For years, researchers have attempted to harness the full potential of gene therapy, a technique that inserts genes into a patient’s cells to treat aggressive diseases such as cancer. But getting engineered DNA molecules into cells is not an easy task.
Operation of the acoustic shear poration (ASP) device in Meacham’s lab. (Courtesy Photo)
J. Mark Meacham, assistant professor of mechanical engineering & materials science at Washington University in St. Louis, leads a team of researchers that has developed a method enabling effective insertion of large molecules — such as DNA, RNA and proteins — into cells and propels them into the cell nucleus. By combining a technique known as Acoustic Shear Poration (ASP) with electrophoresis, the approach uses ultrasound waves and focused mechanical force to create nanoscale holes, or pores, in the cell membrane that are big enough for large macromolecules or nanoparticles to pass into the cell’s interior.
The researchers wrote that so far, ASP has achieved greater than 75 percent delivery efficiency of macromolecules. DNA insertion, or transfection, which is of most interest in gene therapy, is significantly more challenging. Yet the combined application of mechanical and electrical forces pioneered by Meacham and colleagues yields roughly 100 percent improvement in transfection versus pure mechanoporation. Results of the research are published in Scientific Reports.
“We have demonstrated our poration technique using cancer cell lines and patient-derived, primary monocytes, which is an important achievement, but the end goal is to use the new combined method to successfully modify T cells from a patient’s immune system,” Meacham said. “We would take cells extracted from a patient, run them through our device and modify them, then they would be reintroduced to the patient. That’s the Holy Grail of personalized medicine and emerging gene therapies.”
Meacham’s work comes on the heels of an approval in August by the U.S. Food and Drug Administration of the first gene therapy using patient-derived immune cells in the U.S. A second such therapy was approved by the FDA in October. The new immunotherapies, known as CAR-T cell therapies, involve inserting a gene into patients’ own immune cells, which helps those cells home in on and attack cancer cells. The therapies are approved to treat pediatric patients up to age 25 with a form of acute lymphoblastic leukemia (ALL) and adults with certain types of advanced non-Hodgkin lymphoma. Washington University oncologists provide the therapies to patients through Siteman Kids at St. Louis Children’s Hospital and at Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine.
The extreme difficulty associated with delivery of genes into cells for use in these gene therapies has motivated pursuit of effective transfection methods, Meacham said.
“Non-chemical, non-viral approaches, which take advantage of mechanical or electrical stimuli to overcome cellular barriers to gene transfer, are compelling for many reasons, including safety, cost and the potential (or lack thereof) for large-scale manufacturing of therapeutic cells,” he said. “For such methods to be successful, pores must be created in the cell membrane that are large enough and stay open long enough to insert molecules while not damaging or killing the cell and delivering the molecule to the cell’s nucleus where it can work.”
Meacham describes his system, which evolved from a technology he first developed while a doctoral student at Georgia Institute of Technology, as an acoustically-driven droplet generator.
“We apply focused mechanical forces, such as a fluid shear, which are pushing and pulling on the cell using fluid motion,” Meacham said. “We take a suspension of cells and use an ultrasonic field to pump fluid through microscopic constrictions in the tips of the nozzles, or acoustic horns. This results in a kind of spray. The cells that are suspended in the flow experience intense mechanical stimulation as they travel through this confined space and are ejected from the nozzle orifices.”
Experimental results suggest that the device generates pores in the cell membrane of 100 to 150 nanometers, which allow for delivery of even large payloads into the cell. In addition, the pores remain open for up to a minute, which is enough time to deliver molecules to the cell. The technique can be used with almost any cell type and suspension medium, as well as with most biomolecules and nanomaterials, Meacham said.
The device’s unique actuation mechanism allows researchers to explore a much larger range of shear rates and electrical stimulation for cell-specific optimization of treatment parameters, he said.
“We found that the acoustic pressure field and local shear stresses acting on the cell due to the fluid flow are critical to creating pores in the cell membrane,” Meacham said. “The most efficient membrane poration was observed after the cell was exposed to the short duration, high-shear environment at the nozzle constriction as the cell is forced through the nozzles of our device.”
Meacham JM, Durvasula K, Degertekin FL, Fedorov AG. Enhanced intracellular delivery via coordinated acoustically driven shear mechanoporation and electrophoretic insertion. Scientific Reports.
This research was supported by the National Institutes of Health (R44 GM103448 and R44 GM112398) and the National Science Foundation (IIP-1321466). Meacham was principal investigator on two of the grants awarded to OpenCell Technologies Inc., in which he has a financial interest. Research performed under R44 GM112398 as a subcontract to Meacham took place at Washington University in St. Louis. Open Cell Technologies Inc. has exclusive license or ownership of eight issued and two pending patents on the device and method.
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