Molecule may help deliver gene therapies to CF lungs: Study
Thick mucus has been a challenge to delivery, researchers note
Researchers discovered a molecule that may help deliver gene therapies to cells in the thick mucus environment of the cystic fibrosis (CF) lung, a study reported.
Since thick mucus makes gene delivery to the CF lung a significant challenge, “we are encouraged by these results and anticipate that our [molecule] could be used to successfully deliver cystic fibrosis gene therapies in future work,” the authors wrote. The study, “Discovery of peptides for ligand-mediated delivery of mRNA lipid nanoparticles to cystic fibrosis lung epithelia,” was published in Molecular Therapy Nucleic Acids.
In CF, mutations in the CFTR gene lead to no or faulty protein of the same name, which causes the build-up of thick mucus in the lungs and other organs, and the onset of various symptoms. CFTR modulators are a class of therapies designed to improve the functionality of the faulty protein but are only effective in patients with certain CFTR mutations.
Gene therapies represent a potential CF treatment option in which a healthy copy of the CFTR gene is delivered to lung cells to restore the production of working CFTR protein. Such therapies have the potential to work regardless of the CF-causing mutation.
However, the excess thick mucus and penetrating the cell membrane may prevent gene therapies, delivered using harmless viruses or lipid nanoparticles (LNPs), from reaching target cells harboring CFTR mutations. “Given that the thick mucus layer and cell membrane are two of the biggest barriers to successful gene delivery, it is necessary to develop gene-therapy carriers that can overcome these obstacles,” the researchers wrote.
Peptides to penetrate mucus
Scientists at the University of Texas at Austin searched for peptides — chains of amino acids, the building blocks of proteins — that, when incorporated into LNPs, would help them penetrate the mucus in CF lungs.
First, the team inserted a library of genes encoding a wide variety of peptides into a phage, a virus that infects bacteria. Phages were screened against human bronchial epithelial cells (pHBECs), or those that line the airways, from CF patients to identify peptides that help phages penetrate the mucus-rich air-surface interface.
After several rounds of screening, the top 30 phage peptides were analyzed and found to carry a positive charge and readily dissolve in water. The top peptide candidate, dubbed peptide C-LNP, which was significantly better at phage uptake than controls, was successfully incorporated into LNPs.
Next, the team tested whether peptide C-LNP could deliver gene therapy to cells using messenger RNA (mRNA), an intermediary molecule that carries genetic instructions from a gene to the protein-production machinery.
They chose mRNA that coded for luciferase, the enzyme from fireflies that produces light, so cells that successfully incorporated the mRNA would light up. As a comparison, the researchers used LNPs from Spikevax, Moderna’s approved COVID-19 mRNA vaccine. LNPs were then incubated with pHBECs from seven different CF donors.
Results showed that peptide C-LNP was 7.8 times better than the Spikevax LNP at incorporating the luciferase mRNA into pHBECs pooled from all donors. While there was some variability when tested against pHBECs from individual CF patients, all were better than Spikevax. Conversely, peptide C-LNP was 1.7 times worse than the Spikevax LNP at incorporating mRNA into cells derived from human immune macrophages.
LNPs were then administered directly into the windpipe of healthy mice. Like the cell-based experiments, peptide C-LNP was 3.4 times better than the Spikevax LNP at incorporating the luciferase mRNA into the lungs. Moreover, mRNA activity was significantly higher in the lungs versus all other organs, including the heart, brain, kidneys, liver, and digestive tract.
The team assessed whether peptide C-LNP could help edit genes in mice. They chose an mRNA for an enzyme that selectively cuts DNA in modified mice and triggers the production of a red protein. After windpipe administration, peptide C-LNP was 6.3 times better at cutting DNA in cells that lined the airways than immune cells and nearly 30 times better than cells that lined blood vessels. No differences in toxicity were observed between treated and control mice.
“While gene therapy for CF has been a significant challenge due to poor delivery efficacy to the lungs, we are encouraged by our results presented here and see potential for our mucus and cell penetrating targeting peptide to overcome this delivery barrier,” the researchers wrote.
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