Is gene therapy ready for prime time?

A team of researchers in the Netherlands recently published their findings on a gene editing technique called prime editing, which they used to treat the defective CFTR protein that causes cystic fibrosis. Prime editing is essentially a treatment that replaces a faulty gene sequence (for example, a mutated CFTR gene) with the correct one. Led by Hans Clevers of the Hubrecht Institute, the team has shown that the prime editing technique is safe for human treatment. According to the study's first author Maarten Guerts, prime editing is a “safer technique than the conventional CRISPR/Cas9. It can build in a new piece of DNA without causing damage elsewhere in the DNA. That makes the technique promising for application in patients”.

The CF research space has seen some prior efforts at gene editing: mostly attempts to edit the genomes of mice or alter lung cells in the lab. Certainly nothing as effective, though, as the modulator therapies that recently have been approved for many people with CF. At this stage, gene therapy research is focused on improving the biological effectiveness of gene therapy techniques, and first making sure they work in lab-grown cells before risking patient treatment that is ineffective or dangerous.

In their experiment, the Hubrecht Institute scientists employed the prime editing system plus another hot CF research tool – organoids. These are structures grown in the lab from human cells, in this case taken from the digestive tract of CF donors, grown to resemble a human gastrointestinal tract . Prior studies in the Netherlands and elsewhere have used CF organoids as a personalized platform for modulator testing to see if the modulators rescue CFTR in the cells of CF patients with rarer mutations not yet approved for therapy. Hans Clevers himself has an impressive history of using organoids in his research, and now, his team is using organoids in a very similar way, just with gene therapy rather than modulators.

The researchers wanted to correct two different CF mutations – the highly common F508-delta, and the rarer CFTR-R785*. The former has been previously corrected with traditional CRISPR gene editing, and the latter with another gene editing approach called adenine base editing.

How did the prime editing technique perform? It was determined to be safe. The researchers observed no major off-target effects (additional, unwanted mutations caused by the gene therapy) when they scanned the organoids after gene therapy treatment. But they also found, after subjecting a whole batch of organoids to gene therapy, that few of the CFTR-defective organoids were transformed into organoids with working CFTR. Prime editing, in this study, was not significantly more effective than the other forms of gene therapy (adenine base editing or traditional CRISPR gene therapy), but still better than no gene therapy at all.

So is this a dead end for CF gene therapy? Quite the contrary, as the finding that prime editing has proven safe is a very positive result in and of itself. While prime editing may not be a magic bullet, it can serve as another tool in the gene therapy arsenal. And even though its effectiveness may be low, that could certainly improve as the technique is fine-tuned by further research.

Featured Article: Geurts MH, de Poel E, Pleguezuelos-Manzano C, et al. Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids. Life Sci Alliance. 2021;4(10):e202000940. Published 2021 Aug 9. doi:10.26508/lsa.202000940

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Why Antibiotics Fail? (How We Can Make Them Better)

Clinicians have long been stymied by the fact that the common CF pathogen Pseudomonas aeruginosa tends to resist whatever antibiotics they throw at it. What is behind this bad behavior? In a recent review, researchers from Ghent University in Belgium have outlined the many ways that individual patient factors can impact antibiotic effectiveness. Specifically, they focus on the environment that Pseudomonas inhabits within the human lungs.

Researchers have long found that although antibiotics work to kill Pseudomonas in the lab, they are not as effective when actually given to people with CF. Some scientists have focused on developing more accurate lab culture methods - for example, using artificial sputum media that better mimics the airway surface. This work is helping researchers to see more clearly whether antibiotics are actually going to work in the body. But it doesn’t make the antibiotics better.

The Ghent University team, led by Sara Van den Bossche, set out to review a different set of studies - those that explicitly identify the factors in the lungs that prevent antibiotics from working properly. In their review, they have explained how antibiotics can be better designed and delivered to combat antibiotic resistance. Ultimately, designing more effective antibiotic treatment will boost patient outcomes.

The lungs are like a dense forest, playing host to a diverse ecosystem of bacteria, fungi, and viruses. The behavior of each denizen in this ecosystem impacts the others, and they all impact (and are impacted by) the airway environment. That is, factors such as the consistency of mucus, the abundance of resources like iron, sugars, and amino acids (the building blocks of proteins), and the presence of immune system defenders: neutrophils that secrete bacteria-digesting enzymes and macrophages that engulf bacteria for breakfast.

This lung ecosystem looks a lot different for people with CF than it does for other individuals. For example, an overabundance of iron in the CF lungs give bacteria like Pseudomonas a special edge. Pseudomonas is equipped with pincer-like molecular tools called siderophores that steal iron away from host cells, helping it to form antibiotic-resistant biofilms and produce a number of virulence factors. The viscous mucus of the CF lungs also blocks the free movement of antibiotics, making them less likely to reach and kill Pseudomonas. And other CF pathogens play a role too. Staphylococcus aureus, for example, has been found in prior studies to help Pseudomonas resist antibiotics, in some cases by contributing to bigger, bulkier dual-species biofilms.

By outlining all of the factors in the CF lung environment that make antibiotics ineffective, the Ghent University Research team have provided a concise roadmap to improving antibiotic therapy and boost patient outcomes. Researchers are already starting to take these insights about the lung environment and use them to develop better therapy. Knowing, for example, that iron contributes to Pseudomonas biofilm growth has led some scientists to consider drugs that would keep iron away from the bacteria. Knowledge that CF mucus blocks the passage of antibiotics has spurred efforts to develop new delivery vehicles – like nanoparticle capsules – that are better able to breach the mucus barrier. Following a two-pronged approach to antibiotic treatment, finding new antibiotics and modulating the lung environment to make them more effective, offers many opportunities to fight back at antibiotic resistance.

Featured Article: Van den Bossche S, De Broe E, Coenye T, Van Braeckel E, Crabbé A. . The cystic fibrosis lung microenvironment alters antibiotic activity: causes and effects. Eur Respir Rev. 2021;30(161):210055. Published 2021 Sep 15. doi:10.1183/16000617.0055-2021.

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Drug Discovery: Making Highly-Effective CF Modulator Therapy (Even) More Effective

Many people with CF approved for treatment with Trikafta have had considerable improvements in lung function over a very short period of time. With this remarkable success, it’s hard to imagine that things can get much better. But the truth is that Trikafta isn’t perfect. It does help get more CFTR to the cell surface, where it transports cellular ions and water into the lungs and loosens up the sticky airway mucus, but some CFTR still gets stopped along the way. Researchers at the University of Pittsburgh, in collaboration with Pfizer, are looking to make sure that no CFTR gets left behind.

Why is it that the mutated CFTR protein of people with CF doesn’t make it to the cell surface where it belongs? After being cobbled together as a newborn protein, CFTR travels along the endomembrane system on its way out to the cell surface. It consists of multiple, interconnected compartments (the endoplasmic reticulum, or ER, the golgi apparatus) in which the protein is modified (a bit of refolding here, a sugar molecule added there) and readied for export to the cell surface. In the cell, misfolded proteins (including mutated CFTR) are tagged with a molecule called ubiquitin and then broken down and destroyed by proteasomes.

Logically, it would seem that shutting down the proteasomes would allow CFTR to continue on towards the cell surface . Yet prior studies have found that this is actually not the case. CFTR still gets so heavily tagged with ubiquitin that it just sinks into the cell interior and resists correction by modulators.

The Pittsburgh team, led by Jeffrey Brodsky, has a unique approach to this problem – they developed a more powerful inhibitor of ubiquitination. Prior efforts to develop ubiquitination inhibitors have met with unsatisfactory results, but this is largely because the molecules developed were too toxic for human consumption. With this in mind, the research team improved upon the existing drug PYR-41, which is already known to inhibit ubiquitination, and optimized it so that it was far less toxic and even more effective at its task.

The team chose 22 compounds to test (after preliminary screening) and two t were both less toxic and more effective than PYR-41. One of the two, molecule 7134, was further assessed for its ability to improve CFTR activity in lab-grown lung cells. The researchers determined where CFTR could be found along the endomembrane system after treatment with 7134 and a CF modulator (lumacaftor).

In cells treated with lumacaftor alone more CFTR was retained in the ER and the golgi than in non-treated cells, where the CFTR protein was degraded and therefore not present much in either compartment. Furthermore, the team found that cells treated with lumacaftor and molecule 7134 at the same time fared better still – even more CFTR was found in the golgi and the ER. This research suggests that pairing lumacaftor, or perhaps the triple-combo therapy Trikafta, with drugs like 7134 could help to make the CF modulators even stronger. The road to better CF treatment does not end with Trikafta. It stretches on through the development of better, more effective modulators, and to alternative therapies that may eventually meet the needs of all CF patients.

Featured Article: Goeckeler-Fried JL, Aldrin Denny R, Joshi D, et al. Improved correction of F508del-CFTR biogenesis with a folding facilitator and an inhibitor of protein ubiquitination. Bioorg Med Chem Lett. 2021;48:128243. doi:10.1016/j.bmcl.2021.128243.

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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.

The Future of Telehealth for CF: New article discusses the successes and shortcomings of telehealth appointments for people with CF over the past year and a half – and the prospects for CF telehealth care after the COVID-19 pandemic. (Journal of Cystic Fibrosis).

A Gut Feeling: Scientists examine bacteria from the gut of CF infants and find quite a different set of microbes compared to infants without CF. This difference in bacterial community structure further correlates with differences in GI metabolites important for nutrient absorption, possibly explaining why some infants with CF tend to grow more slowly. (BMC Microbiology).

Traditional Medicine as CF Therapy?: Researchers discuss the value of natural compounds to treat CF, focusing on Caffeic acid phenethyl ester (CAPE): a plant-based compound used in traditional Chinese medicine. This review explains why CAPE could be well-suited to treat CF-related inflammation. (Molecular and Cellular Biochemistry).

Emerging CF Pathogen Studied: Study describes the structure of biofilms built by Mycobacterium abscessus, an emerging pathogen causing difficult-to-treat infections in people with CF. Scientists hope that with an understanding of M. abscessus biofilm structure, they can develop better strategies to fight off the bacteria (The Cell Surface).

More CF Gene Therapy: Researchers use a class of gene therapy tools called adenine base editors to treat several CF mutations that aren’t yet approved for treatment with CF modulators. The gene therapy treatment was found to be both safe and effective on lab-grown cells (Nucleic Acids Research).

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Clinical Trial Watch: Moving CF Research Forward

The latest news on CF drug development and clinical trials.

Clinical Trial Recruiting: Roughly 10% of people with CF are still ineligible for highly effective modulator therapy. This trial is looking to close the gap. The approved modulator drugs Symdeko (Tezacaftor+Ivacaftor), Orkambi (Ivacaftor+Lumacaftor), and Ivacaftor itself will be tested on cells possessing certain rare CFTR mutations in the lab. If effective, individuals with these mutations will be eligible to receive the modulators to see if it improves their lung function. (University of Alabama at Birmingham).

Clinical Trial Recruiting: Having a hard time keeping track of CF treatments? There are so many medicines to contend with. This study aims to make life easier by testing a new hand-held pill dispenser (with associated mobile app) that spits out pills at just the right place and time. (Dosentrx Ltd).

Clinical Trial Recruiting: The CF community has been rightfully concerned about the ongoing COVID-19 pandemic. Yet the number of COVID-19 cases for people with CF has been relatively low compared to the general population, and scientists want to know why – is it just that people with CF are naturally good at social distancing, or is there some strange biology at play? This study will look at the proportion of the CF population that possesses SARS-CoV-2 antibodies, which would indicate either prior exposure to the virus or vaccination. The researchers hope to determine whether people with CF might somehow have enhanced immune protection against the coronavirus. (Medical University Innsbruck).

Upcoming Trial: This study is not for CF specifically, but is highly relevant. It will follow a group of COPD patients over several medical appointments. These individuals will wear a non-invasive acoustic sensor clipped onto their chest. The goal is to see how well the sensor can detect lung-related symptoms. If it proves to be a successful diagnostic tool, it would be worth seeing if this device could help monitor CF symptoms as well (Respira Labs, Inc. with El Camino Health and Palo Alto Medical Foundation).

*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.

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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.