On Katie Fitzgerald’s lab bench sat an archaic looking machine—the type that will likely far outlive our fanciest new scientific instruments—with tubing for various liquids that pumped out a pink bubbly liquid foam into a 6-well cell culture plate. Fitzgerald, a research technician in Dr. Matthias Stephan’s lab, explained that this was another experiment further advancing her research on an unsuspecting delivery vehicle for gene therapy, liquid foam. When Fitzgerald joined the lab just over a year ago, her mentor Dr. Stephan pitched her this wild idea for a research project on a foam with the consistency of hand soap foam for delivery of gene therapy vectors to tissues. She explained that the foam would work like “a lazy river, in the way that water squeezes between tubes.” In this analogy, the bubbles are the tubes and the low liquid content in the foam can concentrate any particles or active ingredients that had been added to the foam. Additionally, the liquid foam would stick to cells instead of just moving through. Fitzgerald admits that she initially thought, “it’s such a simple idea, someone should have tried this by now, and there’s a reason people aren’t using it.” But to her surprise, “this beautifully simple idea worked,” and it worked really well.
In a recent Nature Communications study spearheaded by Fitzgerald, the Stephan lab showcased their liquid foam gene delivery method and demonstrated how it can improve both the potency and safety of gene therapy. The first step of this project was making the foam itself. To this end, Fitzgerald tested various substances and ways to mix them to generate the perfect foam. As a researcher with a lot of cell culture experience, Fitzgerald described how in cell culture she was always focused on preventing bubbles, “but here, it’s all about bubbles!” First, Dr. Sirkka Stephan, a chemist in the lab, first developed various foam formulations using compounds that are regarded as safe by the Food and Drug Administration. The researchers settled on a foam formulation based on methylcellulose—an ingredient you’ve probably seen on labels for food, cosmetics or healthcare products due to its emulsifying, adhesive and stabilizing properties. The authors first tested the efficacy of their foam delivery system in vitro by adding a gene expressing luciferase, an enzyme found in fireflies that produces their characteristic bioluminescent glow. Specifically, the researchers used a nonviral mRNA lipid nanoparticle system, similar to Moderna’s COVID-19 mRNA vaccine. Here they demonstrated that the methylcellulose foam outperformed other top foam candidates and a non-foam, standard liquid delivery system by showing increased gene delivery using bioluminescence signal as a read out. Since human tissues are not typically sitting flat, like these cells in a dish, the research team also tested adding the lipid nanoparticles carrying the luciferase gene to cell culture plates placed at an angle, resulting in rapid drainage of the liquid added. Here, only the methylcellulose foam was able to efficiently deliver the gene vector to the cells in a dish, demonstrating that once added, the foam sticks and doesn’t move around much. This would also allow specific topical application of gene therapy vectors delivered by foam, which the researchers creatively demonstrated by using a syringe filled with the lipid nanoparticles in foam and spelling out a bioluminescent “FRED HUTCH” on the petri dish of plated cells.
The researchers next tested whether their liquid foam could effectively be used as a gene therapy vehicle in vivo. Here they tested the efficiency of gene delivery into the abdominal cavity of mice, which could be used to treat organs such as the pancreas, liver, ovaries, and gastrointestinal system. Again, using the lipid nanoparticles with luciferase mRNA to allow for bioluminescence to be used as a read out, the researchers injected these nanoparticles in liquid, or foam, into the abdominal (intraperitoneal) cavity of mice. The liquid foam again outperformed the standard liquid delivery system with over three times the transfection, or gene delivery, rate. Furthermore, liquid foam delivered homogenous gene transfer which was best demonstrated by the entire length of the small intestine being transfected. To support the use of liquid foam as a gene delivery system, the researchers then tested this by now adding lentivirus, which has been used in clinical applications of gene therapy including transducing patient-derived somatic cells ex vivo. Remarkably, liquid foam improved lentiviral mediated in situ gene transfer by over ten-fold.
In addition to demonstrating that liquid foam improves the potency of gene transfer, the researchers also took measures to ensure the safety of their foam delivery system. The foam itself proved to be safe and non-toxic to cells and animals, however it did increase the off-target viral count in the peripheral blood in the case of the lentiviral system, highlighting one potential problematic tradeoff of the increased potency this system offers. Fitzgerald highlighted that in addition to “improving potency and safety, the low cost of foam” could enable the accessibility of gene therapy treatment, which is important to consider for the practical application of this method. While Fitzgerald led this exciting discovery, she acknowledged the role of her mentor Dr. Stephan for his support and coming up with the original idea in addition to Dr. Sirkka Stephan for developing the foam formulation, and Drs. Vicky Wu and Nanxum Ma who helped with the statistical analysis. Fitzgerald also recognized Dr. Lena Schroeder, Director of Cellular Imaging, for her help when it came to the odd task of imaging bubbles. Fitzgerald is continuing to work with foam and is excited to push its clinical application such as for the treatment of diseases like esophageal cancers where treatment is limited. Here the foam could be directly applied to the esophagus and tumor region, in a way that might be as easy as drinking the liquid foam and delivering a potent therapy to the tumor with minimal off target effects.
This work was supported by Fred Hutch Immunotherapy Initiative.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member Dr. Matthias Stephan contributed to this work.
Fitzgerald K, Stephan SB, Ma N, Wu QV, Stephan MT. 2024. Liquid foam improves potency and safety of gene therapy vectors. Nature Communications.