Identifying genetic modifiers of cell function in cystic fibrosis
New drugs for cystic fibrosis (CF) do not treat only the symptoms but also the cause of the disease. These drugs are called CFTR modulators and repair the action of the CFTR protein that does not work well in the cells of people with CF. The extent to which individuals benefit from treatment is variable, even when people have the same mutations as for example, the most common F508del mutation. In this project we tried to find out why people react differently to CFTR modulators and whether that has to do with the CFTR itself. There is still a lot of variation possible in the CFTR between people, even when people have the same mutation. Through using cultured cells from people with CF, we can individually measure the very precise treatment effects of CFTR modulators. In this project we have linked treatment effects at the cellular level to the differences in the gens for CFTR that we find between people. The differences in CFTR between people have been mapped at the so called DNA and RNA level. We are seeing indicators that can better understand the individual treatment effects of CFTR modulators so that in the future treatments can be better targeted at the individual level.
In the current project UMC Utrecht and Cergentis BV studied novel genetic markers and regions that potentially modify CFTR-directed pharmacotherapy, using TLA and intestinal organoids from people with cystic fibrosis. We have found that that heterogeneity in the CFTR gene itself but also CFTR gene expression due to patient-specific genetic variability are linked to heterogeneous CFTR modulator responses. This project is a first step to establish additional genetic tests and define new drug targets that can be used to enable optimal CFTR modulator treatment for individuals with CF.
UMCU has performed organoid experiments, generated TLA haplotype datasets and analysed data. Cergentis has developed tools for CFTR sequencing and CFTR haplotyping, and guidance for TLA sequencing and CFTR haplotyping.
Societal and Economic impact
Societal: currently people with CF show variable response to modulators: this increases uncertainty for the individual about benefit from daily drug use. By developing insights into the mechanisms that regulate individual drug response, future diagnostic and therapeutic approaches can be developed that enable a better individualized treatment (e.g. through drug selection or individualized dosing). The impact of an unclear diagnosis for CF can be huge psychological burden for individuals and leading to inadequate treatment of disease and reduce life span. Current DNA diagnostic kits focus on the most prevalent genotypes and are biased by the Caucasian prevalence of disease. The new TLA protocol sequences the entire gene and is not biased by race or gender and therefor provides a DNA sequence solution for all people with CF.
Economic: Cergentis may be a preferred site for unclear genetic diagnosis in CF, which is needed for approximately 1-5% of CF patients. This would involve approximately 500-2500 patients in the EU alone and a similar number in the US. Better diagnostics may also lead to a more precise use of drugs to increase the cost efficacy of treatment which is needed in the context of the high pricing of these drugs.
Conceptualisation
Targeted Locus Amplification (TLA) is a novel, state-of-the-art genetic technology that uniquely identifies all types of genetic variation in and around a gene of interest, such as the CFTR gene8. TLA haplotypes can be established by linking genetic variation to the correct parental allele, without the need of having parents and sibling materials. The development of the TLA technology for CFTR gene haplotyping has been initiated but needs significant further refinement to encompass all the genetic regulatory elements. We have focused on defining haplotypes in organoids from individuals who only express one responsive F508del allele, with the other allele being a non-functional class I allele (frameshifts, deletions, premature stop codons, consensus splice site mutations), A455E or S1251N allele. This approach has allowed for linkage of genetic variation to functional data. Using the set of organoids, RNA has been collected at three independent culturing time-points (coinciding with functional assessment studies and used to semi-quantitate CFTR gene expression levels, using allele-specific quantitative RT-PCR to selectively amplify F508del, A455E and S1251N. Data have been normalized to two housekeeping genes.
Functional CFTR restoration by VX-770/VX-809 (Orkambi) has been studied in Intestinal organoids that have been grown, followed by forskolin-induced swelling (FIS), performed at three independent culturing time -points using live cell confocal imaging4. Cells have been pre- incubated for 24h with VX-809 (3 μM) and stimulated with 3 concentrations of forskolin (0.128, 0.8, 5 μM)together with 3 μM VX -770. S1251N-containing organoids were only stimulated with forskolin and VX-770. Relative size increase per organoid in 60 min time lapse videos has been established and area under the curve (AUC ) of swelling curves has been established.
Haplotype genetic variability in both the protein coding sequences and the CFTR5’ upstream, intronic and 3’ downstream regulatory elements have been associated with gene expression and functional data obtained from the first steps in the project.
We have used primary cells derived from CF airway and intestinal tissue to profile genetic regions that might be associated with CTFR expression. Depending on the applied culture conditions, airway stem cells could be differentiated towards CFTR-expressing ciliated cells, whereas intestinal stem cells that highly express CFTR could be differentiated into non-CFTR expressing absorptive enterocytes. In the near future histone methylation profiles will be established under both CFTR-expressing and non-CFTR-expressing culture conditions in paired nasal, bronchial and intestinal epithelium from 5 CF donors. We will also perform chromosome conformation capture on chip (4C) on these samples to characterise genomic DNA elements that interact with the CFTR gene promotor11. Genetic regions identified by these approaches will be used to mine available GWAS data sets to identify genetic variability associated with disease severity and drug responses once these become available.
UMCU will perform organoid experiments, generate TLA haplotype datasets and analyse data. Cergentis will develop tools for CFTR sequencing and CFTR haplotyping, and guidance for TLA sequencing and CFTR haplotyping.
Results
Understanding of individual efficacy of modulators: the linkage of mRNA to drug effect shows for the first time a relation between F508del mRNA and drug response. This points out that factors regulating mRNA quantity can be important for development of more precise diagnostics and therapies. We have found relations between DNA sequence and CFTR modulator responses. This needs to be confirmed in larger studies and this may increase precision of diagnostics and personalised medicine.
