Partnering Aptamer and RNAi Technologies (2006)

RNA interference (RNAi) is an endogenous mechanism for post-transcriptional gene regulation. There are two major arms of the RNAi pathway: regulation of translation by micro RNA (miRNA), and directed destruction of mRNA by small interfering RNA (siRNA) [1Aravin A. Tuschl T. Identification and characterization of small RNAs involved in RNA silencing.FEBS Lett. 2005; 579: 5830-5840Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar]. The process of RNAi involves enzymatic cleavage by the RNAse III enzyme, Dicer, of precursor duplexes into siRNAs of between 21-23 bases in length with two-base 3' overhangs. These siRNAs are incorporated into an RNA-induced silencing complex (RISC) that selects the antisense strand that eventually serves as a guide for the identification of complementary target mRNAs. The net result of this process is targeted destruction of an mRNA complementary to the siRNA antisense component. Most eukaryotic cells harbor the machinery for effecting RNAi, and it has thus become one of the more popular choices for sequence-specific inhibition of gene expression. RNAi has rapidly progressed from an interesting phenomenon to an essential epigenetic tool and promising therapeutic [2Hannon G.J. Rossi J.J. Unlocking the potential of the human genome with RNA interference.Nature. 2004; 431: 371-378Crossref PubMed Scopus (961) Google Scholar]. RNAi is now entering the clinic for the treatment of conditions ranging from adult macular degeneration to respiratory syncitial virus (RSV) infection. The major impediment to the widespread use of RNAi as a therapy is delivery of the siRNAs to the appropriate tissues, since negatively charged polymers such as siRNAs do not readily traverse cellular membranes without some protective covering [3Behlke M.A. Progress towards in vivo use of siRNAs.Mol. Ther. 2006; 13: 644-670Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar]. A number of delivery schemes for siRNAs have been proposed, and include cholesterol-conjugated siRNAs [4Soutschek J. et al.Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.Nature. 2004; 432: 173-178Crossref PubMed Scopus (1897) Google Scholar], bi-layer liposomes [5Zimmermann T.S. et al.RNAi-mediated gene silencing in non-human primates.Nature. 2006; 441: 111-114Crossref PubMed Scopus (1178) Google Scholar], transferrin-conjugated β-cycodextrins [6Hu-Lieskovan S. et al.Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma.Cancer Res. 2005; 65: 8984-8992Crossref PubMed Scopus (501) Google Scholar], antibody fragments conjugated to basic protamine [7Song E. et al.Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.Nat. Biotechnol. 2005; 23: 709-717Crossref PubMed Scopus (893) Google Scholar], and chitostatin nanoparticles [7Song E. et al.Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.Nat. Biotechnol. 2005; 23: 709-717Crossref PubMed Scopus (893) Google Scholar]. With the exception of cholesterol, for each of these approaches, the siRNAs must be complexed with the carrier particles, requiring separate preparation of the carrier and the siRNAs. The cholesterol modification allows covalent attachment of the siRNA to a cholesterol moiety. The cholesterol in turn binds to proteins that traffic the complex primarily to the liver or jejunum [4Soutschek J. et al.Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.Nature. 2004; 432: 173-178Crossref PubMed Scopus (1897) Google Scholar]. Two recent publications provide exciting new data expanding the options for cell type-specific delivery of siRNAs by joining the siRNAs to RNA molecules called aptamers [8McNamara II, J.O. et al.Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.Nat. Biotechnol. 2006; 24: 1005-1015Crossref PubMed Scopus (861) Google Scholar, 9Chu T.C. et al.Aptamer mediated siRNA delivery.Nucleic Acids Res. 2006; 34: e73Crossref PubMed Scopus (394) Google Scholar]. Aptamers are nucleic acids derived with SELEX (systematic evolution of ligands by exponential enrichment), which generates high-affinity binding ligands for interactions with other macromolecules [10Tuerk C. et al.RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase.Proc. Natl. Acad. Sci. USA. 1992; 89: 6988-6992Crossref PubMed Scopus (368) Google Scholar]. Depending upon the sequence complexity of the nucleic acid mixture used in the selection process, an astoundingly large variety of shapes are possible, which allows for selection of a specific shape or set of shapes that exhibit high-affinity binding to a selected ligand. Each of the new studies made use of an aptamer selected for high-affinity binding to the prostate-specific membrane antigen (PSMA), a cell surface receptor that is overexpressed in prostate cancer cells and vascular endothelium. Both groups asked whether the PSMA-targeting aptamer could selectively deliver the siRNA to cells expressing this receptor. McNamara et al. used the aptamer to deliver siRNAs targeting polo-like kinase 1 (PLK1) and BCL2, which are survival genes overexpressed in many human tumors [8McNamara II, J.O. et al.Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.Nat. Biotechnol. 2006; 24: 1005-1015Crossref PubMed Scopus (861) Google Scholar]. Chu et al. chose to deliver siRNAs targeting the gene for lamin A/C or GAPDH, two genes routinely used to demonstrate proof-of-principle of new siRNA approaches [9Chu T.C. et al.Aptamer mediated siRNA delivery.Nucleic Acids Res. 2006; 34: e73Crossref PubMed Scopus (394) Google Scholar]. In addition, the two sets of workers used different methods to join the PSMA aptamers to the siRNAs. MacNamara et al. joined the aptamers to the siRNAs covalently, whereas Chu et al. used strepavidin to join biotin end-labeled siRNAs to the aptamer. Both groups used two prostate cancer cell lines, LNCaP and PC3, to demonstrate selectivity in siRNA uptake as a consequence of aptamer binding to PSMA. LNCaP cells express PSMA whereas PC3 cells do not. Both reports demonstrated that the PSMA aptamers only delivered the siRNAs to the LNCaP cells, thereby verifying the selectivity. Both groups of investigators took advantage of the observation that duplex RNAs long enough to be cleaved by Dicer can be effective triggers of RNAi in mammalian cells [11Kim D.H. et al.Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy.Nat. Biotechnol. 2005; 23: 222-226Crossref PubMed Scopus (729) Google Scholar, 12Siolas D. et al.Synthetic shRNAs as potent RNAi triggers.Nat. Biotechnol. 2005; 23: 227-231Crossref PubMed Scopus (368) Google Scholar]. Each group used these longer duplexes to tether the PSMA aptamer with the intent that the aptamer portion would be cleaved off of the siRNA once the complexes or chimeric molecules entered the cytosol where Dicer is present for cleavage. Chu et al. used 27-mer duplexes to attach the aptamer. Two different approaches were tested to join the siRNAs to the aptamer. The first used a 5' biotin on the sense strand, allowing direct interaction with strepavidin conjugated to the aptamer. The other approach used a di-thiol linker between the 5' base and the biotin such that the biotin moiety could be attached by reducing the di-thiols. MacNamara et al. carefully modeled the aptamer secondary structure so it could be covalently joined to the sense strand of a Dicer substrate siRNA. There are pros and cons to each approach. The method of Chu et al. results in four siRNAs per strepavidin, since each molecule of strepavidin can bind four biotins. In theory, this would allow multiplexing of different siRNAs with the same strepavidin-aptamer carrier, but the requirement to join the aptamer to the siRNA by non-covalent linkages could have some drawbacks for clinical applications, such as dissociation of the complex in serum. The covalent linkage of the aptamer with the siRNAs described by MacNamara et al. is a more straightforward method for preparing the chimeric molecules, but requires synthesizing fairly long RNAs containing the aptamer fused to the sense strand. Depending on the size of the aptamer, this could result in poor synthetic yields and high production costs. Nevertheless, it was refreshing to see very similar biological results obtained from the two groups using different aptamer-siRNA complexes. In the study by Chu et al., no backbone modifications for stabilization were utilized, yet the aptamer-siRNA complex was as effective as a cationic lipid encapsulated siRNA in cell culture. The effectiveness of the aptamer-siRNA complex in the LNCaP cells may result from the rapid uptake of these complexes by receptor-mediated endocytosis, which took place within thirty minutes of exposure to cells in culture. MacNamara et al. used 2' fluoro modification of the pyrimidines in the sense strand, which stabilizes the RNAs in serum. They showed that the backbone-modified aptamer-siRNA chimeras were effective in both cell culture and in vivo (see below). Furthermore, in neither case was there any indication that these complexes were activating type I interferon responses [13Schlee M. et al.siRNA and isRNA: two edges of one sword.Mol. Ther. 2006; 14: 463-470Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar]. Both groups demonstrated the specificity of the targeting with the PSMA aptamer based on results in LNcAP versus PC3 cells, although the work by MacNamara et al. went further by demonstrating that downregulation of PSMA gene expression by 5-α-dihydrotestosterone, or by blocking the receptor with an antibody, both dramatically reduced the uptake and biological efficacy of the aptamer-siRNA chimeras. These investigators also conducted extensive FACS analyses of the treated LNcAP cells to verify that the siRNAs targeting the anti-apoptotic genes BCL2 and PLK1 triggered apoptosis. Perhaps the most exciting aspect of the MacNamara et al. study is the demonstration that injection of the PSMA aptamer-Plk1-siRNA chimeras into LNcAP-derived tumors led to tumor regression in a mouse xenograft model. Both of these studies represent important advances in siRNA delivery. The power of SELEX to develop aptamers capable of binding receptors should prompt the widespread use of aptamer delivery of siRNAs via other receptors. The carefully crafted experiments in these two publications provide a powerful proof-of-principle that selective cell- or tissue-specific targeting of siRNAs can be achieved by using the appropriate aptamer/siRNA combinations.