Researchers have discovered a new type of RNA that inhibits a wide range of viral infections

RNA is often described as the single-stranded cousin of DNA, the double-stranded molecule that makes up the genomes of all living organisms. Many types of non-living viruses, however, carry their genetic information in the form of double-stranded RNA (dsRNA). When the human immune system detects the presence of this foreign RNA, it sounds the alarm by increasing the production of protective cytokines called interferons (IFNs), which activate the natural immune response against viral invaders.

Scientists have attempted to harness this natural antiviral response by creating therapeutic dsRNAs that mimic features of viral genomes. But interfering with the innate immune system is a double-edged sword. Analogues of viral RNA can also activate molecular pathways that lead to excessive inflammation in the body, which can do more harm than their antiviral benefits.

Now, a team of researchers at the Wyss Institute for Biological Engineering at Harvard University has discovered a new class of immunostimulatory dsRNAs that potently induce the production of two forms of IFN (IFN-I and IFN-III) while limiting the inflammation normally seen. with previous types of RNA-based immunostimulants.

New dsRNA molecules have dramatically disrupted the ability of many viruses with pandemic potential, including SARS-CoV, SARS-CoV-2, MERS-CoV, and several strains of influenza A, to infect human cells in both traditional cell culture and sophisticated lung organ models a person In a mouse model of COVID-19, dsRNA reduced the amount of virus in the animals by more than 1,000-fold. The results were published today in Molecular therapy – nucleic acids.

“These new dsRNAs are an attractive option for the treatment of COVID-19 because SARS-CoV-2 infection induces an unbalanced immune response in which the protective IFN response is suppressed and the inflammatory response is enhanced. By preferentially increasing IFN-I, our dsRNA has the potential to correct this imbalance and could also be used to treat many other viral diseases,” said co-author Haiqing Bai, Ph.D.

Random discovery, systematic search

For years, Bai and other members of Wyss founding director Don Ingber’s lab have been developing new treatments for viral diseases using their Organ Chip platform. Until 2020, the team worked to identify the genes in human lung cells that regulate their response to infection with influenza A, which at the time was the virus of greatest concern in the pandemic. They began by using CRISPR-Cas9 screening to identify host genes that could inhibit the proliferation of the influenza virus. One of their top candidates during screening was a gene called DGCR5, which makes a long non-coding sequence of RNA rather than a protein. To see how DGCR5 affects infection levels, they “knocked down” the gene by designing three different double-stranded small interfering RNAs (siRNAs) that interfere with gene expression, and then delivering the siRNAs to cultured lung cells.

The researchers found that this mixture reduced DGCR5 levels by more than 80% and inhibited influenza A infection by more than 90%. Excited by these results, they began testing the siRNAs individually as well as some additional DGCR5-specific siRNA sequences. To their surprise, although nine of the ten siRNAs they created significantly reduced DGCR5 gene expression, only one inhibited viral infection. DGCR5 activity did not appear to be a factor in viral infection rates.

When they investigated further, they confirmed that this particular siRNA caused the regulation of several genes involved in the IFN signaling pathway. Additional studies showed that the siRNA exclusively activated one of three signaling pathways that can trigger IFN production, known as the RIG-I pathway, which is an important component of the innate immune response. Researchers began to suspect that they had discovered a new class of dsRNAs that stimulate the immune system through a previously unknown mechanism of action.

They began to systematically generate and test more than 200 variants of the original dsRNA sequence to reveal this mechanism. They identified a particular strand of nucleotides, called a “motif,” that was always present at one end of dsRNA with high IFN-stimulating activity: a cytosine (C) on one strand and three guanines (GGG) on the other strand. Because C binds to one G of the GGG sequence in the motif, each dsRNA has an “overhang” of two Gs at the end. When many copies of the dsRNA are present, the overhanging Gs of one molecule can bind to copies of another molecule through an unusual phenomenon called GG Hoogsteen base pairing . The resulting dsRNA dimers then bind directly to RIG-I very efficiently, causing its activation and subsequent IFN response.

“This was very interesting to us because previous studies have shown that RIG-I can be activated by duplex dsRNAs with ‘blunt ends’ that lack overhangs, and that almost any type of overhang can prevent RIG-I from binding. But the unique GG binding behavior we observed effectively creates blunt-ended dsRNAs that can strongly activate RIG-I, so in this case the overhang is crucial for the activity of our new dsRNA,” said co-author Longlong Xie, Ph.D. , a former Wyss Technology Development Fellow who is now a professor at the Shenzhen Institute of Advanced Technology in China.

From flu to COVID and beyond

To see how effective their newly discovered dsRNAs were in living cells, the researchers tested one of them against a synthetic immunostimulant that mimics viral infections called poly(I:C). When they applied the two treatments to human epithelial cells, they found that dsRNA induced a less inflammatory antiviral response, while the more inflammatory poly(I:C) induced much broader changes in gene expression and affected other biological processes required for normal life. cell functions.

Taking it a step further, the team tested dsRNA in chips of human lung airways and alveoli previously developed at the Wyss Institute to replicate complex human organ tissue and function in vitro. They injected dsRNA into healthy chips and saw that IFN-I expression increased 12-40 times. When they then added the influenza A virus to the organ chips, they found that the dsRNA suppressed the infection by 80-90%.

Then the COVID-19 pandemic hit, and the team shifted their research from influenza to SARS-CoV-2 and the related coronaviruses SARS-CoV, MERS-CoV, and the common cold virus HCoV-NL63. Their dsRNA made a difference, inhibiting MERS-CoV and HCoV-NL63 infection of a monkey-derived mammalian cell line by more than 90%, and SARS-CoV by more than 1,000-fold. It also inhibited SARS-CoV-2 infection of a human epithelial cell line by a staggering 99.99%.

These studies were completed in collaboration with the group of Matthew Freeman of the University of Maryland School of Medicine and Benjamin Tenaver of the Icahn School of Medicine at Mount Sinai (now NYU Grossman School of Medicine).

Finally, they tested the dsRNA in a mouse model of COVID-19 with collaborators Dong Young and Colin Johnson, Ph.D. at the University of Tennessee Health Science Center. When they infected treated mice with SARS-CoV-2, the dsRNA reduced the viral load in the animals’ lungs by more than 1,000-fold compared to animals that received the scrambled dsRNA sequence.

“The COVID-19 pandemic has made it painfully clear that we need broad-spectrum therapeutics capable of attenuating infection with a wide range of viruses, rather than developing ad-hoc treatments for each disease as it occurs. I hope that this therapeutic RNA technology, whose chemical and physical properties allow it to be easily produced on a large scale, will become a widely used approach to fight pandemics in the future,” said Don Ingber, MD, PhD, who is also from is the Juda Folkman Professor of Vascular Systems. Biology at Harvard Medical School (HMS) and Boston Children’s Hospital, and Hansjörg Wies Professor of Bioengineering at Harvard’s John A. Paulson School of Engineering and Applied Sciences.

In addition to treatment viruses, these novel dsRNAs may treat bacterial, fungal, and parasitic infections, as well as diseases such as cancer and autoimmune diseases that may benefit from increased IFN production. They can also be used as an adjuvant to enhance the activity of other vaccines.

The authors say that it is important for future studies to determine the optimal timing of treatment, as too late activation of IFN during infection may worsen inflammation, and to explore the possibility of administering dsRNA directly to the upper airways (eg, via an inhaler) to minimize systemic immune activation.