The most promising initial clinical application of microRNAs might be in cancer diagnostics, recently published research suggests, and several companies are already jumping in to develop microRNA profiling technology.
Todd Golub and his collaborators at the Broad Institute of MIT and Harvard, MIT, the Dana-Farber Cancer Institute, St. Jude's Children's Research Hospital, and Harvard Medical School describe in a paper published in last week's Nature how they were able to classify human cancers by their miRNA expression profiles.
To the researchers, the ability to do so was rather unexpected: "There was no reason to believe, going in, that just a couple of hundred microRNAs would be able to classify anything," Golub told RNAi News this week. "The fact that the major landscape of cancer was somehow captured in the space of microRNA expression profiles was surprising."
Using a bead-based flow cytometric platform from Luminex, the researchers measured the expression of 217 miRNAs in 334 samples, including different human cancers. In tumor samples, they found that miRNAs were generally down-regulated. They were also able to distinguish the development lineage and the differentiation state of the tumors based on their miRNA profiles.
Compared to messenger RNA expression profiles, which have long been explored as potential cancer diagnostics, miRNA profiles appear to offer two distinct advantages that are worth exploring further, according to Golub. One is that the miRNA profiles were able to tell the organ of origin for so-called poorly differentiated cancers, which are difficult to assess using standard pathology methods. "We had previously tried to do this using mRNA profiles, and found …they basically didn't work," Golub said. "[What] we found here, while it wasn't perfect, we were able to do a quite respectable job in classifying those poorly differentiated cancers using microRNAs."
Secondly, the microRNAs seem to be preserved in formalin-fixed paraffin-embedded tissues, the kind that are routinely collected in the clinic, while mRNAs degrade in these samples, he said.
Instead of the glass-slide microarrays used by other researchers to profile miRNAs, Golub's team decided to use Luminex's xMAP beads. These beads carry fluorescent dyes that can yield up to 100 different colors. The scientists coupled one oligo capture probe complementary to an miRNA to each type of bead. After amplifying the miRNAs in the samples and hybridizing them to the capture beads, they analyzed them by flow cytometry. Compared to microarrays, they observed greater specificity, Golub said.
"There is also great flexibility in that as new microRNAs are discovered and they continue to be discovered one can quickly add additional beads to the mixture rather than having to reprint the whole slide," he added. "And it's low cost, which is also advantageous."
However, Golub noted that the sample preparation method, which he would like to automate, still needs improvement. "At present, it has a number of challenging preparative steps, in particular PAGE purification, which we would like to find a way to omit," he said.
Clinical diagnostic applications of the method, which Golub has filed a patent on, are still at least several years away, he said. The first step is to validate the results, which he noted is "an activity not just for a lab but for the community." His group will focus on classifying more undifferentiated cancers, trying to collect large numbers of samples to study.
But there is also considerable commercial interest in developing his miRNA profiling approach further, though Golub remained mum about any companies he has been in touch with, other than saying that "we have had some discussions."
At least three companies have been looking into analyzing microRNAs, some of them for diagnostic purposes.
Earlier this month, Genaco Biomedical Products received a six-month Small Business Innovation Research grant worth $99,000 from the National Cancer Institute to develop a bead-based array system for the specific detection and classification of microRNAs. The company has already developed a method that, like Golub's, is based on the xMAP platform and uses locked nucleic acid technology, according to the grant abstract. Genaco intends to use the system to develop an assay based on 20 cancer-related miRNA targets and to use this prototype assay to study cancer samples.
In addition, Mirus Bio received a one-year, $190,000 grant from the National Institute of General Medical Sciences in April to develop methods for the efficient labeling of microRNA for microarray analysis. The company recently told RNAi News that this grant was supporting work that was expected to result in the market launch of an miRNA-specific labeling product in the next few months (see RNAi News, 6/10/2005).
Meanwhile, Ambion obtained an eight-month, $265,000 grant from the NIGMS in March to develop tools for the analysis of microRNA function.
Jian Han, the CSO of Genaco, told RNAi News this week that he has seen Golub's Nature paper, but that his company's technology called mirMasa takes a different approach to miRNA expression profiling by using locked nucleic acid technology developed by Exiqon. According to the company, this allows for the detection of multiple miRNAs in a single reaction tube with no purification or labeling. Han noted that the company is planning on publishing a paper detailing the technology in greater detail in the near future.
Jim Hagstrom, vice president of scientific operations at Mirus, told RNAi News in an e-mail last week that his company was familiar with Golub's work, but that Mirus is not "working directly with him at this time."
In an e-mail to RNAi News this week, Ambion President Bruce Leander declined to comment on whether his company was in partnership discussions with Golub, but did state that "microRNAs are very interesting … [and] at some point in the not-too-distant future we will want to talk publicly about them."
Julia Karow ([email protected])