Cell-surface receptor specific delivery of short interfering RNAs via bivalent aptamer-siRNA transcripts or covalent antibody-siRNA conjugates (2007)

RNA interference (RNAi) represents an evolutionary conserved mechanism for the posttranscriptional regulation of gene expression first discovered in plants and flatworms. In higher eukaryotes posttranscriptional gene silencing is mediated by short interfering RNAs (siRNAs) that induce the sequence specific degradation of complementary mRNA. For cancer therapy siRNA-based drugs offer the potential to selectively eliminate neoplastic cells by the silencing of genes that are crucial for cell growth and viability. Unlike small molecule drugs siRNAs are not passively taken up by target cells. Therefore the efficient intracellular delivery of functional siRNAs is one of the biggest obstacles which have to be overcome for the widespread therapeutic application of RNA interference based drugs. The first aim of this thesis was to characterize the efficacy of siRNAs directed against eukaryotic elongation factor 2 (EEF2), a key component of the translational machinery. Efficient inhibition of EEF2 should result in the induction of apoptosis and cell death. It was possible to identify potent siRNA sequences that efficiently and sequence specifically mediated EEF2 gene silencing. Furthermore it was proven that the siRNA induced depletion of EEF2 mRNA efficiently induced cell death in various cancer derived cell lines. These data demonstrate the high potential of siRNAs targeting EEF2 to be used as cytotoxic component for siRNA based cancer therapy. The second aim of this thesis was the evaluation of two strategies for the ligand targeted, cell surface receptor specific delivery of functional siRNAs directed against EEF2. Here the affinity of an RNA-aptamer specifically binding to the prominent tumour marker PSMA that is overexpressed on the cell surface of prostate cancer cells was exploited for the cell surface specific delivery of Aptamer siRNAs. Previous studies have demonstrated that by the use of monovalent anti PSMA aptamer it was possible to deliver siRNAs to PSMA expressing LNCaP cells. However the overall activity observed in these experiments was only moderate. Therefore it was aimed to increase the therapeutic efficacy of the monovalent aptamer siRNA transcript. Since the internalization rate of PSMA can be increased upon binding of an anti-PSMA antibody it was predicted that the internalization of the aptamer siRNA transcripts and thus their efficiency could be enhanced by increasing their valency by rational design. Two anti PSMA aptamers were assembled in a way that each binding sequence could independently fold into its active conformation. It was shown that the binding specificity of the bivalent aptamer siRNA transcripts was comparable to that of the monovalent transcript. The binding affinity of the bivalent aptamer siRNAs however was approximately two fold higher. In order to characterize the internalization characteristics of the aptamer siRNAs a new flow cytometric internalization assay was established which allowed to monitor the internalization in a quantitative and time resolved manner. The data obtained showed that bivalent transcripts were internalized up to four fold more efficiently compared to the monovalent xPSM-A3-siEEF2. After 60 min approximately 32% of initially bound bivalent transcripts were internalized wheras only 8% of the of initially bound monovalent aptamer siRNAs were taken up by LNCaP cells. It could be proven that cytotoxic effects of the bivalent aptamer siRNA transcripts could be addressed to the siRNA mediated depletion of EEF2 mRNA and were restricted to PSMA expressing LNCaP cells. Besides that these effects were siRNA sequence specific. The bivalent transcripts exhibited a dramatic increase in cytotoxic efficacy and potency compared to their monovalent counterparts. These data lead to the conclusion that the enhanced biological activity could be addressed to the improved cellular uptake which leads to a higher intracellular concentration of functional siRNAs. The second approach for the cell surface receptor specific delivery of siRNAs involved the generation of covalently bound siRNA-antibody conjugates. Here synthetic siRNAs carrying a reactive amino group at the 3’end of the siRNA sense strand were conjugated to an anti CD30 specific antibody by the application of two different heterobifunctional linkers (SPDP/SMCC). On average three siRNA molecules could be fused to one antibody. The conjugation chemistry applied did not affect the antibody binding characteristics. Finally it was demonstrated that the generated antibody-siRNA conjugates siRNA sequence specifically induced gene silencing and subsequent cell death in CD30 expressing L540cy Hodgkin lymphoma cells. Until now this is the first report describing the specific gene silencing of covalent antibody-siRNA conjugates.