator monotherapy) and Trikafta™/Kaftrio™ (triple combination of correctors and potentiators), which are used to treat approximately 85% of PwCF. For the remaining 15%, there is no CFTR-targeted therapy available yet. The goal however is to bring causal therapies to all PwCF.

In this PhD research, I focused on missense CFTR mutations which might be rescued by existing or novel CFTR modulator (combinations). To this end, I characterized several rare CFTR mutations (E60K, G85E, E92K, A455E and N1303K) with known processing defects. Complementary assays were used to phenotype the maturation, localization and traffic efficiency as well as function of each mutant protein, and the effect of the corrector lumacaftor and/or the potentiator ivacaftor on these processes. Distinct processing and functional defects were identified for the mutations studied. Moreover, all mutations could be rescued by the CFTR modulators tested with the exception of G85E and N1303K. These mutations, therefore, were investigated in more detail in human rectal organoids harboring at least one G85E or N1303K allele. The quantification of CFTR function by FIS in this well-validated primary cell model has been shown to correlate well with clinical outcomes underscoring its translatability. First, I investigated the effect of the CFTR modulator elexacaftor, which had recently become available, alone and in combinations with other types of CFTR correctors and potentiators. G85E was efficiently rescued by elexacaftor, alone but even more in combination with other corrector types. Rescue was mainly through elexacaftor’s corrector mechanism. N1303K was rescued to lower levels, and required the presence of both ivacaftor and elexacaftor. Intriguingly, elexacaftor acted as a co-potentiator for this mutant, on top of the potentiator ivacaftor. Rescue was increased when the co-potentiator apigenin and/or the corrector tezacaftor were added, to levels that were comparable to lumacaftor/ivacaftor rescue in F508del/F508del organoids. This modulator combination is approved for F508del homozygous PwCF and suggests this level of CFTR function in FIS corresponds with modest clinical benefits in PwCF. However, as rescue for N1303K was moderate, we aimed to further improve CFTR functional rescue for this mutant. We hypothesized that increasing the amount of N1303K channels at the PM might result in higher levels of rescue, as more channels would be available for potentiation. Besides, the current CFTR correctors, developed for F508del, only improve N1303K PM expression by ~1.6-fold. A pilot screen of ~3000 small molecules from a repurposing library in a PM density assay for N1303K identified one compound, that improved N1303K functional rescue by Trikafta™/Kaftrio™ combination tezacaftor/elexacaftor/ivacaftor. Evaluation of the mechanism of this compound is ongoing.

Taken together, this work provides a strategy to evaluate by phenotyping rare CFTR mutations in complementary cell models, i.e. laboratory cell lines and rectal organoids from PwCF, in order to identify existing CFTR modulators or combinations for these mutations. This approach further allowed to shift the focus to the N1303K mutation, which was insufficiently rescued by existing approved CFTR modulators. By testing additional CFTR modulator combinations, and a small molecule screen for novel CFTR modulators, its rescue could be improved.