Viral vectors are efficient at transporting desired pieces of DNA into cells, and are used for, among other things, transfecting chimeric antigen receptor (CAR) genes into patient lymphocytes for CAR T cell therapy. But for some gene therapies, vectors come with “a litany of frustrations,” says Masaru Rao, a mechanical engineer at the University of California, Riverside.
Some viral vectors are limited in the size of DNA they can carry, and they integrate that DNA randomly into the genome, risking damaging mutations, Rao explains. The presence of viral particles in the body can, in certain types of gene therapies, induce an innate immune response in patients, he adds. Furthermore, the production of viral vectors, which depends on culturing cell lines, can be difficult to scale up.
“A non-viral transfection method is critical for the field,” says biomedical engineer Abraham Lee of the University of...
Most of the approaches developed so far, however, including electroporation, cell squeezing, and acoustic shearing, indiscriminately disrupt the cells’ membranes to allow the entry of genetic material, says Rao. “The number and size of holes is not well controlled,” he says. As a result, some cells are ripped apart, while others may remain intact but do not take up the DNA. There is often a trade-off between transfection efficiency and cell viability, he explains.
To avoid this problem, Rao and colleagues created a device that generates a single transient pore in each cell, allowing DNA to enter but minimizing the rate of cell death. Using microfluidic manipulations, cells in suspension are guided into individual cell-size wells that are arranged in an array at the bottom of the cell reservoir. Each well houses a single spike, which pierces the cell as it slips into the well. The fluid flow is then reversed to release the perforated cells, which are collected and incubated with the desired DNA before the membrane heals itself.
The team has optimized flow rates to maximize cell viability and tested the device with various human cell types. The researchers achieved transfection efficiencies of greater than 80 percent for a T cell line as well as T cells isolated from blood. An electroporation protocol optimized for the same T cell line, by contrast, yielded an average efficiency of around 20 percent.
The device currently pierces 10,000 cells at a time, but could be scaled up to house a larger array and could be automated for high throughput, says Rao. A typical CAR T therapeutic dose is several million to several hundred million cells. “With their fabrication technique, I believe they could [scale up],” says Lee, who was not involved in the project. “This is an elegant technology and . . . a great addition to the field.” (Nano Lett, 20:860–67, 2020)
|Non-viral transfection technique||How DNA gets in||Cell viability||Transfection efficiency||Number of cells transfected at once|
|Electroporation||A current is passed through a suspension of cells, disrupting the cell membranes.||Varies greatly depending on cell type and electrical current||Varies, but in Rao’s report, using a protocol optimized for a human T cell line, 20 percent||5 million to 10 million|
|Deterministic mechanoporation||Cells are sucked into wells and pierced by a micro-scale spike.||Close to 100 percent||For the same human T cell line, 88 percent||Currently 10,000, but could be scaled up|