For years researchers have been refining methods for recruiting viruses in the fight against cancer. The idea is to harness the harmful effects some viruses exert on host cells and unleash these effects on cancer cells. Typically, a virus is harmful when it replicates to a large number within a cell. Eventually the cell ruptures, spilling viruses which invade surrounding cells, continuing the process. A virus which kills cancer cells very efficiently while sparing normal cells could have great therapeutic potential. Previously, viruses have been engineered to replicate more efficiently in cancer cells than in normal cells, but these have typically shown attenuation - reduced replication rates – compared with wild type virus, even in targeted cancer cells.In a paper to be published Friday in the journal PLoS Pathogens, biologists Ryan Cawood, Hannah Chen, Fionnadh Carroll, Miriam Bazan-Peregrino, and Leonard Seymour of the University of Oxford, in the UK, and Nico van Rooijen of Vrije University, in The Netherlands, report a method of engineering viruses to exhibit reduced replication activity at sites of potential pathology, while maintaining wild type replication at the therapeutic site. To accomplish this, they utilized microRNA technology. MicroRNAs are small non-coding ribonucleic acid sequences, which post-transcriptionally regulate protein expression by binding to messenger RNA in a sequence-specific manner and targeting it for destruction. Many viruses are pathogenic only in certain tissues. This trait can be exploited by the fact that microRNAs are often expressed in a tissue-specific manner. By engineering a binding site for a particular microRNA – known as mir-122 – into an adenovirus gene, the researchers were able to accomplish tissue-specific knockdown of viral replication.
They engineered a virus containing binding sites for mir-122 microRNA, plus a luciferase reporter gene. Luciferase is a bioluminescent enzyme widely used in molecular biology. In the context of a viral replication assay, it works like this. Viruses replicate within infected cells, hijacking cellular machinery to synthesize new copies of the viral genome and new proteins which form the viral capsid, or protein coat. As new viral proteins are manufactured, so are new luciferase molecules. These catalyze light-producing chemical reactions. As viral replication proceeds, luciferase accumulates, resulting in an increase in the intensity of emitted light, which can be measured by a machine.
The researchers infected cells from three different lines with both wild type and mir-122-binding viruses. One of the cell lines expressed mir-122 microRNA. These cells showed a significant decrease in luminescence when infected with microRNA-binding virus compared to wild type virus. The other cell lines did not express mir-122 microRNA. These cells showed no difference in luminescence in response to wild type vs. engineered virus, indicating that the addition of the microRNA-binding sites did not attenuate viral replication in the absence of mir-122 microRNA. This is a key finding, suggesting that such a strategy could maximize the therapeutic value of an engineered virus by allowing it to exert its full wild type effect on targeted cancer cells, while being selectively attenuated in sensitive tissues.
They applied the same bioluminescent imaging technology to live mice, following intravenous injection of both mir-122-binding and wild type adenovirus. Adenovirus is known to cause liver damage in mice, and mir-122 microRNA is highly expressed in mouse liver. As in the in vitro experiments, they observed a significant reduction in luminescence when imaging live mice infected with microRNA-binding virus compared to mice infected with wild type virus.
They also evaluated blood serum levels of two different proteins known to be elevated in response to liver damage. Levels for these markers were 15- to 17-fold lower in mice infected with mir-122-binding virus compared with those infected with wild type virus. Additionally, they performed quantitative PCR using DNA extracted from liver tissues of infected mice to quantify the number of viral genomes present. The wild type virus exhibited a 60-fold increase over the amount injected. By contrast, in mice injected with the microRNA-binding virus, the viral load less than doubled compared to the starting amount, over the same time period. Histological analysis of tissue samples also revealed extensive liver damage in mice infected with wild type virus, and little to none in mice infected with the microRNA-binding virus.
Taken together, these results convincingly demonstrate the feasibility of using microRNAs to eliminate toxicity of a therapeutic virus without diminishing its replicative capacity – and, hence, its effectiveness – in targeted cancer cells. These findings may represent an early step toward more effective virus-based treatments of human cancers, although additional research will, of course, be required before this technology can be tested in humans.
This research was supported by Cancer Research UK, the Bellhouse Foundation, and the New Zealand government.
Ryan Cawood, Hannah H. Chen, Fionnadh Carroll, Miriam Bazan-Peregrino, Nico van Rooijen, Leonard W. Seymour (2009) Use of Tissue-Specific MicroRNA to Control Pathology of Wild-Type Adenovirus without Attenuation of Its Ability to Kill Cancer Cells. PLoS Pathog 5(5): e1000440. doi:10.1371/journal.ppat.1000440
Research Article: http://dx.plos.org/10.1371/journal.ppat.1000440