Feature: RNAi delivers double whammy to cancer

RNA interference (RNAi) was little more than an enigmatic laboratory phenomenon just 15 years ago, but has since been fashioned into a Swiss Army knife, bristling with new blades and widgets for biological and medical research. Eminent cancer researcher, Professor Bryan Williams, Director of the Monash Institute of Medical Research in Victoria, and head of the MIMR's Centre for Cancer Research, marvels at how rapidly the field has evolved.

"RNAi came out of the blue, and an extraordinarily short time elapsed between its discovery to the award of a Nobel Prize in 2006," says Williams.

In the year that Andrew Fire and Craig Mello won their Nobel, Williams was chair of the Department of Cancer Biology at the Lerner Research Institute of the Cleveland Clinic Foundation in Cleveland, Ohio. Synthetic double-stranded RNA (dsRNA) molecules designed to recruit the endogenous RNAi systems of plants, nematode worms and insects had already been shown to silence genes and ward off viral infections in plants and invertebrates. But Williams doubted that short interfering RNA (siRNA) molecules would work in mammals, which would rule out human medical applications, including novel cancer therapies.

When Andrew Fire asked why, Williams cited research by his PhD student, Carol Sledz, that led to a classic co-authored paper in Nature Cell Biology in 2004, cited more than 1000 times since. Sledz' experiments comparing the immune responses of insects and mammals showed that targeted siRNAs did induce specific gene silencing in mice. But, crucially, they also caused non-specific activation of the innate immune system.

"That set the field on its ear," says Williams. "We then tried to figure out the mechanism, which is how I became engaged so early in RNAi research." They described their findings in a follow-up paper in Nature Biotechnology in 2006.

Hitting the trigger

The early-responder sentries and soldiers of the innate immune system - dendritic cells, macrophages and monocytes - express a variety of Toll-like receptors (TLRs) on their surface that detect antigens specific to viruses, bacteria and parasites, via complementary shape-fit interactions. On detecting viral antigens, innate immune system cells block infection by secreting interferons that destroy the virus' RNA-coded genetic blueprint.

Improbably, two receptors, TLR 7 and TLR 8, can directly detect single-and double-stranded RNA molecules that would seem to be too tiny and lack in 3-D structures to trigger the interferon system.

---PB---

In a review article in Nature Biotechnology in 2005, Williams suggested how the problem of targeted siRNAs causing off-target activation of the innate immune response in mammals could be turned into a positive. An siRNA designed to target a specific viral gene could also stimulate the innate immune system's non-specific interferon response, for a double-whammy effect.

The same approach could be employed as a novel anti-cancer therapy: target an oncogene specific to a patient's tumour, and rely on the innate immune system to produce an inflammatory response.

"Inflammation is a bad thing in some circumstances, but it can be a good thing in cancer, because it can reactivate the immune response, including the adaptive immune response, to attack the tumour," says Williams.

"The adaptive immune system commonly fails to recognise cancerous cells, because they down-regulate expression of Class 1 Major Histocompatibility Complex [MHC] molecules that are required to display mutant antigens on the cell surface.

"Interferons up-regulate Class 1 MHC molecules, so if we can activate localised production of interferons in the tumour milieu, it should force cancerous cells to display mutant antigens to the adaptive immune system, activating production of antibody-secreting B-cells and cytotoxic T-cells.

"With Nigel's McMillan's laboratory in Brisbane's, Diamantina Institute, we've confirmed that siRNAs can activate the adaptive immune system in mice. Most of our recent efforts had been directed towards the rational design of siRNAs to build in a strong immunostimulatory response.

"It proved difficult, but we have now demonstrated how biotechnology companies can go about it in a rational way. It involves incorporating sequences that build lumpy structures into the RNA strand. This work was published earlier this year in the journal Molecular Therapy with post-doctoral fellow Michael Gantier as lead author. We've shown that this provides three-dimensional structures that are recognised by the RNA-sensing Toll-like receptors 7 and 8."

Road to the clinic

The rational-design project is funded in part by a $480,000 grant from the National Health and Medical Research Council, with supplementary funding from Roche biotechnology subsidiary, Roche Kulmbach.

Williams says the innate immune system's RNA-sensing Toll-like receptors do not react to single-stranded messenger RNAs because their action is safely isolated within the endosome. His research group is working with Sydney biotechnology company, EnGeneIC, to develop a system targeted to cancerous tissues and to avoid damaging normal tissues.

"One way to avoid exposing normal tissues is to restrict delivery of the siRNAs to the tumour bed, so any non-specific inflammatory reaction is strongly localised," he says. "Tumour-specific targeting can be achieved in a number of ways, most of which involve nanoparticles targeted to cell-surface molecules expressed only by tumour cells. When you deliver them, the molecule flips over and takes the nanoparticle inside the membrane.

---PB---

"We can also get macrophages at the site of the tumour to take up siRNA-loaded nanoparticles targeting any of the standard raft of oncogenes, or genes that prevent cancerous cells activating their apoptosis pathways and programmed to induce local inflammation.

"We're exploring the possibility of delivering multiple immunostimulatory siRNAs targeting different oncogenes in tumours, so we're not relying on a single driver, but we have to be careful because that runs the risk of saturating the cell's RNA-induced silencing complexes. But we think we can deliver up to three different siRNAs per cell type and still get strong activity. The adoption of the technology will depend strongly on successful genetic profiling of individual tumours."

Williams says the reality is that the technology is almost certain to go into the clinic before molecular analysis of tumours becomes commonplace, but a consortium of research centres, headed by the Peter MacCallum Cancer Centre, is currently seeking funding to establish a Victoria-wide tumour-profiling service.

***

Hidden side to vaccines

Could an RNA-triggered innate immune response explain the recent spate of adverse reactions by children under five years old to the seasonal influenza vaccine? Professor Williams says this was his initial suspicion when reports began to emerge of adverse reactions in young children, including elevated temperature, fever and convulsions.

Health authorities are investigating the case of a young Perth girl who developed convulsions and died within hours of being vaccinated with the seasonal flu vaccine.

CSL added antigens from the pandemic H1N1 swine flu strain to antigens from the most common extant strains of influenza A to make this year's seasonal flu vaccine. The company also ruled out ruled out a faulty batch of vaccine as the cause of the girl's death.

Williams says he was planning to design an experiment to determine if dendritic cells, monocytes and macrophages - the innate immune system's early responders - might be responding to 'lumpy' viral RNA residues in the vaccine, detected with their specialised RNA-sensing Toll-like receptors 7 and 8.

---PB---

But Flinders University immunologist Professor Nikiolai Petrovsky had already done the experiment, and shown that an RNA extract from the vaccine triggered what he described as "an enormous reaction" in macrophages in vitro.

The result, which has yet to be replicated by other researchers, challenges to the long-established wisdom that the viral RNA residues in inactivated- and attenuated-virus vaccines play no part in the immune response.

Petrovsky says the experiment leaves "absolutely no doubt" that very high levels of RNA in the seasonal influenza vaccine caused the adverse reactions in children. Petrovsky would not claim to be a disinterested investigator: he is the founder and CEO of Adelaide biotechnology company, Vaxine, which has developed a novel, recombinant vaccine against the H5N1 avian influenza, in collaboration with US-based Protein Sciences Corporation.

The prototype vaccine contains no RNA residues, because it is consists of pure haemagglutin antigen from the virus, produced in recombinant insect cell lines - Protein Science's proprietary technology.

According to Petrovsky, CSL's seasonal influenza vaccines, produced by the 50-year old method of growing the influenza viruses in embryonated chicken eggs, consistently rank near the top of the table for RNA residues.

"It's amazing that the regulatory system still doesn't require manufacturers like CSL to measure the amount of contaminating RNA, despite 50 years of data showing that RNA levels can vary greatly from vaccine to vaccine.

"When you grow the virus in chicken eggs, you're dealing with an interaction between a genetically variable virus, and genetically variable chicken embryos, so the results are inherently unpredictable and unrepeatable. It's a nonsensical way to make a reliable product for human medical use."

Molecular biologist Professor Bryan Williams, Director of the Monash Institute of Medical Research since January 2006, is an acknowledged cancer expert. He won international recognition for his research on Wilms Tumour, a cancer of the kidney that occurs predominantly in children, and for studies on interferons and innate immunity. From 1991 to 2005, he was chairman of the Department of Cancer Biology at the Lerner Research Institute of the Cleveland Clinic Foundation in Cleveland, Ohio. He has specialised in the molecular biology of tumour suppression, and the role of innate immune system in antiviral and anti-tumour surveillance.