Engineers Find A Way To Selectively Activate Gene Therapies In Human Cells

A new RNAA system-based control switch could be used to trigger the production of therapeutic proteins to treat cancer or other diseases.

Researchers from MIT and Harvard University have devised a way to selectively activate gene therapies in target cells, including human cells. Their technology can detect specific messenger RNA sequences in cells, and that detection then triggers the production of a specific protein from a transgene or artificial gene.

Because transgenes can have negative and even dangerous effects when expressed in the wrong cells, the researchers wanted to find a way to reduce the off-target effects of gene therapies. One way to distinguish different types of cells is to read the RNA sequences inside, which differ from tissue to tissue.

By finding a way to produce a transgene only after having “read” specific RNA sequences inside cells, researchers developed technology that could refine gene therapies in applications ranging from regenerative medicine to the treatment of cancer. Cancer. For example, researchers could potentially create new therapies to destroy tumors by designing their system to identify cancer cells and produce a toxic protein just inside those cells, killing them in the process.

Selectively activate gene expression

Researchers at MIT and Harvard University have devised a way to selectively activate gene expression in target cells, including human cells. Their technology can detect specific mRNA sequences (shown in the center of the illustration), which triggers the production of a specific protein (bottom right). Credit: Jose-Luis Olivares, MIT, with figures from iStockphoto

“This brings new control circuits to the emerging field of RNA therapy, opening the next generation of RNA therapies that could be designed to only activate in cell or tissue specific ways,” says James Collins, professor at Termeer. Medical Engineering and Science at the Institute for Medical Engineering and Science (IMES) and the Department of Biological Engineering at MIT and lead author of the study.

This highly targeted approach, which relies on a genetic element used by viruses to control the translation of genes in host cells, could help avoid some of the side effects of therapies that affect the whole body, the researchers say.

Evan Zhao, a researcher at the Wyss Institute for Biologically Inspired Engineering at Harvard University, and Angelo Mao, a post-doctoral fellow and MIT researcher in technology at the Wyss Institute, are the lead authors of the study, which was published on October 28. 2021, in Natural biotechnology.

RNA detection

Messenger RNA molecules (mRNA) are RNA sequences that encode instructions for building a particular protein. Several years ago, Collins and his colleagues developed a way to use RNA detection as a trigger to stimulate cells to produce a specific protein in bacterial cells. This system works by introducing an RNA molecule called a “toehold,” which binds to the ribosome binding site of an mRNA molecule that codes for a specific protein. (The ribosome is where proteins are assembled according to the instructions of the mRNA.) This binding prevents mRNA from being translated into protein because it cannot attach to a ribosome.

The RNA socket also contains a sequence that can bind to a different mRNA sequence that acts as a trigger. If this target mRNA sequence is detected, the toe releases its grip and the mRNA that had been blocked is translated into protein. This mRNA can code for any gene, such as a fluorescent reporter molecule. This fluorescent signal gives researchers a way to visualize whether the target mRNA sequence has been detected.

In the new study, the researchers attempted to create a similar system that could be used in eukaryotic (non-bacterial) cells, including human cells.

Because gene translation was more complex in eukaryotic cells, the genetic components they used in bacteria could not be imported into human cells. Instead, the researchers took advantage of a system viruses use to hijack eukaryotic cells to translate their own viral genes. This system is made up of RNA molecules called internal ribosome entry sites (IRES), which can recruit ribosomes and initiate translation of RNA into proteins.

“These are complicated folds of RNA that viruses have developed to hijack ribosomes because viruses have to find a way to express the proteins,” Zhao explains.

Researchers started with the natural IRES of different types of viruses and designed them to include a sequence that binds to a trigger mRNA. When the modified IRES is inserted into a human cell in front of an exit transgene, it blocks translation of that gene unless the trigger mRNA is detected inside the cell. The trigger causes the IRES to recover and allows the gene to be translated into protein.

Targeted therapeutics

The researchers used this technique to develop tips that could detect a variety of different triggers inside human and yeast cells. First, they have shown that they can detect the mRNA encoding the viral genes of Zika virus and the SARS-CoV-2 virus. One possible application for this could be the design of T cells that detect and respond to viral mRNA during infection, the researchers say.

They also designed molecules capable of detecting mRNA from proteins naturally produced in human cells, which could help reveal cellular conditions such as stress. As an example, they have shown that they can detect the expression of heat shock proteins, which cells make when exposed to high temperatures.

Finally, the researchers showed they could identify cancer cells by creating tips that detect mRNA for tyrosinase, an enzyme that produces excess melanin in melanoma cells. This type of targeting could allow researchers to develop therapies that trigger the production of a protein that initiates cell death when cancerous proteins are detected in a cell.

“The idea is that you would be able to target any unique RNA signature and provide therapy,” says Mao. “It could be a way to limit the expression of the biomolecule to your target cells or tissues.”

The new technique represents “a conceptual quantum leap in controlling and programming the behavior of mammalian cells,” says Martin Fussenegger, professor of biotechnology and bioengineering at ETH Zurich, who was not involved in the research . “This new technology sets new standards by which human cells could be processed to detect and respond to viruses such as Zika and SARS-CoV-2. “

All of the studies performed in this article were performed in cells grown in a laboratory dish. Researchers are now working on delivery strategies that would allow RNA components of the system to reach target cells in animal models.

Reference: “RNA Sensitive Elements for Eukaryotic Translation Control” by Evan M. Zhao, Angelo S. Mao, Helena de Puig, Kehan ​​Zhang, Nathaniel D. Tippens, Xiao Tan, F. Ann Ran, Isaac Han, Peter Q. Nguyen, Emma J. Chory, Tiffany Y. Hua, Pradeep Ramesh, David B. Thompson, Crystal Yuri Oh, Eric S. Zigon, Max A. English and James J. Collins, October 28, 2021, Natural biotechnology.
DOI: 10.1038 / s41587-021-01068-2

The research was funded by BASF, the National Institutes of Health, an American Gastroenterological Association Takeda Pharmaceuticals Inflammatory Bowel Disease Research Fellowship, and the Schmidt Science Fellows program.

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