May 15, 2007 (Vol. 27, No. 10)
Improving Gene Silencing Methodologies Enables Better Understanding of Therapeutics
Analyzing gene expression can provide a roadmap for deciphering the role of specific genes or elaborating critical players in functional metabolic pathways. Companies are pursuing solutions to better understand and define the role of gene expression especially as it relates to therapeutic development. While there is no one-size-fits-all approach, researchers are making strides to integrate genetic, functional, and bioinformatics data as well as to harness powerful new strategies for RNAi.
Systems Biology Approach
Understanding complex diseases and finding therapies requires integrating genetic, functional genomic, and bioinformatics data using a systems biology approach, according to Allan Kuchinsky, principal project scientist at Agilent Technologies (www.agilent.com). “We used schizophrenia as a model to demonstrate how interpreting data in its biological context helps reveal potentially hidden connections and to decipher the story of how genes and proteins interact in a disease process.”
Agilent scientists used a systems approach to identify candidate genes, comparing 66 sibling pairs discordant for schizophrenia. Microarray studies identified differentially expressed genes in the affected set. Additionally, more than 10,000 SNPs were analyzed to discover relationships between genetic variation and schizophrenia. Comparative genomic hybridization was conducted to identify correlations between chromosomal aberrations and the disease. To interpret the data in its biological context, the scientists used an open-source network analysis and visualization software system, public pathway and protein-protein interaction databases, and inference from the scientific literature.
“When we looked at gene expression alone, we saw genes that didn’t differ much between healthy and affected individuals, yet when we analyzed these same genes in the context of biological pathways, they showed up as being connected to genes that were differentiated,” Kuchinsky continues. “These genes are potential drug targets; we could not have identified them had we relied solely upon gene-expression analysis.
“The open-source approach allowed us to collaborate with some of the best people. We belong to the Cytoscape Consortium and helped develop the core system as well as network inference plug-in software.” Agilent scientists continue to enhance Cytoscape capabilities and provide links for curated pathways and literature. “We want to provide complete solutions, not just equipment,” says Kuchinsky. “Agilent supports open standards and open data exchange; Cytoscape, as the emerging standard for systems biology network informatics, accomplishes both.”
RNAi as a Tool
The use of RNAi is fast becoming an important tool to silence gene expression in order to modulate cellular function. To achieve optimal silencing, several factors must be considered, according to Kathy Latham, Ph.D., senior product manager for Ambion (www.ambion.com), an Applied Biosystems business. “The design of siRNA is important. The trick is to find one that efficiently degrades the specific mRNA of interest. We have formulated sophisticated siRNA design and specificity checking algorithms to help with this. These take into account such things as melting temperature and nucleotide locations. Experimentally, the siRNA concentration used is also critical. One should use the least amount in order to minimize off-target effects.”
In addition to siRNA tools, Applied Biosystems also focuses on another player in RNA-directed gene expression, miRNA. The genomes of plants and animals are chock full of these small single-stranded RNAs that impact a variety of important biological processes.
“MicroRNA are naturally occurring regulatory molecules that primarily act at the level of translation. They are believed to regulate 50% or more of all proteins and play critical roles in a variety of different processes from fat metabolism to cancer,” Dr. Latham explains. It is unusual to find a pathway not impacted. But one needs specialized methods to study miRNAs.”
Applied Biosystems provides a host of tools for studying miRNAs. “Our TaqMan® MicroRNA Assays allow one to amplify specific small RNAs to accurately quantify microRNA expression levels and characterize microRNA expression via real-time RT-PCR,” Dr. Latham says.
Aside from studying the expression patterns of miRNAs, one really needs to define the target(s) of the miRNA, explains Dr. Latham. “Bioinformatics can help, but the state-of-the-art right now is imperfect at best. To really understand function, one needs to inhibit or mimic microRNA function. We have developed Pre-miR™ miRNA Precursors that mimic endogenous microRNAs to determine microRNA’s biological effects via gain-of-function experiments. This helps to better understand their impact on the protein you think is being regulated.”
A new methodology developed by Mirus Bio (www.mirusbio.com) involving a simple intravenous injection of large fluid volumes is helping researchers deliver genes or siRNA molecules more efficiently to liver hepatocytes. Richard S. Schifreen, Ph.D., vp of research products, says, “It’s long been desired to have a means to effectively introduce genes into the liver. Our protocol, referred to as hydrodynamic intravascular injection, involves the high-pressure, rapid delivery of sample into the vein using enough volume of nucleic acid solution (naked DNA) to target cells outside of the blood vessel.
“For RNA interference applications in our rodent models, we routinely inject siRNAs into the tail vein and this allows generation of knock-down adult animal models in days without impacting embryonic development.”
Dr. Schifreen says that the inherently simple method also results in low toxicity. “This procedure is quick, only takes a few minutes to perform, and we see 10–40% transfection efficiency into hepatocytes. This is a great improvement over traditional delivery methods that use nonviral DNA or siRNA methodologies. These often only achieve 0–1% efficiency. It is also an exquisite tool in that you can perform both knockdown and then rescue in the same animal. This can help the researcher determine how a gene affects the phenotype in the pathway being studied.”
Mirus Bio is also focusing on the analysis of miRNA expression using microarray analyses. “We have developed a single-step alkylating system to bind the tag to nucleic acids, which allows us to track the nucleic acid throughout various systems, tissues, or even cells. We are applying this to studying microRNA. This labeling system, called Label IT, is sequence independent and does not change the sequence of the labeled microRNA. This is important because microRNAs are small and other commercially available labeling reagents require attachment of additional sequences that result in a failure to detect all of the microRNA species present in the sample.”
miRNAs and Cancer
miRNAs are also being scrutinized in applications related to cancer patient care. “microRNA used to be the new kid on the block, but now it is beginning to grow up,” comments Gary Latham, Ph.D., director of technology development at Asuragen (www.asuragen.com). “In 2001, the human genome was found to encode microRNAs. We are now beginning to see the broad biological impact of this discovery. For example, microRNAs may regulate up to 90% of all gene products. This is an exciting finding with profound implications for both RNA diagnostics and therapeutics.”
Dr. Latham says that Asuragen is pursuing molecular diagnostics with a strong emphasis on RNA, particularly small RNA. “We are focused on oncology. The change in expression of some microRNAs appears to be related to the progression of certain cancers. We have demonstrated proof of principle that differentially expressed microRNAs can distinguish pancreatic cancer from both chronic pancreatitis and normals. This finding sets the stage for additional studies to identify microRNA biomarkers in other tumors using microarrays and quantitative reverse transcriptase PCR (RT-PCR). By constructing ratios of microRNA markers that are up- or down-regulated, we can create quantitative indices that clearly delineate cancer and normal samples.”
Another advantage of miRNAs is their stability. Because of this character, they may be an answer for evaluating challenging clinical samples such as formalin-fixed, paraffin-embedded (FFPE) samples according to Dr. Latham. “The fixation and embedding process is a tortuous procedure that severely compromises mRNA intactness and transcript expression profiling. On the other hand, microRNAs maintain surprisingly good correlations in expression between frozen and fixed tissues. Given that there are an estimated one billion FFPE samples in archives, this opportunity opens up a whole new window for clinical studies.”
Diagnostics and Therapeutics
Because of the prominence of miRNAs throughout the genome, it is no surprise that they also have been implicated in several gene-activation pathways. Zvi Bentwich, M.D., chief scientist for Rosetta Genomics (www.rosettagenomics.com), says, “microRNAs may play a major direct or indirect regulatory role. This makes them an attractive potential target for drug development because they can either activate or suppress other genes.”
Dr. Bentwich notes that miRNAs are associated with at least 50% of all known cancer genes. “So the choice is clear that microRNAs are a powerful diagnostic as well as therapeutic tool. We are developing technologies to more easily extract high-quality microRNAs from fixed tissue as well as from blood serum. Secondly, we are developing technologies for microarrays. Our custom-designed microarray includes more than 700 human microRNAs and positive and negative controls. Additionally, we also have quantitative RT-PCR that allows discrimination of homologous microRNA family members that differ only by a single nucleotide.”
According to Dr. Bentwich, because of their tissue specificity and relatively smaller number, miRNAs constitute “an attractive target for the development of cancer signatures, which are useful in diagnostics—characterizing tumors in general and their tissue origin in particular. The ability to define such genetic signatures is of great value to the clinician and will hopefully save much unnecessary investigation for patients and eventually give a better indication as to optimal treatment. We are focusing on developing such microRNA signatures using data derived from hundreds of samples from about 15 different tumors.
Analyzing High-throughput Data
“It is critical for scientists to be able to put all of their expression data into a meaningful biological context,” advises Stephen Sharp, Ph.D., director of marketing and product management for Ariadne Genomics(www.ariadnegenomics.com). “True biological events should be scrutinized in the discovery phases by analyzing all the probe data from one’s microarrays and not just the genes with major up- or down-expression changes.”
Dr. Sharp says that the company’s Pathway Studio Enterprise provides a complete software solution for pathway analysis of high-throughput data derived from microarrays. “The Enterprise edition analyzes gene expression, metabolomics, and proteomics experiments by employing advanced pathway analysis statistical algorithms.
“For example, our implementation of the Gene Set Enrichment Analysis program takes all microarray gene data and assembles it into sets following biological precepts and functions as well as interactions and pathways. Pathway Studio does this by connecting the identifiers in the experimental data to their biological entities contained in the ResNet database. ResNet Mammalian represents all of the interactions contained in Medline and 47 full-text journals.”