Christopher Lord, Ph.D., staff scientist, and colleagues at the London-based Institute for Cancer Research, are using RNAi technology and high-throughput RNAi screens for target identification and validation in cancer drug discovery. RNAi screens have particular use for identifying drug targets in defined subsets of patients, according to Dr. Lord.
As an example, he describes his work in the identification of therapeutic targets that may be useful in women with breast or ovarian cancer who bear mutations in either the BRCA1 or BRCA2 genes. In 2005, Dr. Lord and colleagues used RNAi technology to demonstrate that cells with mutations in either BRCA1 or BRCA2 are much more sensitive to RNAi knockdown of a DNA repair enzyme, PARP, than are normal cells. This work led directly to a clinical trial of PARP inhibitors in patients.
“The PARP inhibitors are now in a Phase II trial,” says Dr. Lord. “We went very quickly from RNA inhibition, to small molecule inhibitors, to drug-like molecules, to trial.”
The use of siRNA to perform genome-wide screens for target identification and validation has advantages and disadvantages, according to Dr. Lord. RNAi is an attractive tool because it works well in a large variety of cell types and has the potential to knockdown every gene with a relatively high degree of selectivity. Furthermore, commercially available siRNA libraries have made the technology more accessible and user friendly.
Limitations to the application of siRNA screens in drug discovery include the cost of the libraries and the relatively short duration of silencing with transfected siRNA.
An alternative technique for performing gene silencing screens relies on plasmid or viral vectors that deliver shRNA precursors that are processed into siRNA molecules inside a cell. At present, available shRNA libraries are not optimally robust, in Dr. Lord’s view; their potential for recombination compromises their usefulness.
Opportunities for the future include generating more robust genome-wide plasmid libraries and lowering the cost of siRNA libraries. Dr. Lord also describes the advantages to be gained by combining plasmid-based RNAi screens with next-generation sequencing technologies such as Illumina’s Solexa or Applied Biosystems’ SOLiD sequencing systems. This approach could help solve the problems of both price and speed in genome-wide screens.
In her talk, Gwen Fewell, Ph.D., product manager for RNAi at Open Biosystems (www.openbiosystems.com), will describe how the company’s Expression Arrest™ microRNA-adapted shRNA (shRNAmir) libraries overcome some of the drawbacks of siRNA triggers and enable a range of RNAi applications based on the technology’s ability to achieve stable gene knockdown, to perform inducible RNAi experiments, to facilitate in vivo gene silencing, and to enable multiplexed whole-genome screening strategies.
Developed in collaboration with Greg Hannon, Ph.D., professor at Cold Spring Harbor Laboratory, and Steven Elledge, Ph.D., professor at Harvard Medical School, the shRNAmir technology incorporates triggers modeled after primary miRNA transcripts. This strategy allows for processing via the endogenous RNAi pathway, “which has been shown to produce increased and specific knockdown,” says Dr. Fewell.
Libraries targeting the human or mouse genomes include multiple shRNAmir’s per gene packaged in either lentiviral or retroviral vectors. The presence of a TurboGFP marker in the lentiviral vector enables tracking of shRNAmir expression in transfected or transduced cells.
In August, Open Biosystems added inducible TRIPZ lentiviral shRNAmir libraries targeting the human genome to its family of genome-wide, vector-based RNAi products. The TRIPZ lentiviral vector is engineered to be a Tet-On® system, and the shRNA is turned on in the presence of doxycycline. The addition of a TurboRFP marker allows visual tracking of inducible shRNAmir expression.
“Inducible shRNA takes RNAi to the next level,” says Dr. Fewell. “Researchers can now analyze in parallel the on-and-off states of a gene and study its interactions within complex pathways. It also allows researchers to examine the function of essential genes by turning them off temporarily without killing the cells. TRIPZ lentiviral shRNA makes it possible to analyze as well as validate the association between silencing of a target gene and a particular phenotype in the same experiment,” she adds.
Ongoing technology development at Open Biosystems is focusing on enhancing methods for genome-wide screening using multiplexed or pooled RNAi formats.
“Each hairpin expressing vector in our shRNAmir libraries has a unique 60 nucleotide molecular bar code that allows you to track the abundance of a given shRNA in a complex population,” Dr. Fewell explains. Users can pool thousands of different shRNAs, transduce a single population of cells, and perform positive or negative selection screens. The technology is applicable for a variety of biological effects associated with an identifiable and selectable phenotype such as cell survival, increased proliferation, adhesion, migration, or marker expression, reports Dr. Fewell.
“This multiplexed RNAi approach can be set up to study oncogenic pathways, mechanism of action studies, or the effects of drugs on cells sensitized by integrating specific shRNA,” notes Dr. Fewell.
A recent example of the application of multiplexed RNAi for genome-wide screening comes from the laboratory of Michael Green, Ph.D., and colleagues at the University of Massachusetts Medical School. They used a genome-wide pooled shRNAmir approach to identify a pathway of 28 genes required for Ras-mediated epigenetic silencing of the proapoptotic gene Fas. Activation of the Ras oncogene blocks Fas expression, thereby inhibiting Fas-induced apoptosis.