Allele specific inhibition: a novel cancer therapy using genotype specific drugs
01 / 2003 - 01 / 2007
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)
The purpose of this project is to develop highly selective specific inhibitors for cancer cells. Synthetic short interfering RNA (siRNA) has been shown to be a highly selective and potent inhibitor of genes in mammalian cells. In this project we will develop siRNAs as a therapeutic approach for genotype specific inhibition of tumor growth. Cancer cells differ from normal cells in that they have lost large segments of DNA. A subset of these regions with loss of heterozygosity (LOH) are early clonal events in tumorigenesis and these regions with LOH are thought to contain tumor suppressor genes. LOH regions are usually much larger than the tumor suppressor gene and contain many other genes, including genes encoding essential functions. This implies that the cancer cells differ from normal cells in that a subset of essential genes has become hemizygous. This clonal genetic difference can be used to specifically target cancer cells. We have recently shown that inhibition of the allele of an essential gene using allele specific antisense oligonucleotides (ODNs) can inhibit tumor growth in vivo. No major side-effects were detected (Fluiter et al. 2002). Although succesful, the use of conventional antisense phosphorothioate (PS) oligonucleotides as allele specific drugs resulted in non-sequence specific toxicity and a small therapeutic window. The use of the novel ODN chemistry of Locked Nucleic Acid (LNA) as allele specific drugs resulted in a major increase in affinity and efficacy. Because we have shown proof of principle that allele specific inhibition might be a viable therapeutic approach we propose with this project to study the therapeutic use of siRNA as allele specific anti-cancer drugs. The rationale in choosing siRNA for allele specific drugs is that numerous publications have shown that siRNA is very effective in gene knockdown at tenfold lower dosages as normally used with ODNs. In addition, these early reports have shown that the success rate of finding a potent siRNA is much better as compared with finding a potent ODN. Although it is a very novel technique, siRNA is described to inhibit targeted genes very effectively and importantly it can discriminate between single nucleotide mismatches. siRNAs will be tested in their ability for genotype specific inhibition of hemizygous essential genes in tumor cells. The new pSUPER vector system, which directs the synthesis of siRNAs in human cancer cells, will be used to rapidly screen potential siRNA sequences in vitro. This vector system was shown to cause efficient and specific downregulation of gene expression, resulting in functional inactivation of the targeted genes (Brummelkamp et al. 2002). The siRNA sequences will be chosen to overlap single nucleotide polymorphisms (SNPs) in essential genes like RNA polymerase II and the 70 kDa subunit of replication protein A (RPA70). siRNA sequences in the pSUPER vector that show effective knock-down of the targeted protein can easily be subcloned in another vector with a T7 promotor which can be used for in vitro transcription of large amounts of this siRNA. This enzymatically prepared siRNA will be tested for use as genotype specific drug in vivo. The efficacy of the enzymatically prepared siRNA will be compared with conventional synthetic siRNA. siRNA will be tested in vitro using a panel of tumor cell lines with previously determined genotypes of the targeted genes. To test these drugs an established in vivo model will be used. Nude mice carrying two xenografts, each with a different genotype of the target gene, will be used to determine selectivity and efficacy of the treatment. If the treatment shows the expected specificity, one tumor should regress while the other tumor will grow unhindered. We expect that allele specific inhibition will be a promising approach to cancer treatment. In addition, sequence specific targeting of a gene might open the possibility of allele specific inhibition of genes involved in dominant genetic diseases.