Synthetic gene circuits that improve the quality of stem cells

Because iPS cells can be made into just about any type of cell in the body, they hold great promise for cell therapy. A major problem, however, is that not all reprogramming cells successfully become iPS cells, resulting in unwanted cell mixing. In addition, when iPS cells differentiate, some cells are only partially differentiated, again leaving a mixture of unwanted cells. To extract the desired cell types, CiRA researchers report in Scientists progress new synthetic RNA technology composed of ON and OFF switches. These switches control specific genes to kill contaminating cells while leaving desired cells unharmed at rates above standard techniques.

“The standard method of cell purification is to use fluorescence activated cell sorting or magnet activated cell sorting,” said Assistant Professor CiRA Yoshihiko Fujita, one of the study’s authors. “But antibodies are needed, which makes these approaches expensive and the process damages many cells.”

The Hirohide Saito laboratory, of which Fujita is a member, has therefore developed synthetic RNAs which they call “RNA switches” as an alternative. Unlike antibodies, which react with proteins on the cell surface, the RNA switch binds to miRNA naturally expressed inside the cell.

The main function of miRNA is to prevent the translation of RNA into proteins. So the RNA switch, when transfected into a cell, will translate proteins as long as it is not bound to miRNA.

There are several thousand types of miRNA in a cell, but the RNA switch comprises a sequence complementary to a specific miRNA. Therefore, by designing the RNA switch to translate a protein that causes cell death, only cells that strongly express miRNA that binds to the complementary sequence will survive.

In this system, all RNA switches function as OFF switches, in that the presence of high miRNA turns off the switch. These switches can be used to purify cells, but run into problems if done on a large scale, Saito explained.

“There are two problems. First, the amount of RNA switch transfected will influence the purity. Second, there is leakage, which means that some unwanted cells will survive or some wanted cells will perish,” he said. declared.

As a solution, his research team prepared RNA switches that activate when linked by miRNAs. To do this, it took some experimentation on the structure of synthetic RNA that makes up the RNA switch.

In human cells, the translated RNA comprises a poly (A) tail modification. The miRNA ON switch differs from the OFF switch by placing the complementary sequence after this tail rather than before.

“We added the complementary sequence and additional sequence after the poly (A) tail. The additional sequence prevents translation of RNA. When miRNA binds to the sequence, it should cleave the sequence after the poly (A) tail. ), allowing the protein to be translated, ”explained Fujita.

Transfection of cells with the miRNA ON and OFF switches reduced leakage and quantity problems.

“Usually the efficiency of transfection depends on cell type, number of cells, etc. By combining ON and OFF switches, we do not have to worry so much about tightly controlling the amount of RNA for the cell. transfection, ”Saito said.

Saito and his colleagues tested this strategy by designing the RNA ON switch to translate Barnase, a ribonuclease that degrades RNA and also kills cells, and the RNA OFF switch to translate Barstar, a Barnase inhibitor, to eliminate any Barnase expression leak.

In addition, RNA switches were designed to bind to miR-302a-5p, a miRNA strongly expressed in iPS cells, or miR-208a-3p or miR-1-3p, two miRNAs strongly expressed in cardiomyocytes. After transfected cells with these switches, the researchers were able to purify iPS cells over 99% and cardiomyocytes over 95%, numbers that exceed the purity levels achieved with standard methods.

Additionally, given that RNA molecules are relatively safe and that in principle RNA switches can be designed to interact with any type of miRNA, Saito is optimistic about the promise of this technology for research. clinical.

“Our method is applicable to a wide range of cell types which can then be used to study diseases, drugs and cell therapies,” he said.