Response to Thum et al. David M. Patrick, … , Eva van Rooij, Eric N. Olson J Clin Invest. 2011;121(2):462-463. https://doi.org/10.1172/JCI46108. Letter Thum et al. conclude that microRNA-21 (miR-21) is essential for cardiac hypertrophy and fibrosis in response to pressure overload (1). They also claim that our failure to observe a blockade to these processes in mice treated with an 8-mer locked nucleic acid–modified oligonucleot​ ide against miR-21 (called Anti-21) (2) is due to the ineffectiveness of such inhibitors. We wish to point out several caveats to their study regarding the role of miR-21 in cardiac hypertrophy and their conclusions regarding the efficacy of the Anti-21 oligonucleotide. First, we find that Anti-21 inhibits miR-21 with a half-maximal inhibitory concentration of 0.9 nM, indicating the efficacy of Anti-21. Second, Thum et al. do not state the method they used to measure miR-21 inhibition, though we assume it to be quantitative PCR (qPCR). In our hands, qPCR alone is unreliable for measuring miRNA inhibition, especially for 8-mer inhibitors, since they may be displaced during qPCR and thereby give an underrepresentation of miRNA inhibition. To demonstrate functional inhibition of a miRNA, it is important to show data from multiple assays, such as small RNA Northern blots, luciferase reporter assays, and target derepression, as shown in our study (2). Such data are lacking in the Thum et al. rebuttal, which makes comparison of the different chemistries impossible. Thum et al. also state that we measured miR-21 inhibition […] Find the latest version: https://jci.me/46108/pdf letters contrast and consistent with the findings Acknowledgments Address correspondence to: Thomas Thum, reported by Patrick et al., application of We kindly acknowledge the support of the Hannover Medical School, Institute for short 8-mer oligonucleotides against miR- Deutsche Forschungsgemeinschaft (DFG Molecular and Translational Therapeutic 21 did not affect pressure overload-induced TH903/10-1), the BMBF (01EO0802 and Strategies, Carl-Neuberg-Str. 1, 30625 Han- cardiac hypertrophy, fibrosis, and cardiac 01KU0902A), and the Fondation Leducq. nover, Germany. Phone: 49.511.532.5272; dysfunction. Currently, we do not know why Fax: 49.511.532.5274; E-mail: Thum. the reported phenotype of the miR-21–defi- Thomas Thum,1,2 Nelson [email protected]. Or to: Stefan cient mice differs from that of mice that Chau,3 Balkrishen Bhat,3 Engelhardt, Institute of Pharmacology received treatment with long miR-21 inhibi- Shashi Kumar Gupta,1 Peter S. and Toxicology, Technische Universitaet tors. Possible reasons are various means of Linsley,3 Johann Bauersachs,2 Muenchen, Munich, Germany. Phone: genetic compensation upon constitutive and Stefan Engelhardt4 49.89.4140.3260; Fax: 49.89.4140.3261; deletion of the Mir21 gene as well as poten- E-mail: [email protected]. tial off-target effects of anti-miRs that evade 1Institute for Molecular and Transla- current analysis. tional Therapeutic Strategies, IFB-Tx, and J Clin Invest. 2011;121(2):461–462. Taken together, we confirmed that the 2Department of Cardiology and Angiology, doi:10.1172/JCI45938. 8-mer anti–miR-21 is ineffective in pre- Hannover Medical School, Hannover, Ger- 3 venting cardiac disease in a mouse model many. Regulus Therapeutics, San Diego, 1. Patrick DM, et al. Stress-dependent cardiac remod- of left ventricular pressure overload, a find- California, USA. 4Institute of Pharmacolo- eling occurs in the absence of microRNA-21 in ing which is likely due to the modest and gy and Toxicology, Technische Universitaet mice. J Clin Invest. 2010;120(11):3912–3916. 2. Thum T, et al. MicroRNA-21 contributes transient nature of miR-21 suppression by Muenchen, Munich, Germany. to myocardial disease by stimulating MAP 8-mers. For long-term inhibition of miR- kinase signalling in fibroblasts. Nature. 2008; 21 function in vivo, interventions based on Conflict of interest: Thomas Thum, Johann 456(7224):980–984. longer anti-miRs are likely to prove superi- Bauersachs, and Stefan Engelhardt have 3. Liu G, et al. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis. J Exp or, due to their high potency and treatment filed a patent application for the use of miR- Med. 2010;207(8):1589–1597. duration. In contrast, short 8-mer LNA- 21 and have received royalty fees through 4. Levin AA, et al. Basic principles of the pharmaco- modified oligonucleotides against miR-21 the University of Wuerzburg. Nelson Chau, kinetics of antisense oligonucleotide drugs. In: Crooke ST, ed. Antisense Drug Technology: Principles, are of less potency and without therapeutic Balkrishen Bhat, and Peter S. Linsley are Strategies, and Applications. 2nd ed. Boca Raton, Flor- effects in vivo. employees of Regulus. ida, USA: CRC Press; 2007:183–215. Response to Thum et al. Thum et al. conclude that microRNA-21 strate functional inhibition of a miRNA, it Finally, Thum et al. postulate that con- (miR-21) is essential for cardiac hyper- is important to show data from multiple stitutive genetic deletion of miR-21 in mice trophy and fibrosis in response to pres- assays, such as small RNA Northern blots, may not reveal the functions of miR-21 in sure overload (1). They also claim that luciferase reporter assays, and target dere- cardiac disease because of compensatory our failure to observe a blockade to these pression, as shown in our study (2). Such events that mask such functions. If such processes in mice treated with an 8-mer data are lacking in the Thum et al. rebuttal, compensation occurs, it must be specific locked nucleic acid–modified oligonucleo- which makes comparison of the different for the cardiac functions of miR-21, since tide against miR-21 (called Anti-21) (2) is chemistries impossible. miR-21 null mice are resistant to lung due to the ineffectiveness of such inhibi- Thum et al. also state that we measured tumorigenesis (3), consistent with the tors. We wish to point out several caveats miR-21 inhibition on day 2 after dosing documented pro-oncogenic functions of to their study regarding the role of miR-21 with Anti-21, when in fact we measured miR-21. To further address the possibility in cardiac hypertrophy and their conclu- inhibition 3 weeks after dosing. At this of genetic compensation, we have deleted a sions regarding the efficacy of the Anti-21 time point, we observed inhibition of floxed miR-21 allele immediately prior to oligonucleotide. miR-21 in pressure-overloaded hearts at thoracic aortic constriction in mice using First, we find that Anti-21 inhibits miR- a level significantly below that of control a ubiquitously expressed tamoxifen-regu- 21 with a half-maximal inhibitory concen- mice. Thus, their approach for inhibition lated Cre transgene. These animals show tration of 0.9 nM, indicating the efficacy of and/or measurement of miR-21 by their cardiac hypertrophy and fibrosis compara- Anti-21. Second, Thum et al. do not state 8-mer inhibitors differs markedly from ble to that of their Cre-negative littermates. the method they used to measure miR- ours, since we observed robust miR-21 Genetic compensation therefore cannot 21 inhibition, though we assume it to be inhibition 3 weeks after injection, as dem- account for the normal pathological car- quantitative PCR (qPCR). In our hands, onstrated by multiple readouts (2). Using diac remodeling response in miR-21 null qPCR alone is unreliable for measuring mismatched oligonucleotide controls is mice. Moreover, functions of other miRNAs miRNA inhibition, especially for 8-mer also important for interpreting miRNA in heart disease can be revealed by genetic inhibitors, since they may be displaced dur- inhibition studies in vivo, as described in deletion in mice, as shown for miR-208 (4). ing qPCR and thereby give an underrepre- our paper, rather than using PBS as a con- Thus, while 22-mer oligonucleotide inhibi- sentation of miRNA inhibition. To demon- trol, as reported by Thum et al. (1). tors against miR-21 are efficacious in inhib- 462 The Journal of Clinical Investigation http://www.jci.org Volume 121 Number 2 February 2011 letters iting cardiac hypertrophy (1), other loss-of- Boulder, Colorado, USA. J Clin Invest. 2011;121(2):462–463. function approaches appear ineffective. We doi:10.1172/JCI46108. remain enthusiastic about miRNAs as ther- Conflict of interest: Eric N. Olson and Eva apeutic targets and welcome such dialog. van Rooij are co-founders of miRagen Ther- 1. Thum T, et al. MicroRNA-21 contributes to myo- apeutics. Sakari Kauppinen is employed at cardial disease by stimulating MAP kinase signaling in fibroblasts. Nature. 2008;456(7224):980–984. 1 David M. Patrick, Sakari Santaris Pharma. 2. Patrick DM, et al. Stress-dependent cardiac remod- Kauppinen,2 Eva van Rooij,3 eling occurs in the absence of miR-21 in mice. J Clin and Eric N. Olson1 Address correspondence to: Eric Olson, Uni- Invest. 2010;120(11):3912–3916. 3. Hatley ME, et al. Modulation of K-Ras-dependent versity of Texas Southwestern Medical Cen- lung tumorigenesis by microRNA-21. Cancer Cell. 1Department of Molecular Biology, Univer- ter, 6000 Harry Hines Blvd., Dallas, Texas 2010;18(3):282–293. sity of Texas Southwestern Medical Center, 75390-9148, USA. Phone: 214.648.1187; 4. van Rooij E, Sutherland LB, Qi X, Richardson JA, 2 Hill J, Olson EN. Control of stress-dependent car- Dallas, Texas, USA. Santaris Pharma, Hør- Fax: 214.648.1196; E-mail: Eric.Olson@ diac growth and gene expression by a microRNA. sholm, Denmark. 3miRagen Therapeutics, utsouthwestern.edu. Science. 2007;316(5824):575–579. Expression of TNFRSF25 on conventional T cells and Tregs I mmunological tolerance is achieved and Tregs, and our findings are remarkably conclusion is in line with findings obtained through recessive and dominant mecha- different to those reported by Schreiber et using mice that were rendered deficient in nisms.