Engineering of RNA aptamers to selectively target tumor associated extracellular markers: Improved selection against complex targets and for selective cell uptake
Zusammenfassung der Projektergebnisse
During the course of the project a series of aptamers was generated, either by library selection against purified target protein, or by selection on living human cells. The main initial goal of the project, however, to establish a widely applicable selection protocol of aptamer libraries on closely related complex targets could not be achieved. The employed strategy to use trans-well co-culture systems of closely related cells, metastatic and non-metastatic melanoma cell lines established from the same patient, did not result in the identification of aptamers that could discriminate between the two cell types. Instead aptamers that recognize both, cells used for positive selection and cells used for counter selection, were identified. It cannot be ruled out that discriminating aptamers might have been identified even within the selected pools by screening more individual clones. Alternatively, further adaption of the selection strategy might yield more positive results in the future. Some negative observations were made about the aptamer technology in general that are ignored in the literature for unknown reasons. Most importantly relatively high background binding of aptamers can be observed on any mammalian cell type. This background binding is not necessarily due to protein-RNA interactions, since similar aptamer binding to cells stripped for membrane proteins has been observed (own results). It can be assumed that background binding is neither sequence nor structure dependent, but rather due to the intrinsic chemical nature of RNA. It would be interesting to compare the background binding of oligonucleotides of the same sequence, but with different chemical modification to cells side-by-side. An RNA chemistry with much lower background binding that is further compatible with the current aptamer engineering process (e.g. reverse transcription and in vitro transcription) would be a great step forward towards the engineering of aptamers for therapeutic and diagnostic in vivo applications. It should also be noted that a high percentage of published work that deals with aptamer-mediated cell targeting in vitro and in vivo (including highly cited publications in leading scientific journals), cannot be reproduced (personal communications and own results). This supports the assumption that current aptamer technologies are better suited for ex vivo diagnostics or strictly localized in vivo applications. The identification of single stranded 5'ppp-2'fluoro-modified RNA (e.g. aptamer #10F) as a potent PRR agonist that leads to type I IFN-dependent and -indepent apoptosis in melanoma cells should be of general interest. These unrecognized potential side effects should be taken into account whenever in vivo effects of target specific aptamers are evaluated. It has been recently reported that 5'ppp RNA is the substrate for the innate immune sensor RIG-I and that melanoma cells are over sensitized to undergo apoptosis upon prolonged RIG-I signalling (11). However, the dramatic increase in RIG-I agonist activity by incorporation of 2'F-uridine modification into 5'ppp RNA is an interesting novel finding. Single stranded 5'ppp RNA containing 2'F-uridine modification is the most potent RIG-I agonist to date, and might be a novel chemoimmunotherapeutic drug candidate for the treatment of melanoma.
Projektbezogene Publikationen (Auswahl)
- (2010) In vivo selection of tumor-targeting RNA motifs. Nat Chem Biol., 6, 22-24
Mi, J., Liu, Y., Rabbani, Z.N., Yang, Z., Urban, J.H., Sullenger, B.A., Clary, B.M.
- (2011) Retroviral display in gene therapy, protein engineering, and vaccine development. ACS Chem Biol., 6, 61-74
Urban, J.H. and Merten, C.A.
- (2011) RNA in cancer vaccine therapy. Cancer Vaccines: From Research to Clinical Practice (1st edition). Informa Healthcare, London, UK
Nair S., Boczkowski, D., Pruitt, S.K., Urban, J.H.
