A New Delivery System Offers Hope for Cystic Fibrosis

From its first description in 1935 until now, clinical outcomes for patients with cystic fibrosis have undergone a dramatic transformation. While the rare genetic disease was once an early death sentence that often prevented patients from reaching adulthood, advancements in cystic fibrosis therapeutics have greatly extended the lifespans of those suffering from the disease over the years.¹ However, these therapies are not effective in all patients.

In a study published in Nature Communications, scientists reported a novel strategy to deliver the CRISPR-Cas9 gene-editing system into the lungs of a cystic fibrosis mouse model and correct the underlying mutations.² Once developed and tested, this approach could allow clinicians to treat every patient with cystic fibrosis, including those who were previously untreatable.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel on the cell surface that helps control the concentration of salt and water within bodily secretions. Impaired or absent CFTR activity leads to the characteristic thick and sticky mucus associated with the disease and consequently, an increase in the frequency of respiratory infections. Clinicians have developed small molecule drugs, such as Trikafta, that effectively treat 90 percent of patients.³

“The problem is that these drugs are only for symptom management,” said Yehui Sun, a graduate student in Daniel Siegwart’s laboratory at the University of Texas Southwestern Medical Center and author of this study. “[These drugs cannot] cure the root of the disease because it is a genetic disease.” The patient’s cells must produce the CFTR protein for the existing therapies to work, which leaves patients with nonsense mutations without options.

Since its initial discovery, researchers believed that they could use CRISPR-Cas9 to help cure genetic diseases. However, the lack of effective delivery carriers that could target specific organs held back this approach. To solve this problem, Siegwart and his team previously developed an advanced delivery strategy called selective organ targeting lipid nanoparticles (SORT LNP).⁴ By modifying the composition and biophysical properties of these nanoparticles, the researchers selectively targeted cells in the lungs, livers, or brains of mice after intravenous administration.

Building on this work, Sun, Siegwart, and their team further optimized the formulation of their lung SORT LNP and improved its delivery, efficacy, and lung-targeting specificity, while producing minimum toxicity. After encapsulating the CRISPR system components including Cas9 mRNA, mutation-specific single guide RNA, and donor single stranded DNA template, they injected the lipid nanoparticles intravenously into a cystic fibrosis mouse model harboring the nonsense mutation, G542X. Through next-generation sequencing (NGS) of DNA extracted from its lung tissue, the researchers determined that their gene editing strategy successfully corrected the G542X mutation in murine lungs.

While these results were promising, the researchers were unsure if their system could directly reach basal cells, which are resident stem cells in the respiratory epithelium. Because these cells can differentiate into various epithelial cell types, effective CFTR repair in basal cells could potentially lead to long-term improvements in lung function. By treating a transgenic reporter mouse strain with lung SORT LNP carrying Cre mRNA and evaluating the fluorescence of isolated basal cells using flow cytometry, they observed that their lipid nanoparticles could efficiently deliver mRNA to basal cells in vivo.

Efficient delivery of these [genome] editors to target the areas that we want it to work has always been an issue.
-Amy Wong, The Hospital for Sick Children

To test if their approach could also improve CFTR function, Sun and her colleagues grew patient-derived human bronchial epithelial cells expressing the most common mutation in CFTR, F508del, onto a permeable membrane and treated the monolayer with lung SORT LNP. They then evaluated changes in the secretion of chloride ions by applying an electrical current across the epithelial layer and measuring the transepithelial resistance and voltage, as well as the gene editing efficiency through NGS. Although they found that their system repaired the mutation in only four percent of the cells, this correction rate was sufficient to restore the chloride channel’s activity to 71 percent of healthy CFTR function.

“Efficient delivery of these [genome] editors to target the areas that we want it to work has always been an issue,” said Amy Wong, a developmental and stem cell biologist at The Hospital for Sick Children, who was not involved in the study. “Lipid nanoparticles really address the question of delivery.” Wong was particularly excited that the lung SORT LNP reached the basal cells in the lung epithelium. “If you target these stem cells, then you will have a source of cells that can just renew the epithelium and will have the correct CFTR on the plasma membrane,” Wong explained.

Besides treating additional lung diseases, this SORT LNP delivery strategy holds much potential to provide targeted gene editing to cells in other areas of the body. “Cystic fibrosis is not a disease that only affects airways; it affects multiple tissues,” Wong said. “Their intravenous delivery method…could potentially be an avenue to target other organs as well, such as the pancreas [or] the liver.”