Gene-infused nanoparticles used for treating disease work better when they include plant-based relatives of cholesterol because their shape and structure help the genes get where they need to be inside cells, according to scientists from Oregon State University (OSU). The team, which published its study “Plant-based relatives of cholesterol could give boost to gene therapy” in Nature Communications, says its findings are important because many illnesses that can’t be treated effectively with conventional drugs can be treated genetically, i.e., by delivering nucleic acids to diseased cells so they can make the correct proteins needed for health.
Gaurav Sahay, PhD, assistant professor of pharmaceutical sciences in the OSU College of Pharmacy, studies lipid-based nanoparticles as a gene delivery vehicle, with a focus on cystic fibrosis. One faulty gene, the cystic fibrosis transmembrane conductance regulator, or CFTR, causes the disease, which is characterized by lung dehydration and mucus buildup that blocks the airway. Two years ago, Sahay and other scientists and clinicians at OSU and Oregon Health & Science University (OHSU) demonstrated proof-of-concept for a new cystic fibrosis therapy: loading chemically modified CFTR messenger RNA into lipid-based nanoparticles, creating molecular medicine that could simply be inhaled at home.
The mRNA-loaded nanoparticle approach causes cells to make the correct protein, allowing cells to properly regulate chloride and water transport, which is critical to healthy respiratory function. Cholesterol is thought to provide stability in these gene nanocarriers. In the latest study, Sahay and collaborators boosted gene delivery by using plant-based analogs of cholesterol instead. Another plus of these plant-derived sterols is a cardiovascular health benefit, he adds. The type of nanoparticle used to deliver genes in this study has already been clinically approved; it’s being used in a drug, trade-named Onpattro®, given to patients with a progressive genetic condition called amyloidosis, which disrupts organ function through harmful deposits of the amyloid protein.
Sahay and graduate student Siddharth Patel, first author on the study, found that phytosterols (plant-based molecules chemically similar to cholesterol) change the shape of the nanoparticles from spherical to polyhedral and cause them to move faster. That’s important because once inside a cell, the nanoparticles need maneuverability for the escape they need to make: from a cell compartment known as an endosome into the cytosol, where the delivered genes can perform their intended function.
“Endosomal sequestration of lipid-based nanoparticles (LNPs) remains a formidable barrier to delivery. Herein, structure-activity analysis of cholesterol analogues reveals that incorporation of C-24 alkyl phytosterols into LNPs (eLNPs) enhances gene transfection and the length of alkyl tail, flexibility of sterol ring and polarity due to -OH group is required to maintain high transfection,” write the investigators in the Nature Communications paper.
“Cryo-TEM displays a polyhedral shape for eLNPs compared to spherical LNPs, while x-ray scattering shows little disparity in internal structure. eLNPs exhibit higher cellular uptake and retention, potentially leading to a steady release from the endosomes over time. 3D single-particle tracking shows enhanced intracellular diffusivity of eLNPs relative to LNPs, suggesting eLNP traffic to productive pathways for escape. Our findings show the importance of cholesterol in subcellular transport of LNPs carrying mRNA and emphasize the need for greater insights into surface composition and structural properties of nanoparticles, and their subcellular interactions which enable designs to improve endosomal escape.”
“One of the biggest challenges in the delivery of genes is that less than 2% of the nanoparticles reach the cytosol,” said Sahay, who also holds an adjunct faculty position with OHSU. “If you up the dose to get more genes there, now you have problems with toxicity, plus the cost goes higher. But the nanoparticles’ shape changes because of these naturally occurring cholesterol analogs, and the new shape helps them deliver genes better. The analogs boost gene delivery 10-fold and sometimes 200-fold.”
The finding can be used to make inhalable particles that can cross several barriers in the lung in a cystic fibrosis patient, enabling patients to be treated with much higher efficacy, Sahay added.
“In this latest research, we hypothesized that with the analog inclusions, there would be shape changes and changes with how the nanoparticles interact with the cell and how the cell perceives them,” Patel said. “For instance, the sterols might help them get to the ribosomes for translation faster. This opens up a whole new area of research—the shape and structure and composition of the liquid nanoparticles now become quite relevant. We’re just scratching the surface on the way to building LNPs with a rational design to get different properties for treating different diseases with cell-type specificity.”