Aptamer-Based Control of Gene Expression Utilizing Endogenous miRNAs
Authors: Jörg S. Hartig
Journal: Molecular Therapy (2018)

The ability to precisely control the expression of therapeutic or diagnostic transgenes has many important applications in gene and cell therapy. Many transgenes exhibit toxicity in cells other than their target cell population, and the generation of an immune response against both endogenous and exogenous transgenes can counteract the therapeutic intent. In other cases, the ability to control the level and/or timing of transgene expression can be key to effective therapy. In this issue of Molecular Therapy, Mou et al.1Mou H. Zhong G. Gardner M.R. Wang H. Wang Y.-W. Cheng D. Farzan M. Conditional regulation of gene expression by ligand-induced occlusion of a microRNA target sequence.Mol. Ther. 2018; 26 (this issue): 1277-1286Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar present a novel way of controlling transgene expression by making use of an RNA aptamer-ligand interaction to control the function of an endogenous microRNA (miRNA) target site placed into the 3′ intranslated region (UTR) of a transgene. Various systems have been devised to control transgene expression at the transductional, transcriptional, and post-transcriptional levels. Binding of the gene vector to specific cell surface molecules and engaging cell entry mechanisms provides the first level of control. Temporal regulation can be accomplished by placing a gene of interest under the control of transcriptional gene switches. The latter comprise at least two elements: a ligand-dependent transactivator (or transinhibitor) and a transactivator-responsive promoter driving the expression of the transgene. Beyond transcriptional and transductional targeting, miRNA-dependent post-transcriptional suppression of transgene expression has emerged as a powerful technology to increase the specificity of vector-mediated transgene expression. miRNA-based switches that modulate gene expression are useful to many gene therapy applications because they are small and do not require expression of additional proteins. Mou et al.1Mou H. Zhong G. Gardner M.R. Wang H. Wang Y.-W. Cheng D. Farzan M. Conditional regulation of gene expression by ligand-induced occlusion of a microRNA target sequence.Mol. Ther. 2018; 26 (this issue): 1277-1286Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar developed a new type of switch that uses small molecule-induced stem formation to occlude a miRNA-target sequence, thereby activating gene expression. Their switch employs an aptamer, a nucleic acid element that forms specific three-dimensional structures. Aptamers are generated to recognize a specific ligand, such as a small drug-like molecule or a cell surface marker.2Ellington A.D. Szostak J.W. In vitro selection of RNA molecules that bind specific ligands.Nature. 1990; 346: 818-822Crossref PubMed Scopus (7571) Google Scholar, 3Tuerk C. Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.Science. 1990; 249: 505-510Crossref PubMed Scopus (7993) Google Scholar The design of the new miRNA switch is shown in Figure 1. The authors introduced a miRNA target site (red) into the 3′-UTR of a transgene, which allows it to be knocked down by an endogenous cognate miRNA. In addition, they fused a tetracycline aptamer (blue) adjacent to and downstream of the miRNA target site.4Berens C. Thain A. Schroeder R. A tetracycline-binding RNA aptamer.Bioorg. Med. Chem. 2001; 9: 2549-2556Crossref PubMed Scopus (158) Google Scholar, 5Müller M. Weigand J.E. Weichenrieder O. Suess B. Thermodynamic characterization of an engineered tetracycline-binding riboswitch.Nucleic Acids Res. 2006; 34: 2607-2617Crossref PubMed Scopus (123) Google Scholar The aptamer is fused at its other end to an additional sequence partially complementary to the miRNA target site in a way that ligand binding to the aptamer induces formation of a double-stranded region, masking the miRNA target site. In this setup, the presence of tetracycline inhibits miRNA-mediated transgene knockdown, thereby serving as an on-switch for gene expression. The authors tested different designs by transfecting cultured cells and also implemented an example of this mechanism following adeno-associated virus (AAV)-mediated transduction of target cells.1Mou H. Zhong G. Gardner M.R. Wang H. Wang Y.-W. Cheng D. Farzan M. Conditional regulation of gene expression by ligand-induced occlusion of a microRNA target sequence.Mol. Ther. 2018; 26 (this issue): 1277-1286Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar In principle, the approach is modular and should be transferable to any transgene of interest. Moreover, the performance of switching gene expression in response to ligand addition was pronounced and robust in the context of several designs. Two previous studies have also made use of RNAi in combination with aptamers in order to implement ligand-dependent control of gene expression in mammalian cells.6An C.I. Trinh V.B. Yokobayashi Y. Artificial control of gene expression in mammalian cells by modulating RNA interference through aptamer-small molecule interaction.RNA. 2006; 12: 710-716Crossref PubMed Scopus (115) Google Scholar, 7Beisel C.L. Bayer T.S. Hoff K.G. Smolke C.D. Model-guided design of ligand-regulated RNAi for programmable control of gene expression.Mol. Syst. Biol. 2008; 4: 224Crossref PubMed Scopus (103) Google Scholar These approaches differ from the strategy presented here because they introduced theophylline aptamer sequences into RNA interference (RNAi)-triggering short hairpin RNAs (shRNAs). Upon addition of the ligand, the processing of the shRNA into active RNAi sequences was altered. Hence, the RNAi effect was controlled by the aptamer-ligand interaction directly, rather than by controlling the accessibility of an RNAi target site. Although the earlier approaches are interesting, they were less efficient, which might also be due to the use of theophylline instead of tetracycline aptamers. However, together with the present study, these works demonstrate that aptamers can be employed in diverse designs in order to control RNA interference effects. Combined, the results are very interesting and encouraging because they suggest that optimized designs could yield increased performance of controlling gene expression. The new approach presented by Mou et al.1Mou H. Zhong G. Gardner M.R. Wang H. Wang Y.-W. Cheng D. Farzan M. Conditional regulation of gene expression by ligand-induced occlusion of a microRNA target sequence.Mol. Ther. 2018; 26 (this issue): 1277-1286Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar demonstrates high efficiency in switching between on- and off-states and has some distinct advantages. Since endogenous miRNAs are used to control the transgene, there is no need to overexpress regulatory RNA sequences. Such overexpression could potentially be harmful by overloading the RNAi machinery in a process known as saturation. Nonspecific disturbance of RNAi-mediated processes can result in a pronounced cytotoxicity.8Grimm D. Streetz K.L. Jopling C.L. Storm T.A. Pandey K. Davis C.R. Marion P. Salazar F. Kay M.A. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.Nature. 2006; 441: 537-541Crossref PubMed Scopus (1372) Google Scholar This problem is circumvented by the presented strategy of employing endogenous RNAi species. However, the co-opting of a specific endogenous miRNA for this new task could nevertheless drain it away from its natural target site(s). In addition, the use of endogenous miRNAs could potentially be inefficient, as demonstrated in certain cases by the authors of the new study. The authors were able to overcome this potential drawback by overexpression of the RNAi species. This, again, might cause saturation of the RNAi machinery and lead to problems as mentioned above. Nevertheless, the new study demonstrates that aptamers can be utilized in several different configurations within the regulatory elements of transgenes to control their expression. The common factor of such RNA-based strategies is that they need only relatively small additional sequences to achieve regulation. Most importantly, there is no necessity to express foreign regulatory proteins. In addition to regulating RNA interference, other RNA-based strategies have been explored. Aptamer-fused ribozymes that cleave and thereby inactivate a mRNA of interest in a ligand-dependent manner have been utilized.9Felletti M. Hartig J.S. Ligand-dependent ribozymes.Wiley Interdiscip. Rev. RNA. 2017; 8https://doi.org/10.1002/wrna.1395Crossref PubMed Scopus (31) Google Scholar Furthermore, aptamers have been inserted into splice sites to control intron splicing necessary for generating a functional mRNA.10Vogel M. Weigand J.E. Kluge B. Grez M. Suess B. A small, portable RNA device for the control of exon skipping in mammalian cells.Nucleic Acids Res. 2018; (Published online February 6, 2018)https://doi.org/10.1093/nar/gky062Crossref PubMed Scopus (18) Google Scholar The mentioned advantages of using such posttranscriptional mechanisms for expression control, such as small coding space and low immunogenicity, qualify these approaches for future applications in gene therapy. However, as also discussed in the present work, the generation of aptamers for ligands that are pharmacologically more suited than the few examples known to date represents a major challenge—and major opportunity—in the field.11Berens C. Suess B. Riboswitch engineering - making the all-important second and third steps.Curr. Opin. Biotechnol. 2015; 31: 10-15Crossref PubMed Scopus (75) Google Scholar Conditional Regulation of Gene Expression by Ligand-Induced Occlusion of a MicroRNA Target SequenceMou et al.Molecular TherapyFebruary 27, 2018In BriefRNA-based switches that modulate gene expression are useful to many gene therapy applications because they are small and do not require expression of additional proteins. Here, a new class of RNA-based switches is described. These switches use small-molecule-induced stem formation to occlude a microRNA-target sequence, thereby activating gene expression. Full-Text PDF Open Access