The National Institute of General Medical Sciences last month issued two grants worth a combined $305,000 to help two biotechs develop new technologies for microRNA research.
The first grant, to Mirus Bio, is worth $197,636 and will help the company develop a blood-based reporter system designed to allow researchers to determine if and when an miRNA of interest is expressed in the liver of a live mouse.
The second one, to Seattle-based startup Sileretech, is for $107,250 and will support the development of a novel miRNA amplification method.
According to Mirus’ grant abstract, “there is evidence that miRNAs play roles in a wide variety of biological processes including development, differentiation, [and] metabolism,” and computational algorithms suggest that miRNAs may “regulate expression of up to one-third of protein-coding genes in the genome.
“However, target genes have been experimentally assigned to only a handful of miRNAs, and the precise functions of the vast majority of miRNAs remain unknown.”
Current approaches to determine miRNA expression patterns such as Northern blotting and microarray analysis are “labor-intensive and time-consuming, and the small size of miRNAs makes these hybridization-based methods of detection technically challenging,” the abstract notes. “In addition, the animals or cells must be destroyed in order to harvest the miRNAs, making time course studies more difficult and expensive.”
Through its NIH-funded research, Mirus proposes to develop an in vivo system that will allow researchers to monitor miRNA expression over time.
“The system is based on the fact that placement of an exact-match miRNA binding site in an mRNA results in mRNA cleavage via the RNAi pathway,” the company said in the abstract. “Exact-match miRNA binding sites for all known mouse miRNAs will be placed into the 3’ UTR of a non-immunogenic, secreted reporter gene. The reporter gene will be harbored in a plasmid DNA that contains elements necessary for high, long-term expression in mouse liver.”
The company plans to deliver the plasmids using hydrodynamic tail vein injections. “Co-delivery of a plasmid expressing a different, non-immunogenic, secreted reporter gene not under miRNA control will function as a control for variations in delivery efficiency,” it added in the abstract.
Through this approach, the company said it could create large numbers of so-called miRNA sensor mice, of any strain, in a single day.
“These mice will allow the investigator to quickly and easily monitor changes in expression of any miRNA of interest simply by measuring the amount of reporter gene present in the serum,” the company said. “This system will not only allow the investigator to determine if a particular miRNA is functional in the liver, but will also be useful for monitoring changes in miRNA expression over time and under different treatment conditions.”
David Lewis, RNAi program director at Mirus and principal investigator on the grant, told RNAi News that Mirus plans to use the technology to develop a library of plasmids targeting all of the mouse miRNAs in the Wellcome Trust Sanger Institute’s miRBase database.
“We’re shooting to have that completed by December,” he said. “Our plan is then to market these plasmids themselves or mice that have been set up for [detection of] a particular microRNA.”
Mirus’ one-year grant runs until Aug. 6, 2007.
Sileretech’s grant, meanwhile, will support the recently incorporated company’s effort to develop a novel miRNA amplification method.
MicroRNA expression is “highly regulated, and coordinated, different cell types under different physiological condition appear to have a unique miRNA expression pattern,” Sileretech’s grant abstract states. “Increasing evidence suggests miRNA expression pattern could be indicative not only of the physiological state but also the disease state of cell or tissue such as cancer.”
Currently, Northern blot analysis “is the gold standard for detection and quantification of miRNAs,” but it is a “comparatively tedious process with low sensitivity.” As such, “highly sensitive tools are needed for detection and expression profiling of miRNAs.”
“This system will not only allow the investigator to determine if a particular miRNA is functional in the liver, but will also be useful for monitoring changes in miRNA expression over time and under different treatment conditions.”
Because of miRNA’s small size and single-stranded nature, “efficient and faithful amplification by conventional polymerase chain reaction is difficult to achieve. Most current methods for detection and profiling rely on the amplification of signals [but] the sensitivity of those methods is still far below the level [that] can be achieved with PCR amplification.”
To address these issues, Sileretech proposes in its grant project to develop a novel multiplex PCR-based miRNA amplification system “designed to faithfully amplify all miRNAs simultaneously without bias.”
Commercial applications for this amplification system include quantitation of miRNA by real-time PCR for diagnostic purposes, as well as miRNA expression profiling.
Donald Rao, managing director of Sileretech, told RNAi News in an e-mail that his company is working on the project detailed in the grant, but that “we do not have anything to report yet.”
Sileretech’s six-month grant runs until Feb. 14, 2007.