Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method ( “QUSIM”) and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.
References
[1]
Ifediba, M.A.; Moore, A. In vivo imaging of the systemic delivery of small interfering rna. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2012, 4, 428–437, doi:10.1002/wnan.1158.
[2]
Rettig, G.R.; Behlke, M.A. Progress toward in vivo use of sirnas-ii. Mol. Ther. 2012, 20, 483–512, doi:10.1038/mt.2011.263.
[3]
Burnett, J.C.; Rossi, J.J. Rna-based therapeutics: Current progress and future prospects. Chem. Bio. 2012, 19, 60–71, doi:10.1016/j.chembiol.2011.12.008.
Zhou, J.; Swiderski, P.; Li, H.; Zhang, J.; Neff, C.P.; Akkina, R.; Rossi, J.J. Selection, characterization and application of new rna hiv gp 120 aptamers for facile delivery of dicer substrate sirnas into hiv infected cells. Nucleic Acids Res. 2009, 37, 3094–3109, doi:10.1093/nar/gkp185.
[8]
Zhou, J.; Li, H.; Li, S.; Zaia, J.; Rossi, J.J. Novel dual inhibitory function aptamer-sirna delivery system for hiv-1 therapy. Mol. Ther. 2008, 16, 1481–1489, doi:10.1038/mt.2008.92.
[9]
Wheeler, L.A.; Trifonova, R.; Vrbanac, V.; Basar, E.; McKernan, S.; Xu, Z.; Seung, E.; Deruaz, M.; Dudek, T.; Einarsson, J.I.; et al. Inhibition of hiv transmission in human cervicovaginal explants and humanized mice using cd4 aptamer-sirna chimeras. J. Clin. Invest. 2011, 121, 2401–2412, doi:10.1172/JCI45876.
[10]
Ni, X.; Zhang, Y.; Ribas, J.; Chowdhury, W.H.; Castanares, M.; Zhang, Z.; Laiho, M.; DeWeese, T.L.; Lupold, S.E. Prostate-targeted radiosensitization via aptamer-shrna chimeras in human tumor xenografts. J. Clin. Invest. 2011, 121, 2383–2390, doi:10.1172/JCI45109.
[11]
Pastor, F.; Kolonias, D.; Giangrande, P.H.; Gilboa, E. Induction of tumour immunity by targeted inhibition of nonsense-mediated mRNA decay. Nature 2010, 465, 227–230.
[12]
Neff, C.P.; Zhou, J.; Remling, L.; Kuruvilla, J.; Zhang, J.; Li, H.; Smith, D.D.; Swiderski, P.; Rossi, J.J.; Akkina, R. An aptamer-siRNA chimera suppresses HIV-1 viral loads and protects from helper CD4(+) T cell decline in humanized mice. Sci. Transl. Med. 2011, 3, 66–ra6, doi:10.1126/scitranslmed.3001581.
[13]
Zhou, J.; Neff, C.P.; Swiderski, P.; Li, H.; Smith, D.D.; Aboellail, T.; Remling-Mulder, L.; Akkina, R.; Rossi, J.J. Functional in vivo delivery of multiplexed anti-hiv-1 sirnas via a chemically synthesized aptamer with a sticky bridge. Mol. Ther. 2013, 21, 192–200, doi:10.1038/mt.2012.226.
[14]
Tuerk, C.; Gold, L. Systematic evolution of ligands by exponential enrichment: Rna ligands to bacteriophage t4 DNA polymerase. Science 1990, 249, 505–510.
[15]
Ellington, A.D.; Szostak, J.W. In vitro selection of rna molecules that bind specific ligands. Nature 1990, 346, 818–822, doi:10.1038/346818a0.
[16]
Thiel, K.W.; Hernandez, L.I.; Dassie, J.P.; Thiel, W.H.; Liu, X.; Stockdale, K.R.; Rothman, A.M.; Hernandez, F.J.; McNamara, J.O., 2nd; Giangrande, P.H. Delivery of chemo-sensitizing sirnas to her2+-breast cancer cells using rna aptamers. Nucleic Acids Res. 2012, 40, 6319–6337.
[17]
Thiel, W.H.; Bair, T.; Peek, A.S.; Liu, X.Y.; Dassie, J.; Stockdale, K.R.; Behlke, M.A.; Miller, F.J.; Giangrande, P.H. Rapid identification of cell-specific, internalizing rna aptamers with bioinformatics analyses of a cell-based aptamer selection. PLoS One 2012, 7, e43836.
Varkouhi, A.K.; Scholte, M.; Storm, G.; Haisma, H.J. Endosomal escape pathways for delivery of biologicals. J. Control. Release 2011, 151, 220–228, doi:10.1016/j.jconrel.2010.11.004.
[20]
Rockey, W.M.; Hernandez, F.J.; Huang, S.Y.; Cao, S.; Howell, C.A.; Thomas, G.S.; Liu, X.Y.; Lapteva, N.; Spencer, D.M.; McNamara, J.O.; et al. Rational truncation of an rna aptamer to prostate-specific membrane antigen using computational structural modeling. Nucleic Acid Ther. 2011, 21, 299–314.
[21]
Kim, M.Y.; Jeong, S. In vitro selection of rna aptamer and specific targeting of erbb2 in breast cancer cells. Nucleic Acid Ther. 2011, 21, 173–178.
[22]
Lupold, S.E.; Hicke, B.J.; Lin, Y.; Coffey, D.S. Identification and characterization of nuclease-stabilized rna molecules that bind human prostate cancer cells via the prostate-specific membrane antigen. Cancer Res. 2002, 62, 4029–4033.
[23]
Thiel, W.H.; Bair, T.; Wyatt Thiel, K.; Dassie, J.P.; Rockey, W.M.; Howell, C.A.; Liu, X.Y.; Dupuy, A.J.; Huang, L.; Owczarzy, R.; et al. Nucleotide bias observed with a short selex rna aptamer library. Nucleic Acid Ther. 2011, 21, 253–263.
[24]
McNamara, J.O.; Kolonias, D.; Pastor, F.; Mittler, R.S.; Chen, L.P.; Giangrande, P.H.; Sullenger, B.; Gilboa, E. Multivalent 4-1bb binding aptamers costimulate cd8(+) t cells and inhibit tumor growth in mice. J. Clinl. Invest. 2008, 118, 376–386.
[25]
Endo, Y.; Mitsui, K.; Motizuki, M.; Tsurugi, K. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J. Bio. Chem. 1987, 262, 5908–5912.
[26]
Ippoliti, R.; Lendaro, E.; Bellelli, A.; Brunori, M. A ribosomal protein is specifically recognized by saporin, a plant toxin which inhibits protein synthesis. FEBS Lett. 1992, 298, 145–148, doi:10.1016/0014-5793(92)80042-F.
[27]
French, R.R.; Penney, C.A.; Browning, A.C.; Stirpe, F.; George, A.J.; Glennie, M.J. Delivery of the ribosome-inactivating protein, gelonin, to lymphoma cells via cd22 and cd38 using bispecific antibodies. Br. J. Cancer 1995, 71, 986–994.
[28]
Sforzini, S.; de Totero, D.; Gaggero, A.; Ippoliti, R.; Glennie, M.J.; Canevari, S.; Stein, H.; Ferrini, S. Targeting of saporin to hodgkin's lymphoma cells by anti-cd30 and anti-cd25 bispecific antibodies. Br. J. Haematol. 1998, 102, 1061–1068, doi:10.1046/j.1365-2141.1998.00859.x.
[29]
Glennie, M.J.; Brennand, D.M.; Bryden, F.; McBride, H.M.; Stirpe, F.; Worth, A.T.; Stevenson, G.T. Bispecific f(ab' gamma)2 antibody for the delivery of saporin in the treatment of lymphoma. J. Immunol. 1988, 141, 3662–3670.
[30]
Stirpe, F.; Barbieri, L.; Battelli, M.G.; Soria, M.; Lappi, D.A. Ribosome-inactivating proteins from plants: Present status and future prospects. Biotechnology (N Y) 1992, 10, 405–412.
[31]
Barbieri, L.; Bolognesi, A.; Dinota, A.; Lappi, D.A.; Soria, M.; Tazzari, P.L.; Stirpe, F. Selective killing of cd4+ and cd8+ cells with immunotoxins containing saporin. Scand. J. Immunol. 1989, 30, 369–372, doi:10.1111/j.1365-3083.1989.tb01223.x.
[32]
Montecucchi, P.C.; Lazzarini, A.M.; Barbieri, L.; Stirpe, F.; Soria, M.; Lappi, D. N-terminal sequence of some ribosome-inactivating proteins. Int. J. Pept. Protein Res. 1989, 33, 263–267.
[33]
Lappi, D.A.; Esch, F.S.; Barbieri, L.; Stirpe, F.; Soria, M. Characterization of a saponaria officinalis seed ribosome-inactivating protein: Immunoreactivity and sequence homologies. Biochem. Biophys. Res. Commun. 1985, 129, 934–942, doi:10.1016/0006-291X(85)91981-3.
[34]
Lollini, P.L.; Nicoletti, G.; Landuzzi, L.; de Giovanni, C.; Rossi, I.; Di Carlo, E.; Musiani, P.; Muller, W.J.; Nanni, P. Down regulation of major histocompatibility complex class i expression in mammary carcinoma of her-2/neu transgenic mice. Int. J. Cancer 1998, 77, 937–941, doi:10.1002/(SICI)1097-0215(19980911)77:6<937::AID-IJC24>3.0.CO;2-X.
[35]
Nanni, P.; Pupa, S.M.; Nicoletti, G.; De Giovanni, C.; Landuzzi, L.; Rossi, I.; Astolfi, A.; Ricci, C.; De Vecchi, R.; Invernizzi, A.M.; Di Carlo, E.; Musiani, P.; Forni, G.; Menard, S.; Lollini, P.L. P185(neu) protein is required for tumor and anchorage-independent growth, not for cell proliferation of transgenic mammary carcinoma. Int. J. Cancer 2000, 87, 186–194, doi:10.1002/1097-0215(20000715)87:2<186::AID-IJC5>3.0.CO;2-1.
Myers, A.C.; Kovach, J.S.; Vuk-Pavlovic, S. Binding, internalization, and intracellular processing of protein ligands. Derivation of rate constants by computer modeling. J. Biol. Chem. 1987, 262, 6494–6499.
[38]
Tiffany, C.W.; Slusher, B.S. Measurement of glutamate carboxypeptidase ii (naaladase) enzyme activity by the hydrolysis of [(3)h]-n-acetylaspartylglutamate (naag). Curr. Protoc. Pharmacol 2002, Chapter 3, Unit3.10, doi:10.1002/0471141755.ph0310s15.
[39]
Keefe, A.D.; Pai, S.; Ellington, A. Aptamers as therapeutics. Nat. Rev. Drug Discov. 2010, 9, 537–550, doi:10.1038/nrd3141.
[40]
Thiel, K.W.; Giangrande, P.H. Therapeutic applications of DNA and RNA aptamers. Oligonucleotides. 2009, 19, 209–222, doi:10.1089/oli.2009.0199.
[41]
Zhou, J.; Bobbin, M.L.; Burnett, J.C.; Rossi, J.J. Current progress of RNA aptamer-based therapeutics. Front. Genet. 2012, 3, 234.
[42]
Thiel, K.W.; Giangrande, P.H. Intracellular delivery of RNA-based therapeutics using aptamers. Ther. Deliv. 2010, 1, 849–861, doi:10.4155/tde.10.61.
[43]
Zhou, J.; Rossi, J.J. The therapeutic potential of cell-internalizing aptamers. Curr. Top. Med. Chem. 2009, 9, 1144–1157, doi:10.2174/156802609789630893.
