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2004 World Technology Awards Winners & Finalists
Please describe the work that you are doing that you consider to be the most innovative and of the greatest likely long-term significance.
Possibly, the most pronounced technological advance in mammalian research in recent years is the generation of tools to suppress the expression of individual genes through RNA interference (RNAi). The discovery of RNAi whereby double stranded (ds)RNA molecules suppress the expression of complementary genes is known for some years now in lower eukaryotes and plants. However, a much wider interest in this phenomenon was sparked by the discovery that also in somatic mammalian cells dsRNA can selectively suppress gene expression through RNAi. Using short synthetic RNA duplexes (siRNAs) it was possible to induce sequence-specific gene silencing yet avoid the non-selective toxic effects of long dsRNAs. One major limitation of this technique was its transient effect (maximum a week) owing to the limited availability and stability of the synthetic oligonucleotides. To overcome this limitation, we designed a mammalian expression vector that directs the synthesis of siRNA-like transcripts (pSUPER, suppression of endogenous RNA). We used the Polymerase-III H1-RNA gene promoter, as it produces a small RNA transcript lacking a poly-adenosine tail and has a well-defined start of transcription and a termination signal consisting of five thymidines in a row. We designed the gene-specific insert such that it specifies a 19 nt sequence derived from the target transcript, separated by a short spacer of 9 nt from the reverse complement of the same 19 nt sequence. The resulting transcript is predicted to fold back on itself to form a 19 basepair stem-loop structure, resembling that of C. elegans Let-7. By targeting p53 expression we could demonstrate that RNAi vectors such as pSUPER can suppress endogenous protein expression to the extent that it completely abrogates its function. Most importantly, this inhibition was highly specific and maintained over a long period of time.
To demonstrate the powerfulness and applicability of the pSUPER RNAi vector technology in studying cancer we attempted to silence the expression of oncogenes. We chose to inactivate the K-RASV12 oncogene not only because the RAS genes are frequently mutated in human cancers but mainly since it differs from the wild type allele by a single base pair. To allow interference with RASV12 expression in tumor cells we developed a retroviral vector that targets the gene at the site of mutation. We showed in this study that oncogenic alleles of K-RAS could be specifically and stably inactivated in human cancer through the use of a viral RNA interference vector, leading to loss of tumorigenicity. We concluded from this study that by designing target sequences that span common mutations or chromosomal translocation breakpoints found in tumors, RNAi vectors can be use to specifically inhibit the altered transcripts and possibly affect tumorigenicity.
Tumor suppressors are genes found inactivated (by mutations, deletions or epigentic events) in a subset of human cancers. However, each individual tumor contains a large number of genetic alterations, obscuring the relative role that each variation carries towards tumorigenesis. Recently, we have described an approach to directly identify genes whose function is to protect primary human cells from initiating neoplastic transformation. We studied the tumor-suppressive functions of the human INK4A locus, which encodes for the p16INK4A and p14ARF genes and is inactivated in many cancers by several distinct ways. In mouse model systems, p19ARF plays an important protective role from tumorigenicity whereas the loss of p16INK4A gene causes only a limited induction of tumors. In contrast, we find that p16INK4A is the major tumor suppressor of the human INK4A locus. Namely, p16INK4A synergizes with p53 to protect primary cells from unrestricted growth and from oncogenic transformation. p14ARF, on the other hand, regulates through p53 the growth of primary human cells in normal culture conditions but the oncogenic activation of p53 is p14ARF independent. This paper paved the way to study tumor suppressors using a combination of a controlled and well-defined in vitro transformation assay with specific RNAi tools. This allows replacing correlative studies based on already existing tumor material with a direct assessment of novel tumor suppressive activity.
Reuven Agami performed his PhD research (Thesis: Cell cycle and apoptosis control induced by the tyrosine kinase c-Abl) within the department of molecular genetics at the Weizmann Institute of Science, Israel. During his research he identified a novel DNA-damage-induced apoptosis pathway that requires the activation of c-Abl kinase and tyrosine phosphorylation of the p53 homologue- p73?. As a post-doc in the group of Prof. Rene Bernards he initiated his own line of research to identify rapid molecular events that initiate a p53 independent DNA damage response. In September 2001 he started his own group at the Netherlands Cancer Institute, which consists now of three postdocs, three PhD students, a technician and two undergraduate students. He developed an RNAi vector system, which is used extensively in the two main research topics in his group.
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