A rapid quantitative assay to measure cftr function in a primary intestinal culture model

Abstract

The invention relates to an assay for diagnosing a disease or affliction that affects fluid uptake or secretion or for studying the effectiveness of one or more drugs for treating the disease or affliction, wherein the assay comprises measuring swelling of one or more organoids.

Description

FIELD OF THE INVENTION[0001]The invention relates to an assay for fluid and electrolyte homeostasis in an organoid-based culture method.BACKGROUND[0002]Cystic fibrosis transmembrane conductance regulator (CFTR) functions as an anion channel, and is essential for fluid and electrolyte homeostasis at epithelial surfaces of many organs, including lung and intestine. The autosomal-recessive disorder cystic fibrosis (CF) is caused by mutations of the CFTR gene. CF disease is highly variable, and patients have a median life expectancy of approximately 40 years. Loss-of-function mutations cause altered ion and fluid transport, which results in accumulation of viscous mucus in the pulmonary and gastrointestinal tract. This is associated with bacterial infections, aberrant inflammation and malnutrition. Over 1500 mutations have been described, but the most dominant mutation (˜67% of total mutant alleles world-wide) is a deletion of phenylalanine at position 508 (CFTR-delF508). This causes mi...

AI Technical Summary

Benefits of technology

[0007]The invention provides a rapid and simple quantitative assay for CFTR (or other diseases or affliction that affect fluid uptake or secretion) function in a primary intestinal crypt-based culture method15-17. This culture method enables intestinal stem cells to expand into closed organoids containing crypt-like structures and an internal lumen lined by differentiated cells, recapitulating the in vivo tissue architecture. Intestinal CFTR is predominantly expressed at the apical membrane of the crypt cells where its activation drives secretion of electrolytes and fluids18-20. Forskolin21 was found to induce rapid swelling of both human healthy control (HC) and murine wild-type organoids that completely depends on CFTR, as demonstrated by stimulation of intestinal organoids derived from CFTR-deficient mice or CF patients, or upon chemical inhibition of wild-type CFTR. Levels of forskolin-induced swelling by in vitro expanded rectal organoids are comparable with forskolin-induced anion currents measured in ex vivo human rectal biopsies. Temperature and chemical correction of F508del-CFTR function was easily detected by organoid-based fluid transport measurements, and responses to a panel of CFTR-restoring drugs were variable between rectal organoids derived from different F508del homozygous patients. This robust assay is the first functional readout developed in human organoids, and will facilitate diagnosis, functional studies, drug development, and personalized medicine for CF and other related diseases and afflictions.Organoids

[0114]In some embodiments, computer- or robot-assisted culturing and data collection methods are employed to increase the throughput of the screen.

Problems solved by technology

Loss-of-function mutations cause altered ion and fluid transport, which results in accumulation of viscous mucus in the pulmonary and gastrointestinal tract.

This causes misfolding, ER-retention and early degradation of the CFTR protein which prevents function at the plasma membrane.

Other mutations in the CFTR gene that have been found in CF patients also impair protein folding or impair protein production, gating, conductance, splicing and / or interactions with other proteins.

Although these recent developments are very promising, the level of functional restoration of CFTR by these drugs in in vitro model systems is still limited.

In addition, patients show variable responses to these therapies due to yet undefined mechanisms.

The inability to select these non-responding subgroups limits clinical efficacy and drug registration.

Thus far, there are no primary cell models available to screen for compounds that restore mutant CFTR function, only transformed cell lines have been used to identify compounds and their efficiency.

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