Header Ads Widget

A new 'toehold' for cell therapies, RNA therapeutics and diagnostics

RNAs are best known for translating information contained in genes into proteins, which they do in a variety of ways. However, due to its structural complexity and relative stability, RNA has gained a lot of interest as a desirable biomaterial that may be utilised to develop novel sorts of medicines, synthetic diagnostics, and, of course, effective vaccinations, as the COVID-19 pandemic has shown.

Injecting a synthetic RNA molecule into a cell tells it to make a certain protein, which can subsequently be used for therapeutic, diagnostic, and other purposes. The ability to selectively allow cells that cause or are affected by a specific disease to express the protein and not others has been a major issue for researchers. This ability could drastically speed up protein creation in the body while reducing undesired side effects.

eToeholds are small versatile devices built into RNA that enable expression of a linked protein-encoding sequence only when a cell-specific or viral RNA is present, according to a team of synthetic biologists and cell engineers led by James J. Collins, Ph.D. at the Wyss Institute for Biologically Inspired Engineering and Massachusetts Institute of Technology (MIT). The use of eToehold devices opens up new possibilities for more targeted RNA therapy, in vitro cell and tissue engineering, and the detection of various biological dangers in humans and other higher species. Nature Biotechnology published the findings.

Collins' team, in collaboration with Wyss Core Faculty member Peng Yin, Ph.D., developed toehold switches for bacteria that are expressed in an off-state and respond to specific trigger RNAs by turning on the bacterial protein synthesising machinery to produce a desired protein. However, because of their more intricate architecture and protein synthesis equipment, the bacterial toehold design cannot be applied in more complex cells, such as human cells.

"In this study, we took IRES [internal ribosome entry sites] elements, which harness the eukaryotic protein translating machinery and engineered them from the ground up into versatile devices that can be programmed to sense cell or pathogen-specific trigger RNAs in human, yeast, and plant cells," Collins explained. "eToeholds could be employed as tools in basic research and synthetic biology to enable more targeted and safer RNA therapeutic and diagnostic techniques not only in humans but also in plants and other higher species."

Internal ribosome entry sites, or IRESs, are control elements found in viral RNA that enable the host cell's protein-synthesizing ribosomes access to a portion of the viral genome next to a sequence encoding a viral protein. When ribosomes latch on to RNA, they begin scanning the protein's encoding sequence while also building the protein by successively adding amino acids to its expanding end.

"We reverse-engineered IRES sequences by inserting complementary sequences that bind to each other to build inhibitory base-paired structures that block the ribosome from binding the IRES," stated Evan Zhao, Ph.D., a Postdoctoral Fellow on Collins' team. "In eToeholds, the hairpin loop-encoding sequence element coincides with certain sensor sequences that are complementary to known trigger RNAs. The hairpin loop breaks open when the trigger RNA is present and binds to its complement in eToeholds, allowing the ribosome to switch on and create the protein."

In the eToehold project, Zhao collaborated with co-first author and Wyss Technology Development Fellow Angelo Mao, Ph.D., to combine their fields of expertise in synthetic biology and cell engineering to break new ground in the manipulation of IRES sequences.

They were able to create and optimise eToeholds that were functional in human and yeast cells, as well as cell-free protein-synthesizing assays, through a rapid iteration approach. In comparison to control RNAs, they were able to induce up to 16-fold expression of fluorescent reporter genes related to eToeholds exclusively in the presence of their corresponding trigger RNAs.

"We built eToeholds that precisely recognised Zika virus infection and the presence of SARS-CoV-2 viral RNA in human cells, as well as additional eToeholds triggered by cell-specific RNAs such as an RNA that is only expressed in skin melanocytes," Mao explained. "Importantly, eToeholds and the sequences encoding desired proteins attached to them may be encoded in more stable DNA molecules, which are then transformed into RNA molecules that are customised to the type of protein expression we seek when introduced into cells. This increases the number of target cells that eToehold can reach."

The researchers believe that their eToehold technology could assist focus RNA and gene therapies to certain cell types, which is crucial because many of these therapies are impeded by off-target toxicities. Ex vivo differentiation techniques that guide stem cells through developmental paths to generate specific cell types for cell treatments and other uses could also be facilitated. eToeholds could aid in enriching desired cell types by converting stem cells and intermediate cells along several differentiating cell lineages.

"This study demonstrates how Jim Collins and his team on the Wyss Living Cellular Device platform are developing innovative tools that can advance the development of more specific, safe, and effective RNA and cellular therapies, and thus positively impact the lives of many patients," said Wyss Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children's Hospital, and Professor of Molecular Medicine at Harvard Medical School

Post a Comment