Drugs that restore the shape of the errant protein behind cystic fibrosis (CF) have, over the past eight years, helped the majority of patients, who have certain mutations. Gene-corrected stem cells might offer a “mutation agnostic” option to CF.
CF results from a glitch in a glycoprotein with the unwieldy name “cystic fibrosis transmembrane conductance regulator”, or CFTR. The proteins normally fold into channels that regulate the flow of ions into and out of cells, controlling the balance of water and salts in linings and barriers of the respiratory tract, pancreas, intestines, and elsewhere. If the proteins can’t fold correctly, or can’t migrate to the cell’s surface and then open and stay that way, the resulting ion imbalance allows too much water into lining cells and secretions thicken. CF symptoms ensue, such as difficulty breathing and digesting. The Cystic Fibrosis Foundation has a helpful video (see below) both on why CF develops and the promise of gene-editing.
The most common CF mutation, F508del, removes just one of the protein’s 1,480 amino acids (a phenylalanine), and that’s enough to wreck the ion channels. Ninety percent of patients have at least one F508del variant. Researchers have identified more than 2,000 variants in the CFTR gene, about 350 of which are pathogenic.
A Brief History of Cystic Fibrosis Treatments
Historically, CF treatments tackled symptoms: pounding on the back to break up mucus, vibrating vests, sprinkling digestive enzymes on applesauce, fighting inflammation and infection.
Discovery of the CFTR gene in 1989, by Lap-Chee Tsui, Francis Collins, and their colleagues, revealed the molecular defect. A patent application filed a week before the two papers in Science reporting the discovery were published proposed gene therapy. It would introduce a functional CFTR gene into epithelial cells in general, and airway epithelial cells specifically, to counter the predominant symptoms.
The patent language was either purposely general, or perhaps the complex histological landscape of airway epithelium and turnover rates of the constituent cell types weren’t yet appreciated, because attempts at gene therapy hit roadblocks starting in the early 1990s. Effects were absent or transient, or the gene therapy induced inflammation or injury.
Gene therapy for CF hasn’t been abandoned, however. ClinicalTrials.gov lists 49 studies. But another sector of the biotech toolbox has found a better way: small molecules that interact with CFTR protein.
FDA approved the first targeted drug, Kalydeco (ivacaftor), in 2012, for the 5 percent of patients with a certain mutation, G551D. Their CFTR proteins lodge at the cell surface, stuck closed like a clogged drinking straw.
In 2018 adding a second drug broadened the patient pool to include those with two copies of F508del. The dual drug unfurls and refolds the ion channels correctly so that they stay at the cell membrane long enough to work.
Then in 2019 came yet a third drug, which adds patients who have one copy of F508del plus another mutation. It enables cells to override mutations that shorten the ion channel protein.
But the trio of small molecule drugs doesn’t help 7 percent of patients.
Enter Stem Cells
An excellent review article in Human Gene Therapy, “Correction of Airway Stem Cells: Genome Editing Approaches for the Treatment of Cystic Fibrosis,” from Nicholas E. King and Brian R. Davis at the Center for Stem Cell and Regenerative Medicine, University of Texas Health Science Center and colleagues, suggests that the years of gene therapy attempts may have underestimated the complexity of the proximal airway lining.
Today single-cell RNA sequencing (scRNA-Seq) is describing the functions of the different cell types of the airways to complement analyses of the distribution and abundance of CFTR glycoproteins lodged in their cell membranes. At this level of scrutiny, the upper airway is not a homogeneous sheet of seeping cells like rolled up bathroom tile, but a dynamic surface with a complex topography.
Their paper is more than a review, for it is also a look ahead, sprinkled with “would” and “potential,” and “future.” But the idea to deploy patients’ stem cells with corrected CFTR genes makes sense for all patients.
A Complex Cellscape
RNA-Seq reveals 4 cell types in the proximal airway lining, identified in mice and in human lung organoids called bronchospheres.
Basal stem/progenitor cells hug basement membranes and so don’t face the airway lumen. Experiments that seed basal cells onto denuded rat tracheas and transplanting them into the flanks of mice revealed their fates. The cells self-renew and give rise to ciliated cells, secretory cells, and ion-pumping cells called ionocytes. A few CFTR channels dot the secretory cells; they’re most abundant on the ionocytes, but these cells are sparse.
Basal cells seem the “preferred target cells for long-term efficacious CFTR editing,” the team writes. But RNA-Seq has also captured diversity among the basal cells. Distinct gene expression patterns drive basal cells in the throes of cell division, differentiating into other airway cell types, or adhering to other cells. A challenge is to identify and isolate the true stem cells, not the ones already committed to the more restricted fate of a progenitor cell.
Stem Cells Provide Continuity; Gene Editing, Precision
Swapping in a functioning CFTR gene could work for any mutation. It could replace the missing amino acid of the F508del patients, as well as trade places with more elaborate alterations, like splice site mutations or extensive deletions. Gene editing is like writing over whited-out print, for those who remember white-out.
Gene therapy adds a gene. It delivers a gene to an episome (a loop of DNA outside a chromosome) or integrates it randomly into a chromosome. In contrast, gene editing fixes a mutant gene.
Editing of CFTR has been done using zinc finger nucleases (just the gene) in 2012 and CRISPR/Cas9 (in organoids) in 2013.
A 2020 paper in Cell Stem Cell describes editing F508del in basal stem cells from the upper airway and bronchi of ten CF patients. CFTR functioned in 20% to 50% of the cells. The researchers embedded the corrected upper airway stem cells in membrane from a pig’s small intestine to show that the cells maintain their stemness and the ability to spawn specialized daughter cells. “This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF,” they conclude.
Variations on the Autologous Theme for Cystic Fibrosis
King, Davis, and co-workers describe several routes to correcting CFTR mutations: primary cells, induced pluripotent stem (iPS) cells, and in vivo correction.
Primary Cells Airway basal stem cells can be collected from the nose or with a bronchoscope and expanded in culture. These primary cells are of two types: some that cease dividing quickly, and others that do so up to 50 times, which are the better target. Tweaking culture conditions to maximize cell division and learning how to recognize those cells that will maintain stemness through the most doublings are priorities. Then the cells must be tested to see that they do what they’re intended to do – create normal airway lining – without turning cancerous.
iPS Cells In a second strategy, iPS cells made from skin fibroblasts or white blood cells are reprogrammed to recapitulate lung development from endoderm and specialize into airway basal stem cells. After ex vivocorrection of the CFTR mutation and perhaps a tweak to provide a transient selective advantage, the cells would be transplanted back into the patient. A 2015 paper describes this approach.
Compared to primary cells iPS cells divide indefinitely, but present risks and challenges.
Will they veer into a teratoma or cancerous state, or incompletely differentiate due to skewed epigenetic regulation or off-target effects during reprogramming?
How many corrected iPS cells are required to restore airway function and where should they be placed amid the environs of “tight junctions and ceiling of mucus,” as the review paper puts it?
Will “pre-conditioning” be necessary, like in a bone marrow transplant, to make room for the introduced cells?
How will the corrected stem cells fit in with resident uncorrected stem cells in the airway?
In vivo Correction This third strategy is farther out. Researchers need to learn more about the choreography of the turnover of cell types in the airway lining, and what might happen if cells other than the stem cells are corrected.
The bottom line: Gene-edited basal stem cells, no matter how they are delivered, could provide a “one time, long-term functional ‘cure’ for all those affected by CF, irrespective of the particular mutation,” King and colleagues conclude.