By Pauline Parry
August 15, 2003 | There comes a time in the evolution of every new molecular technique when the practitioners stop including an explanatory “how-it-works” slide in their presentations. By that measure, RNA interference (RNAi) has clearly arrived. At the recent Beyond Genome conference*, only one of 18 presenters discussing RNAi bothered to show such a slide.
RNAi (see Silence Is Golden, July 2003 Bio-IT World, page 26) is post-transcriptional gene silencing induced by the introduction of small interfering RNA (siRNA) molecules into a cell. The RNAi sessions focused on new technical developments and potential applications in functional genomics, drug development, cancer, and antiviral therapeutics.
siRNA can be introduced into cells as synthetic sequences or as part of a bacterial plasmid. Both methods have their pros and cons, but the pendulum is swinging toward the use of chemically synthesized molecules. However, the efficiency with which these synthetic siRNA molecules are introduced varies by cell type, and constitutes the limiting step in the control of gene-silencing efficiency.
Amgen research scientist Ren Xu illustrated the development of optimized procedures but cautioned that they were cell- and siRNA-specific and that each system would require rigorous validation. In addition, the stability of siRNA is system-specific. For example, Premlata Shankar, junior investigator and assistant professor of pediatrics at The Center for Blood Research at Harvard Medical School, achieved siRNA-mediated inhibition of HIV replication in macrophages for up to 15 days, while the siRNA did not persist in uninfected cells. Kathrin Heermeier, senior scientist at Aventis Pharmaceuticals, found that siRNA was stable for about three days. William Marshall, R&D vice president at Dharmacon, reported degradation of siRNA molecules within 10 seconds of transfection in the systems that they studied, and argued that judicious selection of the RNAi sequence does extend the stability.
Pioneering the therapeutic role of siRNA, Jo Milner, professor of cell biology at University of York, England, is beginning to search for willing American clinicians to explore the use of siRNA in patients. She demonstrated that siRNA effectively silences the human papillomavirus (HPV) in cervical cancer cell lines by selectively inducing apoptosis (programmed cell death) exclusively in infected cells. Apoptosis is induced by targeting the E7 transcript of HPV.
Milner’s studies so far have been in vitro, but she is "utterly convinced" that cervical tumors, because of their relative clinical accessibility, could be readily treated by topical application of siRNA. Given the general stability profile of siRNA, developing such a therapeutic will not be trivial. Finding an appropriate method of delivery and addressing the associated formulation and pharmacology issues will be challenging, as no evidence currently supports a topical route of administration.
Recent findings of nonspecific effects of siRNA molecules in experiments using synthetic siRNA to silence genes in the human cervical cancer cell line (Nature Biotechnology 21, 6, 635-637; 2003) did little to reduce attendees’ enthusiasm. Some researchers argued that there could not be a single protocol for RNAi, and its application would have to be refined on a case-by-case basis. Others, such as Kathy Fosnaugh, senior scientist at Sirna Therapeutics, were unfazed. "I believe that siRNA is the therapeutic of the future," she said.
In contrast to RNAi, using proteomics to discover new biomarkers is still being done with something of a “frontier approach,” according to Brad Guild, director of biomarker discovery proteomics at Millennium Pharmaceuticals. Guild and Beyond Genomics chief technology officer Steven Naylor emphasized that these are early and exciting days for this application. The stability of the proteome is still undetermined. They and other groups, including Fred Regnier’s chemistry group at Purdue University, have distinguished proteome profiles, which can be separated by age, sex, and ethnicity. However, a process bias (how the samples were collected and handled) should not be overlooked in data analysis.
Clarissa Desjardins, vice president of corporate development at Caprion Pharmaceuticals, presented an approach to overcome the sample-to-sample variation in the proteome. Caprion pre-fractionates samples into their subcellular structures and organelles before proteome analysis. This results in samples with stable, comparable proteomes: 95 percent of peptides from two healthy, age- and sex-matched individuals vary by less than 5 percent. Caprion is searching for tumor immunotherapy targets in lung and colon tissues. Ten percent of proteins are upregulated three- to tenfold or more in tumors compared to normal colon samples. Caprion’s successes also reflect its simplification of the proteome by effectively preselecting classes of proteins for study, removing the high-abundance species to reveal scarcer -- and potentially more interesting -- species.
Proteomics includes much unexplored territory, but it does have significant federal support. Susan Old, leader of the bioengineering and genomics applications group in the Division of Heart and Vascular Diseases at the National Heart, Lung, and Blood Institute, discussed the Initiative to Develop and Apply Innovative Proteomic Technologies, showing a plan reminiscent of the early stages of the genome project. Begun in September 2002, the initiative has established 10 centers of excellence, to the tune of $197 million over seven years.
Data management was another recurring theme at Beyond Genome. The European Bioinformatics Institute (EBI) is doing for proteomics what has been done for genomic and sequencing data. The EBI’s Proteomics Standard Initiative aims to create a set of universal standards for the presentation and handling of proteomic data. Although still in the planning stages, it promises to create a necessary common currency for the community.
*Beyond Genome 2003 (Cambridge Healthtech Institute): San Diego, Calif.,
June 15-19, 2003