Novel Mouse Models
Animal models can provide invaluable tools for assessing therapeutics. TaconicArtemis has developed in vivo technology platforms to help researchers gain insights into drug and target-related disease mechanisms. Christine L. Olsson, Ph.D., commercialization scientific director, reported on the company’s inducible/ reversible RNAi technology.
“We have developed mouse models that are transgenic for custom shRNAs that can be turned on and off because of an inducer element cloned along with the shRNA. This allows us to see changes in protein levels based on RNAi message levels as a measure of phenotypic parameters.”
Such models allow for the mimicking of how drugs function. “The RNAi can decrease the amount of a specific protein in different tissues, to a different degree,” Dr. Olsson said. This differential decrease in target is similar to the action of an antagonistic drug. These drugs decrease the amount of activity of their target depending upon bioavailability, etc.
“In the RNAi case, we are determining if the anticipated drug target is an actual drug target. One can reduce the amount of protein in the cells by giving the inducer, measuring the in vivo effect, removing the inducer, giving the drug of interest, and seeing if you have the same effect.”
According to Dr. Olsson, the transgenic technology begins by cloning a specific shRNA into an inducible cassette and then placing it into modified embryonic stem cells (ESC). “As in typical transgenic technologies, these are injected into normal mouse blastocysts. During embryogenesis, the ESC incorporate into the blastocyst by an unknown mechanism to create a chimeric mouse. Further genetic screening of subsequent offspring creates strains for specific RNAi.”
The process can be complicated since not all tissues have the same level of expression. “The levels of knockdown can vary. That’s why the inducer is valuable. We can change the level of induction using a tetracycline derivative that can be given in the feed and water, for example, depending on the study. You can also vary the duration of knockdown in that way.”
Taconic has also created in vivo models for reversible kinase switches that provide a way to identify biological roles of specific kinases and possible side effects that result from their inhibition. “The novelty of both technologies is in their ability to preferentially allow inhibition or induction. They are valuable models whose uses are only limited by the imagination of the investigator.”
Cilia and Cellular Phenotypes
High-throughput RNAi (HT-RNAi) screening provides a means to discover new therapeutic targets. Pedro Aza-Blanc, Ph.D., director of functional genomics resources at the Sanford-Burnham Medical Research Institute, heads a core facility that performs siRNA screens.
“HT-RNAi screening allows forward-genetic approaches in tissue culture cells by providing rapid genetic screens for cellular phenotypes. This can be applied to multiple fields including cancer, virology, stem cell biology, and metabolism. You can address any cellular phenotype as long as you have a high-throughput amenable method to detect it. To perform a screen, individual siRNAs are transfected into cells in individual wells in a high-throughput manner. The effect is measured in an assay system such as a high-throughput microscope or a plate reader.”
According to Dr. Aza-Blanc, there are two major applications for HT-RNAi screening. “The most common application is target discovery toward the treatment of diseases that can be associated with cellular phenotypes. An emerging application is to profile compound activity in the same way that genetic screens in yeast have been used in the past.
“Although it is unclear how widely applicable it will be, one can envision this technology being used to classify compounds such as those directed against the same molecular target but displaying different activities in live cells. This will help in the drug discovery pipeline as you can get an early snapshot of the compound’s mechanism of action.”
Dr. Aza-Blanc described work performed in collaboration with Joon Kim and Joseph Gleeson to understand the dynamics of primary cilia. “In recent years, we’ve seen the importance of these organelles in regulating intracellular signals involved in diverse processes from embryonic development to cancer. The disruption of their structure or function can have profound phenotypic consequences.
“We have used siRNA-screening techniques to identify modulators of cilia formation. This work exemplifies the use of siRNA libraries. Such screening can cover the entire genome as long as you have an assay to measure an effect. Other types of screens may employ library subsets of the genome such as proteases or kinases.”
HT-RNAi screening will continue to emerge as a major player in therapeutics on several fronts, Dr. Aza-Blanc said. “HT-RNAi screening can identify genetic networks involved in disease, as well as help identify an agent’s target and detect unexpected off-target activities. Overall, this approach allows one to make better informed decisions early on in the drug discovery process.”
RNAi therapeutics possess enormous potential. Researchers are just beginning to scratch the surface of this young and vibrant field. Many challenges remain, but progress continues to encourage further development.