Cystic Fibrosis Research News № 3
Alternative Chloride Channels: The Next Frontier in CF Therapy?
At the recent North American Cystic Fibrosis Conference (NACFC), a key theme of the plenary sessions was the “path to a cure” for the 10% of CF patients who don’t benefit from CF modulators. For those patients who do benefit from modulators, many presenters also considered how better therapies can be developed. Among the many studies cited at the conference, the research effort led by Professor Mads Larsen at the University of Pittsburgh School of Medicine stands out. Their study aimed to identify alternative chloride channels that work similarly to CFTR. The idea is that boosting the function of these alternative channels will recapitulate and strengthen the effects of CFTR modulation.
In this study, the chloride channel of interest is called SLC26A9. Potentially, one might target this channel with molecules that bind to it, alter its structure, and make it more permissible to chloride ions - much as the CF potentiator ivacaftor makes the CFTR channel more permissible to chloride ions. Even though SLC26A9 is likely to be functional in CF patients (it is not mutated like CFTR), the idea was to coax the SLC26A9 channel to transport even more chloride across the cell membrane in order to compensate for the lack of chloride channeled by CFTR.
In an early effort to understand the potential of SLC26A9 therapy, the study set out to validate the hypothesis that SLC26A9 hyper-activation is a realistic treatment route. As a preliminary step, the research team grew lung cells in the lab and demonstrated that SLC26A9 mRNA was indeed present in the cells.
The study then proceeded to show that SLC26A9 does function fully in the absence of CFTR by knocking down CFTR (substantially reducing the amount of CFTR protein) and observing that the amount of chloride ions channeled through SLC26A9 was not diminished. Just to make sure, the researchers also treated cells with a CFTR small molecule inhibitor and arrived at the same conclusion: SLC26A9 still worked okay.
These experiments were important to perform because in the absence of functional CFTR, the SLC26A9 channel might not operate effectively. A 2017 study (led by Carol Bertrand, also at the University of Pittsburgh) had shown that CFTR and SLC26A9 physically interact within the cell interior before arriving at the cell membrane, and that cells with the Delta-F508 CFTR mutation exhibit trafficking defects in both CFTR and SLC26A9. By finding that at least some SLC26A9 does make it to the cell membrane in the absence of CFTR, however, the Larsen study offers hope that SLC26A9 activation therapy is still a possibility for those who aren’t eligible for modulator drugs. Furthermore, Larsen’s study reinforces the notion that modulator therapy and SLC26A9 activation therapy may work synergistically.
If SLC26A9 therapy is successful, it might move enough chloride into the airways to rehydrate them and thin out the sticky mucus that makes the CF lungs congested and receptive to infection by dangerous bacteria. This is not a silver bullet solution, however. SLC26A9 hyper-activation could have unintended side effects, and may not make the airways completely normal. SLC26A9, unlike CFTR, is not thought to be a major player in channeling bicarbonate – which is a molecule that keeps the airways from becoming too acidic (because CF patients lack CFTR, they have abnormally acidic airways that render the immune system less effective at fighting off bacteria). That being said, there is reason to be hopeful that alternative chloride channel therapy can move us further along the path to a cure - if not all the way down the road.
Featured Article: Bertrand CA, Mitra S, Mishra SK, et al. The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9. Am J Physiol Lung Cell Mol Physiol.2017;312(6):L912-L925. doi:10.1152/ajplung.00178.2016.
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Individual CF Bacterial Communities Drive Disease Progression
At a fundamental level, CF is a relatively simple disease: it’s a monogenic illness caused by a single defective protein. Yet clinical outcomes for CF patients are incredibly diverse. Different individuals develop different symptoms to different degrees. For example, only a fraction of CF patients develop CF-related diabetes or CF liver disease. Some CF patients see a rapid decline in lung function from early adolescence on, while others maintain a relatively stable lung function into middle age. CF researchers have long attempted to understand what factors drive these divergent clinical symptoms.
Currently a multinational research team (with members spanning the globe from Austria to Arkansas) is investigating the composition of the unique bacterial community that inhabits the lungs of each person with CF.
Throughout the study, the researchers gathered 818 sputum samples from 109 CF patients. After analyzing which bacterial genera were present (using 16S RNA sequencing), the researchers classified patients – and their bacterial communities – into 8 classes, or “pulmotypes”. Each pulmotype had a unique profile. For example, pulmotype 3 is Staphylococcus (Staph) dominated, while pulmotype 7 had an even mix of Pseudomonas and Staph. The study showed that certain pulmotypes are associated with more or less severe disease: for example, pulmotypes 4 and 6 (Streptococcus and Pseudomonas-dominated respectively) are associated with moderate disease while pulmotype 2 (with a more even mix of Streptococcus, Prevotella, and Pseudomonas) is associated with more severe disease.
In addition, the researchers found that certain bacterial community types are more stable, while others tend to morph into different community structures. This is important because it relates to findings in previous studies that a more stable bacterial community is associated with more stable health. In other words, a patient who transitions between three different pulmontypes over the span of a single year is likely to see sharper lung function decline and more hospitalizations than a patient who maintains a single pulmotype. In the present experiment, the researchers found support for this theory with the finding that pulmotype 6 was clearly the most stable of all pulmotypes, and as mentioned above, is associated with mild disease.
This research is valuable not only because it provides a clearer picture of CF lung disease and the factors that impact clinical outcomes, but also because it can help provide a roadmap for clinical care. For clinicians, understanding what bacteria are living together in the CF lung, and how these bacteria contribute to disease severity, gives them more power to choose the right regimen of antibiotics, and allows them to counsel patients more accurately about how their individual experience with CF is likely to unfold.
Featured Article: Widder S, Zhao J, Carmody LA, et al. Association of bacterial community types, functional microbial processes and lung disease in cystic fibrosis airways. ISME J. 2021;10.1038/s41396-021-01129-z. doi:10.1038/s41396-021-01129-z.
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Making CF Modulators Work with Tricky Class I Mutations
For the minority of people with CF still waiting for effective modulator therapy, a potential new therapeutic approach is well on its way. Working with a cocktail of drugs, an international team of researchers from the Netherlands and Germany have identified a five-molecule combination that could help individuals who produce a truncated version of the CFTR protein that can’t be rescued by the CFTR modulators.
These particular patients possess so-called class 1 CFTR mutations, which are considered to be the most severe. The common Delta-F508 mutation, in contrast, is a less severe class II mutation. For people with class 1 mutations, the DNA encoding the CFTR protein is altered in such a way that when the protein is transcribed into mRNA, the resulting message contains a premature stop signal (or stop codon) that prevents protein translation from proceeding all the way through. Such an alteration of CFTR is known as a “nonsense mutation”. The resulting incomplete protein is often degraded by the cell and thus can’t be rescued by CFTR modulators.
The solution to this problem is to instruct the ribosomes, which translate proteins in the cell, to simply ignore the premature signal and produce the full-length protein. Other research teams have already found and tested drug candidates that accomplish this. The compound ELX-02, developed by Eloxx Pharmaceuticals in Watertown, Massachusetts, has already made it into phase 2 clinical trials for CF patients.
But the team behind this study, lead by Eyleen de Poel of Wilhelmina Children's Hospital at Utrecht University, believe that more powerful therapy for patients with premature stop codons can be developed by combining multiple drug candidates, and administering them simultaneously with Trikafta or other approved CF modulator drugs.
To test this theory, the Utrecht researchers developed a platform with 12 distinct intestinal organoid cultures, each possessing a different rare CFTR mutation that gives rise to a premature stop codon in mRNA. These organoids are lab-grown structures, built from human cells, which mimic the structure of the intestinal lumen. When treated with drugs that improve the function of CFTR, they swell up in size, as one of the major roles of the CFTR protein is to channel water out of the cell and hydrate the epithelial surface. This swelling provides a clear indicator of drug efficacy.
The drug cocktail they used was a quintuplet combination, composed of :
- the three CFTR modulators present in Trikafta,
- a compound that address the premature stop codon problem by stopping the cell from carrying out nonsense-mediated mRNA decay (NMD), a process by which mRNAs containing premature stop codons are chewed up before they get to be translated, and
- a compound that induces translational readthrough, telling the ribosome to ignore the premature stop codon and produce the rest of the protein.
The research team found that not only were the mutant CF organoids swelling more when the drug cocktail was applied to them, but also that CFTR mRNA levels were boosted in these treated organoids, illustrating that premature stop codon drugs were working with the modulators to rescue CFTR function and that CFTR mRNA was not being degraded by the cell before translation. These experimental results are a strong sign that people with CF still waiting for effective therapy have new options coming their way.
Featured Article: de Poel E, Spelier S, Suen SWF, et al. Functional Restoration of CFTR Nonsense Mutations in Intestinal Organoids. J Cyst Fibros. 2021;S1569-1993(21)01425-9. doi:10.1016/j.jcf.2021.09.020.
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Featured Five CF Stories
It’s impossible to list all of the amazing research that is on-going for CF. Below is a quick list of a few fascinating articles that seem to show significant promise.
Getting Gene Therapy into the Lungs: New study investigates the ability of nanoparticle carriers to help gene therapy drugs breach the sticky mucus barrier in the CF lungs. Early findings in mouse models point to success – gene therapy carried by nanoparticles is more effective. (BMJ Thorax).
CFTR Modulators Boost Pancreatic Function Too: Scientists have found that CF modulator treatment is associated with roughly a 65% reduction in hospitalizations for pancreatitis. The study that produced this finding followed over 10,000 CF patients from 2012-2018, 1800 of whom had received modulator therapy. (American Journal of Gastroenterology).
New Biomarkers for Pseudomonas Biofilms: Noting the importance of identifying chronic Pseudomonas infection earlier so that it can be treated properly, researchers identify new biomarkers that differentiate chronic vs. newly-colonizing strains of Pseudomonas. (Nature Scientific Reports).
Options for Modulator-Ineligible Patients: A suite of new drugs are in the early stages of development for patients with rare, yet-untreatable CFTR mutations. These include read-through agents for nonsense mutations, RNA/DNA-based therapies, and cell-based therapies (Cells).
CF and COVID-19: New data collected from multiple CF registries around the world affirms that people with CF are not more likely to acquire SARS-CoV-2 than the general population, and that most individuals tend to experience mild symptoms, with the exception of those who have undergone lung transplant and/or have relatively low function (Current Opinion in Pulmonary Medicine).
Clinical Trial Watch: Moving CF Research Forward
The latest news on CF drug development and clinical trials.
Clinical Trial Recruiting: Now that many people with CF have begun highly-effective modulator therapy, there are questions about the need to keep performing other daily treatment tasks. The SIMPLIFY study will test whether discontinuing either hypertonic saline or pulmozyme (both often paired with airway clearance and taken daily) has any serious negative consequences for individuals on Trikafta. (University of Washington & Dartmouth Hitchcock Medical Center).
Clinical Trial Recruiting: At Yale University, researchers are testing the ability of phages – viruses that target bacteria – to reduce the load of Pseudomonas in the sputum of CF patients. The hope is that phage therapy might serve as a tool for clinicians to fight back against chronic Pseudomonas infection. (Yale University).
Clinical Trial Recruiting: After recently establishing that Trikafta is safe for CF patients ages 6-11 that possess one or two copies of the common mutation Delta-F508, Vertex Pharmaceuticals is new recruiting children ages two to five to see if the drug should be prescribed even earlier. (Vertex Pharmaceuticals).
Clinical Trial Recruiting: A new trial is underway to test the safety of a drug called brensocatib in CF patients over a four-week period. If effective, brensocatib will help quiet pro-inflammatory immune enzymes that contribute to CF lung disease. (Insmed Incorporated – Tyler, Texas).
*Note: If you or a family member are interested in entering clinical trials that are currently recruiting, please click the link to ClinicalTrials.gov and See ‘Contacts and Locations’ for participating research institutions, and speak to your clinician for guidance.
A Call to Action
Cystic fibrosis (CF) research is very much dependent on the strength of the CF community. It’s not simply an effort carried out by scientists in white lab coats - although there are many of them, and their work has enormous impact. Advances in research also depend on the technicians and engineers who operate the laboratory equipment that enables drug discovery, and the industrial machinery that allows drug development. Research depends on both business and marketing professionals, those who make biopharma companies viable and promote clinical trials. Successful research further depends on clinical trial coordinators, who carry out studies and work tirelessly to recruit and support patients throughout the complicated trial process. Particularly for rare diseases like cystic fibrosis, research depends on the work of foundations and patient advocates, which includes in the United States organizations such as the CF Foundation, Emily’s Entourage, CFRI, and the Boomer Esiason Foundation, as well as countless other across the globe, and hundreds of committed clinicians and researchers. Most importantly, research depends on people with CF and their devoted families and friends.
There can be no progress in CF research without patients willing to participate in clinical trials: not only to test new drugs, but also to provide, quite literally, their flesh and blood. It is with the help of patient samples that scientists can understand the damage that CF inflicts upon the human body, and also how drugs developed by the research community can remedy these damages.
This newsletter aims to pull all of these threads together; allowing the CF community to more fully appreciate how well the aims of its many members are aligned (and it extends an invitation to all readers not yet a part of the CF community, to embrace the cause and take up the task of pushing CF research forward). There’s something here for everyone - those interested in the clinical side of CF care, or in drug development, or the technical work performed in CF-centered laboratories. The newsletter also has as its objective to showcase new clinical trials; an opportunity for patients and clinicians to take part. Wherever and whoever you are in the world, you too may push CF research forward - either by direct participation, or simply by reading and sharing this newsletter with others.