Preclinical characterization of antagomiR-218 as a potential treatment for myotonic dystrophy
2021; Cell Press; Volume: 26; Linguagem: Inglês
10.1016/j.omtn.2021.07.017
ISSN2162-2531
AutoresEstefanía Cerro-Herreros, Irene González-Martínez, Nerea Moreno, Jorge Espinosa-Espinosa, Juan M. Fernández‐Costa, Anna Colom-Rodrigo, Sarah J. Overby, David Seoane-Miraz, Javier Poyatos‐García, Juan J. Vílchez, Adolfo López de Munaín, Miguel A. Varela, Matthew J. A. Wood, Manuel Pérez‐Alonso, Beatriz Llamusí, Rubén Artero,
Tópico(s)Mitochondrial Function and Pathology
ResumoMyotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by expansion of unstable CTG repeats in a non-coding region of the DMPK gene. CUG expansions in mutant DMPK transcripts sequester MBNL1 proteins in ribonuclear foci. Depletion of this protein is a primary contributor to disease symptoms such as muscle weakness and atrophy and myotonia, yet upregulation of endogenous MBNL1 levels may compensate for this sequestration. Having previously demonstrated that antisense oligonucleotides against miR-218 boost MBNL1 expression and rescue phenotypes in disease models, here we provide preclinical characterization of an antagomiR-218 molecule using the HSALR mouse model and patient-derived myotubes. In HSALR, antagomiR-218 reached 40–60 pM 2 weeks after injection, rescued molecular and functional phenotypes in a dose- and time-dependent manner, and showed a good toxicity profile after a single subcutaneous administration. In muscle tissue, antagomiR rescued the normal subcellular distribution of Mbnl1 and did not alter the proportion of myonuclei containing CUG foci. In patient-derived cells, antagomiR-218 improved defective fusion and differentiation and rescued up to 34% of the gene expression alterations found in the transcriptome of patient cells. Importantly, miR-218 was found to be upregulated in DM1 muscle biopsies, pinpointing this microRNA (miRNA) as a relevant therapeutic target. Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by expansion of unstable CTG repeats in a non-coding region of the DMPK gene. CUG expansions in mutant DMPK transcripts sequester MBNL1 proteins in ribonuclear foci. Depletion of this protein is a primary contributor to disease symptoms such as muscle weakness and atrophy and myotonia, yet upregulation of endogenous MBNL1 levels may compensate for this sequestration. Having previously demonstrated that antisense oligonucleotides against miR-218 boost MBNL1 expression and rescue phenotypes in disease models, here we provide preclinical characterization of an antagomiR-218 molecule using the HSALR mouse model and patient-derived myotubes. In HSALR, antagomiR-218 reached 40–60 pM 2 weeks after injection, rescued molecular and functional phenotypes in a dose- and time-dependent manner, and showed a good toxicity profile after a single subcutaneous administration. In muscle tissue, antagomiR rescued the normal subcellular distribution of Mbnl1 and did not alter the proportion of myonuclei containing CUG foci. In patient-derived cells, antagomiR-218 improved defective fusion and differentiation and rescued up to 34% of the gene expression alterations found in the transcriptome of patient cells. Importantly, miR-218 was found to be upregulated in DM1 muscle biopsies, pinpointing this microRNA (miRNA) as a relevant therapeutic target. IntroductionIn contrast to most genetic diseases, which stem from lack of a protein function due to a mutation in the encoding gene, myotonic dystrophy type 1 (DM1) originates from the depletion of Muscleblind-like 1 (MBNL1) and 2 (MBNL2) proteins due to sequestration by toxic RNAs forming microscopically visible ribonuclear structures known as foci.1Miller J.W. Urbinati C.R. Teng-Umnuay P. Stenberg M.G. Byrne B.J. Thornton C.A. Swanson M.S. Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy.EMBO J. 2000; 19: 4439-4448Crossref PubMed Scopus (698) Google Scholar,2Fardaei M. Larkin K. Brook J.D. Hamshere M.G. In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts.Nucleic Acids Res. 2001; 29: 2766-2771Crossref PubMed Scopus (177) Google Scholar This is favorable for therapy, since protein depletion can be compensated by degradation of toxic RNA3Lee J.E. Bennett C.F. Cooper T.A. RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1.Proc. Natl. Acad. Sci. USA. 2012; 109: 4221-4226Crossref PubMed Scopus (120) Google Scholar,4Pandey S.K. Wheeler T.M. Justice S.L. Kim A. Younis H.S. Gattis D. Jauvin D. Puymirat J. Swayze E.E. Freier S.M. et al.Identification and characterization of modified antisense oligonucleotides targeting DMPK in mice and nonhuman primates for the treatment of myotonic dystrophy type 1.J. Pharmacol. Exp. Ther. 2015; 355: 329-340Crossref PubMed Scopus (85) Google Scholar or enhanced expression of genes encoding the depleted proteins,5Zhang F. Bodycombe N.E. Haskell K.M. Sun Y.L. Wang E.T. Morris C.A. Jones L.H. Wood L.D. Pletcher M.T. A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I.Hum. Mol. Genet. 2017; 26: 3056-3068Crossref PubMed Scopus (21) Google Scholar,6Chen G. Masuda A. Konishi H. Ohkawara B. Ito M. Kinoshita M. Kiyama H. Matsuura T. Ohno K. Phenylbutazone induces expression of MBNL1 and suppresses formation of MBNL1-CUG RNA foci in a mouse model of myotonic dystrophy.Sci. Rep. 2016; 6: 25317Crossref PubMed Scopus (25) Google Scholar which remain perfectly functional in patients, in an approach termed therapeutic gene modulation. DM1 is a rare autosomal dominant neuromuscular disease that is characteristically multisystemic, with highly variable clinical phenotypes encompassing the central nervous system (CNS), heart, and skeletal and visceral musculature.7Ashizawa T. Gagnon C. Groh W.J. Gutmann L. Johnson N.E. Meola G. Moxley 3rd, R. Pandya S. Rogers M.T. Simpson E. et al.Consensus-based care recommendations for adults with myotonic dystrophy type 1.Neurol. Clin. Pract. 2018; 8: 507-520Crossref PubMed Scopus (75) Google Scholar Skeletal muscle alterations include myotonia and muscle weakness and atrophy, which contribute significantly to disease morbidity and mortality due to respiratory distress, dysphagia, and immobility.8Smith C.A. Gutmann L. Myotonic Dystrophy Type 1 Management and Therapeutics.Curr. Treat. Options Neurol. 2016; 18: 52Crossref PubMed Scopus (37) Google Scholar The mutation causing DM1 is an expansion in the number of CTG trinucleotide repeats in the 3′ untranslated region (UTR) of DM1 protein kinase (DMPK9Brook J.D. McCurrach M.E. Harley H.G. Buckler A.J. Church D. Aburatani H. Hunter K. Stanton V.P. Thirion J.P. Hudson T. et al.Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member.Cell. 1992; 69: 385Abstract Full Text PDF PubMed Scopus (10) Google Scholar,10Mahadevan M. Tsilfidis C. Sabourin L. Shutler G. Amemiya C. Jansen G. Neville C. Narang M. Barceló J. O'Hoy K. et al.Myotonic dystrophy mutation: an unstable CTG repeat in the 3′ untranslated region of the gene.Science. 1992; 255: 1253-1255Crossref PubMed Scopus (1409) Google Scholar). Above ∼50 repeats, they become transcribed into mutant DMPK transcripts; these fold into CUG RNA hairpins that sequester MBNL1 and 2 proteins. This gives rise to molecular defects and phenotypes similar to loss of MBNL1 and 2 functions, as demonstrated in mice by knockout of Mbnl111Kanadia R.N. Johnstone K.A. Mankodi A. Lungu C. Thornton C.A. Esson D. Timmers A.M. Hauswirth W.W. Swanson M.S. A muscleblind knockout model for myotonic dystrophy.Science. 2003; 302: 1978-1980Crossref PubMed Scopus (582) Google Scholar or Mbnl212Charizanis K. Lee K.-Y. Batra R. Goodwin M. Zhang C. Yuan Y. Shiue L. Cline M. Scotti M.M. Xia G. et al.Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy.Neuron. 2012; 75: 437-450Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar or the compound loss of both.13Lee K.-Y. Li M. Manchanda M. Batra R. Charizanis K. Mohan A. Warren S.A. Chamberlain C.M. Finn D. Hong H. et al.Compound loss of muscleblind-like function in myotonic dystrophy.EMBO Mol. Med. 2013; 5: 1887-1900Crossref PubMed Scopus (115) Google Scholar This hypothesis is further supported by experiments in which phenotypes produced by targeted expression of CUG repeats expanded in the skeletal muscles driven by human skeletal actin transgene (HSALR14Mankodi A. Logigian E. Callahan L. McClain C. White R. Henderson D. Krym M. Thornton C.A. Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat.Science. 2000; 289: 1769-1773Crossref PubMed Scopus (555) Google Scholar) are rescued by adeno-associated virus-15Kanadia R.N. Shin J. Yuan Y. Beattie S.G. Wheeler T.M. Thornton C.A. Swanson M.S. Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy.Proc. Natl. Acad. Sci. USA. 2006; 103: 11748-11753Crossref PubMed Scopus (271) Google Scholar or transgene-mediated overexpression of Mbnl1,16Chamberlain C.M. Ranum L.P.W. Mouse model of muscleblind-like 1 overexpression: skeletal muscle effects and therapeutic promise.Hum. Mol. Genet. 2012; 21: 4645-4654Crossref PubMed Scopus (58) Google Scholar and also by candidate drugs that rescue disease-like phenotypes through upregulation of MBNL1 gene expression in human cells and mouse and Drosophila models.5Zhang F. Bodycombe N.E. Haskell K.M. Sun Y.L. Wang E.T. Morris C.A. Jones L.H. Wood L.D. Pletcher M.T. A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I.Hum. Mol. 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Cell Biol. 2005; 7: 1240-1247Crossref PubMed Scopus (105) Google Scholar MBNL1 plays a primary role in controlling alternative splicing in skeletal and cardiac muscle, and MBNL2 serves a related function in the CNS.12Charizanis K. Lee K.-Y. Batra R. Goodwin M. Zhang C. Yuan Y. Shiue L. Cline M. Scotti M.M. Xia G. et al.Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy.Neuron. 2012; 75: 437-450Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar Genetic redundancy is well illustrated by the fact that deletion of only one paralog resulted in the upregulation of the other and occupancy of its binding sites.13Lee K.-Y. Li M. Manchanda M. Batra R. Charizanis K. Mohan A. Warren S.A. Chamberlain C.M. Finn D. Hong H. et al.Compound loss of muscleblind-like function in myotonic dystrophy.EMBO Mol. Med. 2013; 5: 1887-1900Crossref PubMed Scopus (115) Google Scholar Whereas MBNL proteins promote adult-type alternative splicing patterns, CUGBP Elav-like family member 1 (CELF1), which is abnormally activated in skeletal muscle in DM1, promotes the fetal type,30Wang E.T. Ward A.J. Cherone J.M. Giudice J. Wang T.T. Treacy D.J. Lambert N.J. Freese P. Saxena T. Cooper T.A. Burge C.B. Antagonistic regulation of mRNA expression and splicing by CELF and MBNL proteins.Genome Res. 2015; 25: 858-871Crossref PubMed Scopus (123) Google Scholar as similarly described also for heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1).31Li M. Zhuang Y. Batra R. Thomas J.D. Li M. Nutter C.A. Scotti M.M. Carter H.A. Wang Z.J. Huang X.-S. et al.HNRNPA1-induced spliceopathy in a transgenic mouse model of myotonic dystrophy.Proc. Natl. Acad. Sci. USA. 2020; 117: 5472-5477Crossref PubMed Scopus (15) Google Scholar Maintaining a fetal alternative splicing pattern in muscle transcripts in adults results in unfit proteins that cause specific symptoms, as demonstrated by chloride voltage-gated channel 1 (CLCN132Charlet-B N. Savkur R.S. Singh G. Philips A.V. Grice E.A. Cooper T.A. Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing.Mol. Cell. 2002; 10: 45-53Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar) or bridging integrator 1 (BIN133Fugier C. Klein A.F. Hammer C. Vassilopoulos S. Ivarsson Y. Toussaint A. Tosch V. Vignaud A. Ferry A. Messaddeq N. et al.Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy.Nat. Med. 2011; 17: 720-725Crossref PubMed Scopus (232) Google Scholar) transcripts. Genome-wide alterations in the transcriptome of patients have recently been reported in detail.34Wang E.T. Treacy D. Eichinger K. Struck A. Estabrook J. Olafson H. Wang T.T. Bhatt K. Westbrook T. Sedehizadeh S. et al.Transcriptome alterations in myotonic dystrophy skeletal muscle and heart.Hum. Mol. Genet. 2019; 28: 1312-1321Crossref PubMed Scopus (50) Google ScholarWe have previously demonstrated that hsa-miR-218-5p (hereafter miR-218) is a natural MBNL1 and 2 repressor in human myotubes and mice and that its inactivation through antisense oligonucleotide (antagomiR)-mediated blocking leads to miRNA depletion and MBNL1 and 2 protein upregulation.19Cerro-Herreros E. Sabater-Arcis M. Fernandez-Costa J.M. Moreno N. Perez-Alonso M. Llamusi B. Artero R. miR-23b and miR-218 silencing increase Muscleblind-like expression and alleviate myotonic dystrophy phenotypes in mammalian models.Nat. Commun. 2018; 9: 2482Crossref PubMed Scopus (41) Google Scholar Here we expand on these observations by addressing the dose response, duration, and toxicity of antagomiR-218 treatments as well as characterizing their effect at the transcriptome level and biodistribution. We also report that miR-218 is significantly overexpressed in DM1 muscle samples and disease models, thus potentially contributing to disease phenotypes through MBNL1- and 2-independent pathways. These results underline the therapeutic potential of miR-218 blocking oligonucleotides as potential treatments for DM1.ResultsMBNL1 is a direct target of miR-218We previously showed that miR-218 directly regulated MBNL2 stability and expression through 3′ UTR luciferase sensor constructs, but the potential regulation of human or mouse MBNL1 was not formally addressed.19Cerro-Herreros E. Sabater-Arcis M. Fernandez-Costa J.M. Moreno N. Perez-Alonso M. Llamusi B. Artero R. miR-23b and miR-218 silencing increase Muscleblind-like expression and alleviate myotonic dystrophy phenotypes in mammalian models.Nat. Commun. 2018; 9: 2482Crossref PubMed Scopus (41) Google Scholar Specifically, in mice, according to the bioinformatics tools miRDB, miRanda, and TargetScan, there are four predicted target sites for miR-218, whereas in humans only two are conserved (Figures S1A and S1B). When combining miR-218 with mouse Mbnl1 3′ UTR wild-type sensor constructs in C2C12 cells, a decrease in luciferase is observed, but when using the versions with the mutated target sites, only the one with the mutation in site 3 stops responding. Thus, the active repressor site for miR-218 is number 3 (Figure 1A). Consistently, perfect-match (PM) constructs for all four candidate sites confirm a decrease in luciferase and verify that the plasmid is expressing miR-218. Finally, in HeLa cells, we confirmed the functional conservation of mouse miR-218 site 3 in human MBNL1 3′ UTR sensor constructs, using a synthetic agomiR-218 (Figure S1C).Using a similar approach, we transfected HeLa cells with a luciferase 3′ UTR MBNL1 fusion construct containing the wild-type predicted binding sites for miR-218 according to miRecords35Xiao F. Zuo Z. Cai G. Kang S. Gao X. Li T. miRecords: an integrated resource for microRNA-target interactions.Nucleic Acids Res. 2009; 37: D105-D110Crossref PubMed Scopus (1127) Google Scholar (Figure S1C). A preexisting construct containing a perfect match for miR-218 in the MBNL2 3′ UTR served as a positive control. Co-transfection of reporter constructs and a synthetic miR-218 agomiR demonstrated a robust reduction of Gaussia luciferase expression, whereas a negative control agomiR with a scramble sequence (SC) failed to repress the construct (Figure S1C). Thus, these results demonstrate the direct recognition of at least one miR-218 binding site in the MBNL1 3′ UTR.Similar antagomiR-218 efficacy via subcutaneous and intravenous administrationTo account for potential differential effects depending on the administration route, we tested the activity of 12.5 mg/kg of antagomiR-218 by subcutaneous and intravenous injection in HSALR model mice, which we have used previously in similar analyses and are readily available in our premises. Four days post injection, gastrocnemius and quadriceps muscles were processed to quantify miR-218, and Mbnl1 at the RNA and protein levels (Figures S2A–S2E). miR-218 levels were under 50% of normal amounts, and Mbnl1 transcripts and proteins were upregulated, yet differences between subcutaneous and intravenous administration were negligible in both muscles except for Mbnl1 transcripts in quadriceps. Both administration types rescued weight-normalized grip strength, but intravenous delivery yielded significantly greater rescue than subcutaneous delivery, whereas myotonia was only rescued when antagomiR-218 was subcutaneously provided (Figures S2F and S2G). Taking these results together, we detected no difference in miRNA and Mbnl1 protein levels, whereas functional rescues were more consistent with subcutaneous delivery. Several parameters were analyzed in total blood obtained before sacrifice to check whether administration routes differentially altered blood biochemistry. These parameters showed more alterations in intravenous administrations than subcutaneous treatment and normalized to PBS (Table S1, Tab A). Amylase and total bilirubin were significantly increased compared with mice injected subcutaneously with antagomiR-218. The scramble (SC) negative control also showed alterations in lipase after intravenous injection. However, the levels achieved were reported as not clinically relevant.36Shen W. De Hoyos C.L. Migawa M.T. Vickers T.A. Sun H. Low A. Bell 3rd, T.A. Rahdar M. Mukhopadhyay S. Hart C.E. et al.Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index.Nat. Biotechnol. 2019; 37: 640-650Crossref PubMed Scopus (125) Google Scholar Unaltered weight and visual necropsy confirmed no major toxic effects in the treated mice. Analysis of the white blood cell differential count from mice treated with antagomiR-218 via the two administration routes found no significant differences, with a similar conclusion in mice treated with SC (Table S1, Tab B). These results confirm that both administration routes are effective as therapy. However, the ability to compare with previous studies together with the experimental ease of subcutaneous injections prompted us to use this delivery method in all subsequent experiments.The effects of antagomiR-218 depend on the dosageAntagomiR-218 was previously tested at a single dose of 12.5 mg/kg,19Cerro-Herreros E. Sabater-Arcis M. Fernandez-Costa J.M. Moreno N. Perez-Alonso M. Llamusi B. Artero R. miR-23b and miR-218 silencing increase Muscleblind-like expression and alleviate myotonic dystrophy phenotypes in mammalian models.Nat. Commun. 2018; 9: 2482Crossref PubMed Scopus (41) Google Scholar but this failed to confirm that its molecular and in vivo effects depended on the concentration of the oligonucleotide, or whether this response was linear or otherwise. To this end, we subcutaneously injected antagomiR-218 at a low (3 mg/kg), intermediate (12.5 mg/kg), or high (40 mg/kg) concentration and recovered quadriceps and gastrocnemius muscles (Figures 1B–1E). miR-218 levels were not significantly different from normal at 3 mg/kg, but both intermediate and high concentrations similarly lowered miR-218 to slightly below 50% of normal levels. Of note, the SC negative control nonspecifically increased miR-218 levels at the highest concentration. In contrast to the plateau detected for miR-218 levels at 12.5 and 40 mg/kg, Mbnl1 transcripts and protein amount increased steadily with dose in both muscles, although the statistical differences were more or less significant depending on the magnitude of the error. In general, 3 mg/kg was too low to significantly enhance Mbnl1 expression at the transcript or protein level, except in quadriceps, which exhibited the most robust response in terms of Mbnl1 protein amounts, and both muscles showed a similar pattern of response to antagomiR-218 administrations. Importantly, treatment did not significantly alter HSA transgene expression (Figure S3A). Thus, two direct molecular readouts of antagomiR-218 activity (Mbnl1 mRNA and protein levels) respond to the antagomiR dose in an approximately linear fashion (Figures S3B–S3E), whereas miR-218 levels seem to plateau at the highest concentration tested. Nevertheless, miRNA levels get further reduced if compared to negative control SC, which unspecifically increases miR-218 levels (Figure S3F).Enhanced expression of Mbnl1 in HSALR model mice was confirmed to rescue characteristic exon inclusion events, myotonia, and grip strength in a dose-dependent manner (Figure 2; Figure S4). Nfix and Clcn1 transcripts showed abnormally increased inclusion of exon 7 in HSALR mice, but this recovered to ∼40%–50% of normal values at 12.5 mg/kg in muscles. The low dose was unable to significantly improve mis-splicing, whereas the high dose only marginally enhanced exon inclusion rescue compared with the intermediate dose values. Turning to functional measurements, myotonia grade was 4 for DM1 mice and progressively dropped to almost 2 (myotonic discharges at only half of the electrode insertions) at the highest dose tested. Weight-normalized grip strength was ∼70% of normal in control-treated HSALR mice but significantly improved at all concentrations tested and reached close to normal values at intermediate and high doses. In summary, antagomiR-218 was shown to rescue the characteristic mis-splicing defects described in the model mice and improved myotonia and grip strength in a dose-dependent manner, but with a trend toward a plateau effect at 40 mg/kg.Figure 2Subcutaneous antagomiR-218 improved mis-splicing of Mbnl-dependent transcripts, myotonia, and force in a dose-dependent fashion in HSALR miceShow full caption(A) RT-PCR analyses of the splicing of Nfix exon 7 and Clcn1 exon 7a in gastrocnemius (gt) and quadriceps (qd) muscles. PBS1 control corresponds to 12.5 mg/kg treatment, and PBS2 corresponds to 3 and 40 mg/kg treatments. Exon inclusion levels of healthy control mice are shown for FVB. Quantification of these representative exon inclusion results is provided in Figure S4. (B) Heatmap representing the percentage of normal splicing recovery for Nfix and Clcn1 in gt and qd in treated HSALR mice according to the color scale provided. (C and D) Myotonia grade (C) and percentage of normal grip strength (D) were analyzed before injection (BI) and 4 days after injection (AI). N indicates the number of mice used for each experiment. Each treatment was statistically compared with its respective PBS control via Student's t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Different biochemical parameters were analyzed in total blood obtained before sacrifice to check for potential deleterious effects in response to increasing doses. Neither antagomiR-SC nor antagomiR-218 treatments caused significant alterations in quantified tissue damage biomarkers compared with PBS (Table S1, Tab C). Mouse weight also showed no significant changes between the different treatments, and visual necropsies were normal at all dosages tested. Nevertheless, ANOVA and Kruskal-Wallis analyses revealed statistically significant differences in urea levels and body weight between Friend Virus B (FVB) and HSALR mice treated with PBS, and lipase levels were also significantly different between FVB and HSALR mice treated with PBS, SC, and antagomiR-218. Therefore, these alterations seem more closely associated with disease state than experimental treatment, consistent with previous reports.37Cerro-Herreros E. González-Martínez I. Moreno-Cervera N. Overby S. Pérez-Alonso M. Llamusí B. Artero R. Therapeutic Potential of AntagomiR-23b for Treating Myotonic Dystrophy.Mol. Ther. Nucleic Acids. 2020; 21: 837-849Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Analyzing white blood cell differential count, there were no significant treatment- or dose-related differences between the groups analyzed (note the lack of clustering among treated mice in the dendrogram of Table S1, Tab D). Overall, these data support that even the highest concentration tested caused no significant modification of blood biochemistry parameters.One single injection of antagomiR-218 produces weeks-lasting molecular and phenotypic improvementsOur previous description of antagomiR-218 activity was at 4 days,19Cerro-Herreros E. Sabater-Arcis M. Fernandez-Costa J.M. Moreno N. Perez-Alonso M. Llamusi B. Artero R. miR-23b and miR-218 silencing increase Muscleblind-like expression and alleviate myotonic dystrophy phenotypes in mammalian models.Nat. Commun. 2018; 9: 2482Crossref PubMed Scopus (41) Google Scholar but a similar antagomiR was recently reported to bring about functional recoveries lasting up to 45 days in the HSALR mouse model.37Cerro-Herreros E. González-Martínez I. Moreno-Cervera N. Overby S. Pérez-Alonso M. Llamusí B. Artero R. Thera
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