Plasmid gene therapy may work across CF mutations, new study suggests
Approach boosted mature CFTR protein in lab tests
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Gene therapy using circular forms of DNA called plasmids may help treat lung disease in cystic fibrosis (CF), based on early lab studies, regardless of the disease-causing gene mutation, a study suggests.
Optimized plasmids boosted production of the fully mature CFTR protein, which is missing or not working properly in CF, nearly 15 times in lab-grown airway cells derived from a CF patient, compared with healthy airway cells.
This approach “may provide promising prospects for the development of a novel and effective treatment for lung cystic fibrosis,” the scientists wrote in the study, “Development of CpG-Depleted CFTR Plasmid-Based Nanoparticles for Nonviral Gene Therapy in Lung Cystic Fibrosis Disease,” published in The Journal of Gene Medicine.
CFTR mutations drive disease, limit treatment options
In CF, mutations in the CFTR gene disrupt the function or production of the CFTR protein, leading to the buildup of abnormally thick mucus that affects various organs, including the lungs.
CFTR modulators are a class of medications that have transformed the management of CF. They can improve lung function, reduce exacerbations (flare-ups), and extend survival, but they work only for patients with certain gene mutations, leaving roughly 10% of patients without effective options.
Gene therapy offers a different approach by introducing a healthy copy of the CFTR gene into diseased cells. Unlike CFTR modulators, this approach works regardless of the patient’s mutation. Preclinical research suggests that restoring just 5%–10% of normal CFTR protein or gene activity may be enough to provide clinical benefit.
One promising tool for delivering the CFTR gene is a CFTR plasmid — a small, circular piece of DNA. Plasmids are easier to manufacture, less costly, and stable during storage. Moreover, unlike some gene therapies delivered by viruses that integrate the therapeutic gene into a patient’s DNA, plasmids remain separate from the cell’s chromosomes.
Despite this potential, plasmids have two key drawbacks: their effects are temporary, and certain DNA sequences within them, called CpG sequences, can trigger an immune response.
To address these limitations, a team led by scientists at University College Dublin in Ireland first deleted the CpG sequences from a CFTR plasmid. They also incorporated a sequence called S/MAR, which is used in gene therapy to help maintain gene activity without integrating into chromosomes.
Researchers test optimized plasmids in CF airway cells
Candidate plasmids were then compacted into tiny DNA nanoparticles and combined with HPAEs, a type of biodegradable, chain-like molecule designed to help deliver genes efficiently. Each plasmid was introduced into CF bronchial epithelial cells (CFBE41o-), and CFTR protein levels were measured.
After 48 hours, one plasmid, dubbed pOP-CFTR2, which combined a CpG-free backbone with an optimized CFTR sequence to maximize protein production, achieved a 4.1-fold increase compared with a plasmid carrying the naturally occurring CFTR sequence (pWT-CFTR1).
Importantly, pOP-CFTR2 produced about 14.5 times more of the mature, fully processed CFTR protein (known as Band C) compared with healthy airway cells. Further tests showed higher overall CFTR protein levels in pOP-CFTR2 compared with all other plasmids tested.
“Given its superior CFTR expression levels, particularly in the mature Band C form, pOP-CFTR2 emerges as the most promising candidate for further investigation,” the team wrote.
The researchers then tested two promoters (hCMV and hEF1-alpha), which are DNA segments upstream of the gene that help control gene activity. After seven days, the plasmid with the hEF1-alpha promoter produced about 1.8 times more CFTR protein than hCMV.
Plasmid design boosts CFTR gene activity and protein levels
The team also measured levels of CFTR messenger RNA (mRNA), the intermediate molecule that serves as a template for protein production.
The hEF1-alpha-driven pOP-CFTR2 reached 9.7 and 13.4 times normal mRNA levels at 24 and 48 hours, respectively. In comparison, the CMV-driven version reached much higher levels of 66.4 and 46.4 times normal levels at those same time points. By day 7, however, mRNA levels for both plasmids had dropped to less than 5% of normal levels.
Despite that drop, CFTR protein levels remained meaningful across all time points for both plasmids. pOP-CFTR2-hEF1-alpha produced about 2.3 times normal CFTR protein levels at seven days, and pOP-CFTR2-CMV produced about 1.2 times normal levels at the same time point.
The researchers noted that the system achieved CFTR levels above those thought to be clinically meaningful and maintained expression for up to seven days. “The newly developed CpG-depleted CFTR plasmid demonstrated the potential for high CFTR protein expression in the CFBE41o- cell line,” the scientists concluded. “Moreover, using the HPAEs complexed to the CpG-depleted CFTR, the system indicated a 10% increase in CFTR protein expression up to 7 days after transfection, overcoming the hurdle of the transient CFTR expression in gene replacement therapies.”
