November 2021 ERCC webinar by MIT's Michael Segel

The November 2021 ERCC webinar was by Michael Segel from MIT.

This blog originated as a press release from MIT News. Thanks to them for allowing us to repost it here.

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

eToehold press releaseResearchers at MIT and Harvard University have designed a way to selectively turn on gene expression in target cells, including human cells. Their technology can detect specific mRNA sequences (represented in the center of the illustration), which triggers production of a specific protein (bottom right).
Image: Jose-Luis Olivares, MIT, with figures from iStockphoto

Researchers at MIT and Harvard University have designed a way to selectively turn on gene therapies in target cells, including human cells. Their technology can detect specific messenger RNA sequences in cells, and that detection then triggers 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 off-target effects from gene therapies. One way of distinguishing different types of cells is by reading the RNA sequences inside them, which differ from tissue to tissue.

By finding a way to produce transgene only after “reading” specific RNA sequences inside cells, the researchers developed a technology that could fine-tune gene therapies in applications ranging from regenerative medicine to cancer treatment. 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.

“This brings new control circuitry to the emerging field of RNA therapeutics, opening up the next generation of RNA therapeutics that could be designed to only turn on in a cell-specific or tissue-specific way,” says James Collins, the Termeer Professor of Medical Engineering and Science in MIT’s Institute for Medical Engineering and Science (IMES) and Department of Biological Engineering and the senior author of the study.

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

Evan Zhao, a research fellow at the Wyss Institute for Biologically Inspired Engineering at Harvard University, and Angelo Mao, an MIT postdoc and technology fellow at the Wyss Institute, are the lead authors of the study, which appeared recently in Nature Biotechnology.

RNA detection

Messenger RNA (mRNA) molecules are sequences of RNA that encode the 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 based on mRNA instructions.) This binding prevents the mRNA from being translated into protein, because it can’t attach to a ribosome.

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

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

Because gene translation is more complex in eukaryotic cells, the genetic components that they used in bacteria couldn’t be imported into human cells. Instead, the researchers took advantage of a system that viruses use to hijack eukaryotic cells to translate their own viral genes. This system consists 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 need to find some way to express protein,” Zhao says.

The researchers started with naturally occurring IRES from different types of viruses and engineered them to include a sequence that binds to a trigger mRNA. When the engineered IRES is inserted into a human cell in front of an output 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 toeholds that could detect a variety of different triggers inside human and yeast cells. First, they showed that they could detect mRNA encoding viral genes from Zika virus and the SARS-CoV-2 virus. One possible application for this could be designing T cells that detect and respond to viral mRNA during infection, the researchers say.

They also designed toehold molecules that can detect mRNA for proteins that are naturally produced in human cells, which could help to reveal cell states such as stress. As an example, they showed they could detect expression of heat shock proteins, which cells make when they are exposed to high temperatures.

Lastly, the researchers showed that they could identify cancer cells by engineering toeholds that detect mRNA for tyrosinase, an enzyme that produces excessive melanin in melanoma cells. This kind of targeting could enable researchers to develop therapies that trigger 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 deliver a therapeutic,” Mao says. “This could be a way of limiting expression of the biomolecule to your target cells or tissue.”

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

All of the studies done in this paper were performed in cells grown in a lab dish. The researchers are now working on delivery strategies that would allow the RNA components of the system to reach target cells in animal models.

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

Reference

Zhao EM, Mao AS, et al. RNA-responsive elements for eukaryotic translational control. (2021) Nat Biotechnol AOP 2021 Oct 28. doi: 10.1038/s41587-021-01068-2 PMID: 34711989.

Reprinted with permission of MIT News.

This blog originated as a press release from the Max Planck Institute for Medical Research in Heidelberg. Thanks to them for allowing us to repost it here.

Scientists create synthetic exosomes with natural functionalities and present their therapeutic application.

Scientists from the Max Planck Institute for Medical Research in Heidelberg and colleagues at the DWI Leibniz Institute for Interactive Materials in Aachen have engineered synthetic exosomes that regulate cellular signaling during wound closure. The synthetic structures are built to resemble naturally occurring extracellular vesicles (EV) that play a fundamental role in communication between cells during various processes in our bodies. The scientists uncovered key mechanisms in the regulation of wound healing and the formation of new blood vessels. They designed and built programmable fully-synthetic EVs from scratch rather than isolating natural EVs from cells. Inspired by the roles of the natural counterparts, the scientists demonstrate for the first time that fully synthetic exosomes with therapeutic functions can be constructed.

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This blog originated as a press release from the Technical University of Denmark (DTU). Thanks to them for allowing us to repost it here.

DTU Health Tech researchers have developed a method for detection of SARS-CoV-2 RNA that can be adapted to detect other diseases.

Current SARS-CoV-2 RNA detection methods recommended by the World Health Organization profoundly rely on the roles of biological enzymes. High cost, stringent transportation and storage conditions, as well as a global supply shortage of enzymes, limit large-scale testing. The result is that most countries have to prioritize testing on vulnerable cases, which creates delay in diagnostics and identification of positive cases, which again can hamper pandemic mitigation and suppression.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is still the gold standard for whole genome detection and has been playing a key role in controlling the COVID-19 pandemic. However, the sample-to-result time for qRT-PCR is several hours, and the method requires a complex thermocycler instrument to raise and lower the temperature of the reaction in discrete steps.

Simpler and less expensive

Non-enzymatic isothermal amplification methods, being simpler and faster, have shown promising potential to substitute for qRT-PCR. Although these methods perform very well when the target gene is short, they are yet to function efficiently for detection of whole viral genomes (long DNA or RNA targets).

During the COVID-19 pandemic, the Euro area alone experienced a 3.8% drop in GDP within the first quarter of 2020 (Eurostat 2020). Thus, developing a lower-cost methodology for pathogen detection would be highly beneficial for both patients and the healthcare systems aiming to battle future pandemics.

Associate Professor Yi Sun and Postdoc researcher Mohsen Mohammadniaei at DTU Health Tech have invented a one-pot assay, which they have named NISDA (Non-enzymatic Isothermal Strand Displacement and Amplification assay). The assay is for rapid detection of SARS-CoV-2 RNA without the need for the RNA reverse transcription step of the qRT-PCR methodology. Being one-pot enables a single step detection routine. The user only needs to add the sample into a single tube, place it in the instrument, and wait for 30 minutes to obtain the result.

The assay works at constant temperature, requires no enzymes, and is based on the toehold-mediated strand displacement (TMSD) approach. TMSD is an enzyme-free molecular tool from which one strand of DNA or RNA (output) is displaced by another strand (input) to form a more stable duplex structure.

High accuracy and sensitivity

The NISDA assay was able to detect a very low concentration of RNA (10 copies/µL) in only 30 minutes. In collaboration with Hvidovre Hospital and Bispebjerg Hospital, the research team clinically validated the NISDA assay, acheiving 100% specificity as well as 96.77% and 100% sensitivity when setting up in the laboratory and hospital, respectively.

Associate Professor Yi Sun elaborates, “We exploited the TMSD approach and designed three DNA probes. One probe exchanged the whole genome to a short DNA strand and the other two probes utilized the exchanged short DNA for triggering a fluorescence signal amplification cascade reaction. The beauty of NISDA assay is its simplicity. We removed the usage of enzymes to reduce the assay cost and enhance its robustness at room temperature.”

In the assay workflow, the extracted RNA from throat swab samples is added to the reaction mixture and incubated at 42°C for 30 minutes. The next step is fluorescence measurement, and samples with significantly higher fluorescence signals than that of the control samples are considered positive.

Schematic of NISDA assayThe NISDA assay comprises a single tube containing three DNA probes. After the addition of the extracted RNA from swab samples and incubation at 42°C for 30 min, positive samples show higher fluorescent signals than negative samples.

Towards a multiple disease diagnostics tool

“Being directly involved in improving people’s health is the ultimate dream of a biomedical researcher and we believe that the NISDA assay has given us this wonderful chance to attain that ambition”, Postdoctoral Researcher Mohsen Mohammadniaei says.

“The next step is to further design the NISDA assay for detecting different pathogens and develop a point-of-care diagnostic device for multiple disease diagnostics. Another advantage of the NISDA assay is its ability to be designed for short RNA targets such as cancer biomarker microRNA. We are currently exploring different schemes for the commercialization of the NISDA assay and we are certain that the NISDA assay will become widely-known in the near future”, Associate Professor Yi Sun finishes.

Reference

Mohammadniaei M et al., A non-enzymatic, isothermal strand displacement and amplification assay for rapid detection of SARS-CoV-2 RNA. (2021) Nat Comm 12: 5089. doi: 10.1038/s41467-021-25387-9 PMID: 34429424.

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