Cell-Penetrating RNAs: New Keys to the Castle
Authors: Donald H Burke
Journal: Molecular Therapy (2012)

The plasma membrane represents the walls of the cellular fortress. It maintains cellular integrity by separating the insides from the outside, and the gatekeepers within these walls are very selective about which molecules are allowed in or kept out. Since the pioneering work on bacterial transformation by Griffith, Avery, and others more than half a century ago,1Griffith F The significance of pneumococcal types.J Hyg. 1928; 27: 113-159Crossref PubMed Scopus (652) Google Scholar,2Avery O Macleod C McCarty M Studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus Type III.J Exp Med. 1944; 79: 137-158Crossref PubMed Scopus (1799) Google Scholar a series of technologies has been developed to deliver payloads across membranes. In a forthcoming article in Molecular Therapy, Magalha˜es et al. describe a new way to unlock one of the fortress gates in the form of aptamers they dub "c1" and "Otter."3Magalha˜es MLB Byrom M Yan A Kelly L Li N Furtado T et al.A general RNA motif for cellular transfection.Mol Ther. 2015; (in press)Google Scholar These aptamers bind and penetrate into a diverse collection of mouse and human cells, including primary cells. Although there are many unanswered questions about the biology of these aptamers, it seems plausible that they could prove useful as tools for delivering molecular payloads for basic research and therapeutic applications. A wide range of strategies and reagents deliver molecules into the cell in a manner that is appropriate for in vitro studies. Cationic and lipidic transfection reagents are standard components for delivering small interfering RNA (siRNA) and plasmid DNA. Membrane permeabilization by the glycoside digitonin allows uptake of antibodies and other proteins, and shock treatments such as heat, low osmolarity, or high-voltage pulses can stimulate uptake. These methods are relatively invasive and disruptive and can potentially obscure the underlying biology one wishes to observe. They are also relatively inefficient for most primary cells, and they are not generally suitable for therapeutic or diagnostic use. Recent years have seen the rapid adoption of a gentler alternative approach involving the use of structured nucleic acid aptamers that recognize specific cell surface molecules. These can be derived from extremely diverse combinatorial libraries (~1014 species) through an iterative process of exponential enrichment (SELEX) involving selective capture and amplification of active species. To do so, a synthetic, random-sequence DNA library is either used directly or transcribed in vitro into RNA, often with 2′-fluoro (2′-F) substitutions in the pyrimidines so as to enhance stability against degradation by serum nucleases. The modified library is then incubated with a pure target protein, and the subset of the library that binds the target is captured on beads or nitrocellulose filters, amplified by reverse transcription and polymerase chain reaction, and then retranscribed for the next cycle of selection. Each cycle enriches the library many fold for species that bind the target protein until nearly all the surviving species bind the molecular target. Aptamers that bind surface proteins in their native cellular contexts can subsequently be internalized through receptor-mediated endocytosis or through membrane protein recycling. The aptamers and their cargo are thereby delivered into endosomes and (ideally) into the cytoplasm. Even when the relevant cell markers are not known or cannot be conveniently purified, the complex cell surfaces can themselves serve as the positive selection targets. This process of Cell SELEX has been used to generate aptamers that differentiate type A and type B red blood cells4Morris K Jensen K Julin C Weil M Gold L High affinity ligands from in vitro selection: complex targets.Proc Natl Acad Sci USA. 1998; 95: 2902-2907Crossref PubMed Scopus (292) Google Scholar and that target numerous tumors in situ.5Bayrac AT Sefah K Parekh P Bayrac C Gulbakan B Oktem HA et al.In vitro selection of DNA aptamers to glioblastoma multiforme.ACS Chem Neurosci. 2011; 2: 175-181Crossref PubMed Scopus (91) Google Scholar,6Van Simaeys D López-Colón D Sefah K Sutphen R Jimenez E Tan W Study of the molecular recognition of aptamers selected through ovarian cancer cell-SELEX.PLoS One. 2010; 5: e13770Crossref PubMed Scopus (100) Google Scholar,7Cerchia L Esposito C Jacobs A Tavitian B de Franciscis V Differential SELEX in human glioma cell lines.PLoS One. 2009; 24: e7971Crossref Scopus (53) Google Scholar An attractive feature of using aptamers is the ease with which they can be adopted by nonspecialists to deliver siRNA and other cargo into cells by simply lengthening the transcript and base-pairing the cargo with the appended nucleotides. This approach has been used successfully to deliver siRNA to cells that express viral or tumor markers, resulting in messenger RNA downregulation that induced tumor regression and sensitization,8Pastor F Kolonias D Giangrande P Gilboa E Induction of tumour immunity by targeted inhibition of nonsense-mediated mRNA decay.Nature. 2010; 465: 227-230Crossref PubMed Scopus (199) Google Scholar,9Ni X Zhang Y Ribas J Chowdhury WH Castanares M Zhang Z et al.Prostate-targeted radiosensitization via aptamer-shRNA chimeras in human tumor xenografts.J Clin Invest. 2011; 121: 2383-2390Crossref PubMed Scopus (121) Google Scholar,10Thiel K Giangrande P Intracellular delivery of RNA-based therapeutics using aptamers.Ther Deliv. 2010; 1: 849-861Crossref PubMed Scopus (53) Google Scholar,11Li N Nguyen H Byrom M Ellington AD Inhibition of cell proliferation by an anti-EGFR aptamer.PLoS One. 2011; 6: e20299Crossref PubMed Scopus (145) Google Scholar or that suppressed replication of HIV-1 and herpes simplex virus (HSV) in humanized mouse models.12Wheeler LA Trifonova R Vrbanac V Basar E McKernan S Zu Z et al.Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras.J Clin Invest. 2011; 121: 2401-2412Crossref PubMed Scopus (190) Google Scholar,13Zhou J Li H Zhang J Piotr S Rossi J Development of cell-type specific anti-HIV gp120 aptamers for siRNA delivery.J Vis Exp. 2011; (23 June pii: 2954)Google Scholar,14Zhou J Rossi J Aptamer-targeted RNAi for HIV-1 therapy.Methods Mol Biol. 2011; 721: 355-371Crossref PubMed Scopus (32) Google Scholar In these cases, the aptamer provided cell-type specificity for these targeted therapeutics, acting as a homing module both in cell culture and in animals. By contrast, Magalha˜es et al.3Magalha˜es MLB Byrom M Yan A Kelly L Li N Furtado T et al.A general RNA motif for cellular transfection.Mol Ther. 2015; (in press)Google Scholar identify what may best be described as a general-purpose, cell-penetrating RNA (CPR) with little apparent specificity. Random-sequence 2′-F RNA was first incubated with adhered human or mouse cells. Nonbound RNA was then washed off, and RNA that remained on the surface was removed by aggressive nuclease treatment, leaving behind only the RNAs that had become internalized. Total RNA was then recovered for reverse transcription–polymerase chain reaction amplification and further cycles. The resulting c1 and Otter aptamers internalize into eight different transformed human cell lines, five different transformed mouse cell lines, and two primary cell/tissues, including mouse mucosal cells deep in the lamina propria following vaginal delivery (Table 1). This versatile targeting is fortuitous, because the selection was not designed to demand multitarget recognition.Table 1Cell types with demonstrated ability to take up cell-penetrating RNA aptamers c1 or OtterMouse cell linesHuman cell lines MIN6B1 (MOUSE β -CELLS) HELA (CERVICAL ADENOCARCINOMA) BTC3 (MOUSE PANCREATIC INSULINOMA) JURKAT (T-LYMPHOCYTE) BEND. 3 (IMMORTALIZED MOUSE BRAIN ENDOTHELIUM) A549 (ADENOCARCINOMA OF ALVEOLAR BASAL EPITHELIUM) SOL8 (MOUSE MYOGENIC CELLS) PC3 (PROSTATE CANCER) C2C12 (MOUSE MYOBLAST) LNCAP (PROSTATIC ADENOCARCINOMA)Primary cells and tissues HEK293 (HUMAN EMBRYONIC KIDNEY) NORMAL HUMAN PRIMARY BRONCHIAL EPITHELIAL CELLS HEK-EBNA (HEK293-EXPRESSING EPSTEIN-BARR VIRUS NUCLEAR ANTIGEN 1) MOUSE VAGINAL LAMINA PROPRIA HBEC (HUMAN BRONCHIAL EPITHELIUM) Open table in a new tab Both c1 and Otter are small (~40–45 nucleotides) and strikingly similar, with relatively simple structures in which two base-paired stems are interrupted by a large, symmetrical, pyrimidine-rich internal loop with 9 (c1) or 11 (Otter) nucleotides in each strand of the internal loop (Figure 1a). The similarity is best explained by convergent evolution for recognition of very similar molecular targets. Cross-contamination during the selections is unlikely because the libraries used for mouse and human cells used different primer-binding sequences and carried different-sized random regions. To test the convergence model, the workers labeled each aptamer with a different dye and titrated one against the other. The two aptamers competed for cell entry, again suggesting that they recognize the same unidentified molecular target. Magalhães et al. also demonstrate through pathway-specific inhibition that uptake occurs through clathrin-mediated endocytosis. The potential implications of these results are broad and far-reaching. A general-purpose, off-the-shelf aptamer that can be quickly adapted for use with any cell type would be an empowering tool for research and therapeutic applications. In principle, an investigator could append a suitable 5′ or 3′ extension onto c1 or Otter, anneal the extension with a suitable oligo, and thereby deliver siRNAs or other cargo to his or her favorite cell type, all without having to generate new aptamers for each cell type or to learn specialized conjugation chemistry. This gentle delivery may be especially well suited for preserving the underlying biology of the cells, and it could even be engineered to carry multiple cargoes (Figure 1b), provided that the appendages do not interfere with recognition of the molecular target. Eventual generalization of c1 and Otter will depend, in part, on the ease with which different cargoes can be delivered, on species and cell type specificity, and on off-target effects. First, the only "cargoes" utilized in the present study are a series of fluorescent dyes. It will be important to demonstrate that uptake and cytoplasmic release are compatible with other cargoes, such as siRNA and antisense, antibodies, radiopharmaceuticals, magnetic resonance imaging contrast agents, proteins, and nanoparticles. Second, it is not yet known whether any cell types exist for which the castle gates are barred against entry by c1 or Otter, or whether they can internalize into cells from species other than mouse or human. Because the saturable molecular target has not been identified, its expression patterns and evolutionary conservation cannot be guessed. It is not yet possible to say whether these aptamers will prove to be specific to placental mammals, to vertebrate animals, to eukaryotes, or to some other phylogenetic grouping. Third, any overt cytotoxicity associated with these aptamers is not yet known, nor is there any indication of whether they activate innate immune responses, saturate RNA transport or processing pathways, or have other adverse effects. It will be important to keep in mind the lessons from a decade of siRNA studies, which have revealed multiple avenues to unintended consequences. The broad range of cell types that can be targeted by the CPRs is reminiscent of a class of compounds known as cell-penetrating peptides (CPPs). These are short, positively charged peptides that readily cross cell membranes, carrying with them whatever cargo may be attached.15Frankel A Pabo C Cellular uptake of the tat protein from human immunodeficiency virus.Cell. 1988; 55: 1189-1193Abstract Full Text PDF PubMed Scopus (2323) Google Scholar,16Green M Loewenstein P Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein.Cell. 1988; 55: 1179-1188Abstract Full Text PDF PubMed Scopus (1276) Google Scholar The mechanism by which CPPs enter cells is not based on binding to specific receptor proteins but has instead been variously modeled as involving direct penetration of the membrane through electrostatic interactions, nonspecific endocytosis, and formation of transitory structures. There are many chemistries for conjugating CPPs to various cargoes, but even this modicum of synthetic chemistry can be an obstacle for research labs whose expertise lies elsewhere. In moving from in vitro studies to whole-animal and clinical contexts, the most straightforward applications of agents such as CPRs and CPPs are likely to be those that involve surface or localized delivery. Examples include aerosolized delivery to lung epithelium, direct injection into solid tumors, or topical application to epidermis or to sites of injury. Interestingly, the low immunogenicity of nucleic acids could provide further advantages for in vivo studies. Disseminated cellular targets are more problematic because any reagent attached to a CPR or CPP is likely to be taken up by the cells of the vascular endothelium long before it reaches its targets. Indeed, there are enough unknowns in CPR technology that it would be premature to throw out the siRNA transfection reagents just yet, but clearly we now have a new "key to the castle" that appears likely to find applications both in vitro and, possibly, in the clinic.