Positive INTERFERENCE

RNAi’s prevalence and importance in basic medical research

By Deborah Komlos

Although their therapeutic applications may only come to light some years from now, particular small RNA molecules continue to command a keen interest from the scientific community.
Called small interfering RNAs (siRNAs), these 21-23 nucleotide double-stranded molecules are being used extensively to study gene function. Of interest is their ability to perform the process of RNA interference (RNAi), which permits the silencing of gene expression. While this capability holds great therapeutic promise, there is still much groundwork to cover regarding its underlying mechanism.
“RNAi is a process that exists in most eukaryotic organisms, and so it’s very important to learn how this process goes on, and why it is so important and so conserved among species,” explains Patrick Provost, PhD, a researcher with the Centre de Recherche en Rhumatologie et Immunologie (Ste-Foy, QC) and assistant professor at Laval University (Quebec City, QC).
Provost aims to improve current understanding of the RNAi pathway. In RNAi — a process that acts as a cellular defence mechanism — double-stranded RNAs (dsRNAs), either produced endogenously or introduced exogenously, activate the degradation of homologous messenger RNAs (mRNAs). The consequence: inhibiting the expression of encoded proteins.

Cutting Edge
It was during his post-doctoral fellowship at the Karolinska Institutet (Stockholm, Sweden) that Provost says a chance discovery led to his identification of Dicer, a key enzyme in the RNAi pathway.
“It was really an accident because my main research focus during my post-doctoral studies was on an enzyme called 5-lipoxygenase, which is involved in inflammation,” he recalls. “And so, while studying that enzyme I was searching for cellular proteins that could interact with 5-lipoxygenase. Then I had identified Dicer as one of these proteins.”
His group was the first worldwide to express and characterize a recombinant protein involving RNA silencing, Provost says.
According to current understanding, Dicer cuts dsRNA into siRNAs, which are believed to then guide a multi-component complex known as RISC (RNA-induced silencing complex) to destroy specific mRNAs. RISC either cleaves the mRNA, if the siRNA is perfectly complementary to that sequence, or stays there and blocks translation, Provost says. Details on how this blocking occurs remain unknown, he adds.
Provost says a current strong focus in the RNAi community is on the relationship between diseases and tiny endogenously produced single-stranded RNAs called microRNAs (miRNAs), of which humans have about 200 to 250.
In mammals, he says, miRNAs do not lead to mRNA cleavage. Rather, they just repress translation. (Fig. 1, pg. 24)
“The thing that’s interesting about these miRNAs is the fact that we had them under our eyes for decades because these RNA are very, very small — between 19 and 25 nucleotides. And so, given the knowledge at that time in the ’50s and ’60s and ’70s, you couldn’t have meaningful small RNAs . . . they were considered as degradation products,” he explains.
While about five per cent of the human genome encodes for protein, at least one per cent encodes miRNA, Provost says. That one per cent regulates up to 10 per cent of the genome, which is significant, he says, because several proteins are involved in any cell process or signalling pathway at a given time. “So, that makes it likely that at least one component of each pathway may be regulated by
miRNAs.
“If there is a defect, a mutation in the transcription of one miRNA, then you can suspect that one or more genes will turn on,” he says. “Whereas this miRNA should have kept them off. So, dysfunction of the RNAi pathway is likely to give rise to serious diseases.”
To Provost’s knowledge, only a handful of researchers in Canada are currently studying the mechanistic aspects of RNAi. More commonly, he notes, RNAi is being used as a tool. A survey released last October by BioInformatics LLC (Arlington, VA) attests to this popular usage.
The Market for RNA Interference Products: Challenges and Opportunities polled more than 500 scientists worldwide who currently use siRNA, and more than 360 who plan to use it within the next year. Of the 51 per cent of respondents who expect their lab to increase the number of RNAi experiments over the next year, more than half expect an increase of 50 per cent or more.
Robin Rothrock, PhD, director of Market Research at BioInformatics, says assigning gene function was found to be the top research objective (40 per cent) for RNAi experiments, with in vivo knockouts as second most common usage (22 per cent).
“I think one reason why it’s been so widely adopted is that it allows researchers to leverage tools that they already know how to use,” Rothrock explains. “You can order siRNAs that have been synthesized: they’re primers — synthetic oligonucleotides. Then, people have been doing transfection for years.
“It’s not starting from a complete unknown,” she adds. “It’s not hard to do and so I think that the barriers to entry, from the perspective of a scientist, are pretty low.”
But challenges remain, Rothrock mentions, including developing more efficient delivery systems.
“No matter how well designed your siRNA is,” she says, “it doesn’t mean anything if you can’t get it into the target cell — and that will be increasingly important when you think about using siRNA as a therapeutic.”

Fundamental Links
Eileen Denovan-Wright, PhD, an assistant professor in the department of pharmacology at Dalhousie University (Halifax, NS), is investigating the use of adeno-associated viral vectors as a vehicle for siRNA delivery.
Along with collaborator Ron Mandel, PhD at the University of Florida (Gainesville, FL), Denovan-Wright is introducing siRNA to knock down huntington, a protein that accumulates in the brain of patients with the progressive neurodegenerative disorder Huntington’s disease (HD).
The researchers are working with mouse models, assessing fundamental questions such as whether or not the siRNA can reduce HD severity, and when is the most effective time point in the mouse lifespan to introduce the molecules.
“The idea of RNAi is really quite simple,” Denovan-Wright explains. “You want to put in a molecule that will actually specifically target the message, leave the other ones alone, use the cell’s machinery that’s already inside the cell, and in combination with the RNAi, it should lead to a decrease in the amount of a specific message and in doing so, may be useful for understanding the disease or having a therapy.”
Targeting a disease gene’s expression may result in slowing the disease process, but Denovan-Wright cautions of a number of caveats.
“There are issues about what’s the best way to get these molecules into patients because it’s fairly easy to get them into cells and you don’t do a whole lot of damage putting them into cells,” she explains. “But we still don’t know what would be the best tissue that you would have to target. Does it have to be in a specific part of the brain, or all over the brain? Does it also have to be in the body?”
A perceived advantage of using adeno-associated viral vectors, she says, is being able to deliver the material and have it expressed for a long time in the cells, avoiding the need for continual external delivery.
Denovan-Wright emphasizes that the work is still at a very early stage. “It’s a long, long, long way from therapy, and there are many things to be thought of and understood before there would be a therapy,” she says.

Alternative RNAi Avenues
In the lab of Dr. Wei-Ping Min, PhD, an assistant professor in the department of surgery, microbiology and immunology at the University of Western Ontario (London, ON), RNAi is vital to research on a novel therapeutic technology.
His lab is applying its findings for the purposes of organ transplantation and for preventing autoimmune diseases, such as rheumatoid arthritis.
When the body recognizes a transplanted organ as foreign, an immune response occurs, which can result in organ rejection. To pre-empt such a reaction, immune-suppressive medication can be used. But such treatment “is like a nuclear bomb, it disrupts everything,” Min says. “So our body will eventually get cancer or infections.”
In an effort to control the immune response, Min’s group has designed a type of “smart” siRNA that only targets dendritic cells (DCs), which play a critical role in determining whether an immune response is activated or suppressed. By modifying the signal sent by these antigen-presenting DCs to T-cells, the immune response is suppressed. Min says his group is the first in the world to use siRNA to silence immune-associated genes in immune cells. He hopes to further develop the technology through ToleroTech Inc., a company that he founded as an early stage spin-off from the Lawson Health Research Institute (London, ON).
Thus far, the team has demonstrated success in preclinical work, Min says, showing survival of more than 50 days following heart transplant in a mouse model; without treatment, the organ is rejected in about two weeks.
Although the research is in its early stages, Min says he is positive regarding the benefits of an immune-modulation approach.
“In the future of autoimmune diseases and transplantation patients, we will never use any immune-suppressant drugs,” he says. Instead, he adds, immune modulation will be employed as a “more natural” and non-toxic approach. “Tolerance means this immune-modulation procedure has started and is maintained in your body without any external immune drugs.”
Also offering a pioneering approach to RNAi is DNP Pharmaceuticals Inc. (DNPP). With research facilities at the State University of New York (SUNY) at Buffalo (Buffalo, NY), DNPP has developed an RNA-based platform technology involving chemical modification of RNA by DNP derivatization.
The work is built upon the research of Jui H. Wang, PhD — DNPP’s chairman and CSO, and Einstein Professor of Science, SUNY at Buffalo — who has been investigating gene-silencing RNAs since the early ’90s, explains Toronto, Ont.-based president and CEO Nancy Dudgeon.
DNPP has found promising results from its in vivo testing of DNP-derivatized single-stranded antisense RNA (DNP-ssRNA), showing sequence-specificity and dose-dependence.
In the human breast cancer SCID mouse xenograft, DNP-ssRNA was found to significantly improve prognosis, reduce tumour growth, improve mortality, and confer 100-per-cent prevention of metastasis, Dudgeon says.
Applying DNP-ssRNA in a murine leukemia model for AIDS, “we not only eliminated the viremia, but also eliminated any trace of integrated viral DNA,” she says. These findings were also seen in a duck model for hepatitis B, for which a further finding was recovery of the duck liver to normal histology upon pathological examination.
Dudgeon says the gene silencing effect that DNPP’s technology has produced is likely not occurring through RNAse H cleavage, as normally happens with antisense.
“We believe it is the same mechanism as RNAi,” she says. “It has higher binding affinity than previous (antisense) varieties. It even has higher binding affinity than siRNA.” Other attributes Dudgeon lists are greater stability in the presence of ribonucleases, higher affinity, and more effective gene silencing at both the mRNA and protein levels.
Another advantage is that, unlike siRNA, the platform does not require plasmids or vectors for delivery, Dudgeon says. Moreover, the firm has demonstrated successful oral delivery of DNP-ssRNA in its leukemia model.
DNPP currently has a few preclinical candidates in its pipeline, Dudgeon says, and has a long-term goal of seeing its platform applied therapeutically and diagnostically.
Despite siRNA’s great promise and considering the RNAi realm is relatively young — the first paper on RNAi was published in 1998 — Provost emphasizes that many challenges remain to be addressed, including means of administration, and stability and specificity issues.
“It’s hard to balance the new knowledge that we get about the mechanism and the development of therapeutics based on RNAi because you can’t go wrong,” Provost says. “You can’t assume certain things that later you found out that it doesn’t work that way.”
Provost says there are several firms looking to develop siRNAs as therapeutics, and while they can work to improve their reagents and tools by themselves, they still don’t have the complete picture. “There are important aspects coming out from mechanistic studies that they are not performing and they are waiting for,” he says. “So there are several questions remaining and they cannot move further or quicker because there are some unknowns.
“That’s why it’s really important to know how the RNAi pathway works . . . and that would help us develop therapeutics much more efficiently.”