Create a new "foothold" for RNA therapy, cell therapy and diagnostics

Delivering a synthetic RNA molecule into the cell essentially instructs the cell to produce the proteins it needs, which can then perform therapeutic, diagnostic, and other functions
.

A key challenge for researchers is to allow only cells that cause or are affected by specific diseases to express this protein, and not other cells

Now, a team of synthetic biologists and cell engineers led by Dr.
James J.
Collins of the Wyss Institute of Bioengineering and the Massachusetts Institute of Technology (MIT) has developed eeehold, a small multi-functional DNA built into RNA.
The functional device can express the linked protein coding sequence only when there is cell-specific or viral RNA
.

The eehold device provides multiple opportunities for more targeted types of RNA therapy, in vitro cell and tissue engineering methods, and the perception of diverse biological threats to humans and other higher organisms

In 2014, Collins' team collaborated with Dr.
Peng Yin, a member of Wyss Core, and successfully developed a bacterial "foot switch" that is expressed in the off state and turns on the synthesis of required proteins through the bacterial protein synthesis mechanism in response to specific triggers.
rna
.

However, bacterial footpoint design cannot be used for more complex cells, including human cells with more complex structures and protein synthesis devices

"In this research, we put the IRES (internal ribosome entry site) element, a common virus in a certain control element, using the eukaryotic protein translation machine, and planning them from the ground to the multifunctional device, which can plan to feel Cell or pathogen-specific triggers RNA in human, yeast, and plant cells," Collins said
.

"Eehold can make more specific and safer RNA therapy and diagnostic methods not only in humans, but also in plants and other higher organisms, and be used as tools for basic research and synthetic biology

Control elements are called "internal ribosome entry sites", or IRESs for short.
They are sequences found in viral RNA that allow the protein synthesis of host cells to synthesize ribosomes close to a segment of the viral genome that is next to the viral protein.
Sequence
.

Once attached to the RNA, the ribosome begins to scan the protein's coding sequence and at the same time synthesizes the protein by sequentially adding the corresponding amino acids to its growing end

"We introduced complementary sequences to combine with each other to form an inhibitory base-pair structure, so as to forward engineer IRES sequences to prevent ribosomes from binding to IRES," said co-first author Dr.
Evan Zhao, a postdoctoral researcher in the Collins team
.

"The hairpin loop coding sequence element in eToeholds is designed to overlap with a specific sensor sequence that is known to trigger RNA complementation

Zhao and Wyss technology development researcher Dr.
Angelo Mao collaborated in the eehold project, which allowed them to combine their respective expertise in the fields of synthetic biology and cell engineering to achieve new breakthroughs in the operation of IRES sequences
.

In the process of rapid iteration, they were able to design and optimize etoe that functions in human and yeast cells, as well as cell-free protein synthesis assays
.

Compared with the control RNA, they achieved a 16-fold induction of the fluorescent reporter gene linked to eeehold only in the presence of an appropriate triggering RNA

Professor Mao said: "The eehold we designed can specifically detect Zika virus infection and the presence of SARS-CoV-2 viral RNA in human cells, as well as other eehold triggered by cell-specific RNA, such as a type of melanocytes that only affect the skin.
expression of RNA
.

"" importantly, the desired protein sequence can be encoded in a more stable and eeehold encoding DNA molecule, when introduced into a cell, the DNA molecules are converted into an RNA molecule, it is our expectation protein The expression type is tailor-made

The researchers believe that their eehold platform can help targeted RNA therapies and some gene therapies for specific cell types, which is important because many such treatments are hampered by excessive off-target toxicity
.
In addition, it can promote in vitro differentiation methods and guide stem cells to generate specific cell types along the developmental path for cell therapy and other applications
.
Transformation of stem cells and intermediate cells along many differentiated cell lines is often not very effective, and eehold can help enrich the required cell types
.

"This research highlights that Jim Collins and his team are developing innovative tools on the Wyss live cell device platform, which can advance the development of more specific, safe, and effective RNA and cell therapies, which will have a positive impact on the lives of many patients.
Impact,” said Dr.
Donald Ingber, the founding director of Wyss, who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and the Professor of Bioengineering at the Harvard John A.
Paulson School of Engineering and Applied Sciences
.

The other authors of the study are Helena de Puig, PhD, Kehan ​​Zhang, PhD, Nathaniel Tippens, PhD, Little Tan, MD, F.
Anpao, PhD, Wyss Research Assistant Isahan, Dr.
Peter Ruan, Emma Dr.
Chory, Tiffany Hua, Dr.
Pradeep Ramesh, Wyss scientist Dr.
David Thompson, Crystal Yuri Oh, and Max Eric Zigon English
.
This research was funded by BASF, the National Institutes of Health (at #RC2 DK120535-01A1), and the Weiss Institute of Bio-Inspired Engineering
.

Journal Reference :

1. 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, James J.
   Collins.
   RNA-responsive elements for eukaryotic translational control .
   Nature Biotechnology , 2021; DOI: 10.
   1038/s41587-021-01068-2 332.
   htm (accessed October 28, 2021).