Utilización de microRNAs como dianas terapéuticas en Distrofia Miotónica

La distrofia miotónica tipo 1 (DM1) se debe a una expansión del triplete CTG en 3´UTR del gen DMPK, mientras que, la distrofia miotónica tipo 2 (DM2) se origina por la de la repetición CCTG en el intrón 1 del gen CNBP. Son dos enfermedades multisistémicas con manifestaciones patológicas en músculo esquelético (miotonia, degeneración y debilidad muscular), corazón (defectos en la conducción cardiaca) y sistema nervioso central. Los transcritos DMPK y CNBP mutantes se pliegan en horquillas que secuestran factores de splicing alternativo, principalmente Muscleblind-like 1 (MBNL1; y su parálogo MBNL2). La falta de la proteínas MBNL origina problemas en el splicing de transcritos musculares y conduce a la miopatía en ratones knock out para estos genes. Mientras que la sobreexpresión de MBNL1 en músculo esquelético de ratones HSALR modelo de DM1, suprime fenotipos típicos de DM1 tales como alteraciones en el splicing alternativo, miotonía y secuestro de MBNL1 en foci ribonucleares. A pesar del intenso trabajo de investigación de la última década, que ha aclarado muchos aspectos del mecanismo de fisiopatogénesis de la DM y ha permitido experimentar con tratamientos potenciales, no se ha transferido a la práctica clínica una terapia efectiva para la enfermedad. Una nueva aproximación terapéutica desarrollada en esta tesis y avalada por nuestros resultados en modelos animales en Drosophila y ratón HSALR DM1 y mioblastos de pacientes de DM1, es potenciar la expresión endógena de MBNLs mediante el silenciamiento de microRNAs represores de su expresión para así compensar su secuestro y mejorar algunos de los fenotipos característicos de la enfermedad como son la espliceopatía o la miopatía. En el caso de Drosophila la desrepresión de Muscleblind provocada por el silenciamiento de dme-miR-277 y dme-miR-304 en moscas modelo de la enfermedad, es suficiente para rescatar de forma significativa diferentes eventos de splicing alterados en DM1 y características funcionales tan importantes como la capacidad de vuelo y la vida media. Esta aproximación terapéutica también puede ser testada en un nuevo modelo de DM2 en Drosophila generando dentro del marco de esta tesis. Por otro lado, el silenciamiento de miR-23b y miR-218 mediado por antagomiRs en mioblastos DM1 y ratones HSALR desencadena un aumento de los niveles de las proteínas MBNL1/2 rescatando eventos de splicing típicamente alterados en la patología y mejora fenotipos musculares típicos de la enfermedad

Bioengineered in vitro 3D model of myotonic dystrophy type 1 human skeletal muscle

Myotonic dystrophy type 1 (DM1) is the most common hereditary myopathy in the adult population. The disease is characterized by progressive skeletal muscle degeneration that produces severe disability. At present, there is still no effective treatment for DM1 patients, but the breakthroughs in understanding the molecular pathogenic mechanisms in DM1 have allowed the testing of new therapeutic strategies. Animal models and in vitro two-dimensional cell cultures have been essential for these advances. However, serious concerns exist regarding how faithfully these models reproduce the biological complexity of the disease. Biofabrication tools can be applied to engineer human three-dimensional (3D) culture systems that complement current preclinical research models. Here, we describe the development of the first in vitro 3D model of DM1 human skeletal muscle. Transdifferentiated myoblasts from patient-derived fibroblasts were encapsulated in micromolded gelatin methacryloyl-carboxymethyl cellulose methacrylate hydrogels through photomold patterning on functionalized glass coverslips. These hydrogels present a microstructured topography that promotes myoblasts alignment and differentiation resulting in highly aligned myotubes from both healthy and DM1 cells in a long-lasting cell culture. The DM1 3D microtissues recapitulate the molecular alterations detected in patient biopsies. Importantly, fusion index analyses demonstrate that 3D micropatterning significantly improved DM1 cell differentiation into multinucleated myotubes compared to standard cell cultures. Moreover, the characterization of the 3D cultures of DM1 myotubes detects phenotypes as the reduced thickness of myotubes that can be used for drug testing. Finally, we evaluated the therapeutic effect of antagomiR-23b administration on bioengineered DM1 skeletal muscle microtissues. AntagomiR-23b treatment rescues both molecular DM1 hallmarks and structural phenotype, restoring myotube diameter to healthy control sizes. Overall, these new microtissues represent an improvement over conventional cell culture models and can be used as biomimetic platforms to establish preclinical studies for myotonic dystrophy

Proof of concept of peptide-linked blockmiR-induced MBNL functional rescue in myotonic dystrophy type 1 mouse model

Myotonic dystrophy type 1 is a debilitating neuromuscular disease causing muscle weakness, myotonia, and cardiac dysfunction. The phenotypes are caused by muscleblind-like (MBNL) protein sequestration by toxic RNA in the DM1 protein kinase (DMPK) gene. DM1 patients exhibit a pathogenic number of repetitions in DMPK, which leads to downstream symptoms. Another disease characteristic is altered microRNA (miRNA) expression. It was previously shown that miR-23b regulates the translation of MBNL1 into protein. Antisense oligonucleotide (AON) treatment targeting this miRNA can improve disease symptoms. Here, we present a refinement of this strategy targeting a miR-23b binding site on the MBNL1 3ʹ UTR in DM1 model cells and mice by using AONs called blockmiRs. BlockmiRs linked to novel cell-penetrating peptide chemistry showed an increase in MBNL1 protein in DM1 model cells and HSALR mice. They also showed an increase in muscle strength and significant rescue of downstream splicing and histological phenotypes in mice without disturbing the endogenous levels of other miR-23b target transcripts

Preclinical characterization of antagomiR-218 as a potential treatment for myotonic dystrophy

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 2weeks 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.This work was funded by research grants from Instituto de Salud Carlos III, including funds from FEDER, to M.P.-A. and B.L. (PI17/00352) and HR17-00268 (TATAMI project) from the “la Caixa” Banking Foundation to R.A. I.G.-M. was funded by the Precipita Project titled “Desarrollo de una terapia innovadora contra la distrofia miotónica,” E.C.-H. and J.M.F.-C. were supported by the post-doctoral fellowships APOSTD/2019/142 and APOSTD/2017/088 from the Fondo Social Europeo for science and investigation, while J.E.-E. was the recipient of a Santiago Grisolia fellowship (Grisolip2018/098) from the Generalidad Valenciana. Part of the equipment employed in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020). Antibody MB1a (4A8) was provided by MDA Monoclonal Antibody Resource

Two enhancers control transcription of Drosophila muscleblind in the embryonic somatic musculature and in the central nervous system.

The phylogenetically conserved family of Muscleblind proteins are RNA-binding factors involved in a variety of gene expression processes including alternative splicing regulation, RNA stability and subcellular localization, and miRNA biogenesis, which typically contribute to cell-type specific differentiation. In humans, sequestration of Muscleblind-like proteins MBNL1 and MBNL2 has been implicated in degenerative disorders, particularly expansion diseases such as myotonic dystrophy type 1 and 2. Drosophila muscleblind was previously shown to be expressed in embryonic somatic and visceral muscle subtypes, and in the central nervous system, and to depend on Mef2 for transcriptional activation. Genomic approaches have pointed out candidate gene promoters and tissue-specific enhancers, but experimental confirmation of their regulatory roles was lacking. In our study, luciferase reporter assays in S2 cells confirmed that regions P1 (515 bp) and P2 (573 bp), involving the beginning of exon 1 and exon 2, respectively, were able to initiate RNA transcription. Similarly, transgenic Drosophila embryos carrying enhancer reporter constructs supported the existence of two regulatory regions which control embryonic expression of muscleblind in the central nerve cord (NE, neural enhancer; 830 bp) and somatic (skeletal) musculature (ME, muscle enhancer; 3.3 kb). Both NE and ME were able to boost expression from the Hsp70 heterologous promoter. In S2 cell assays most of the ME enhancer activation could be further narrowed down to a 1200 bp subregion (ME.3), which contains predicted binding sites for the Mef2 transcription factor. The present study constitutes the first characterization of muscleblind enhancers and will contribute to a deeper understanding of the transcriptional regulation of the gene

Sequence and melting temperature (Tm) of PCR primers used for cloning genomic DNA.

<p>Restriction sites are highlighted in bold.</p

Genomic organization of the human MBNL1 gene.

<p>(A) Scale representation of 198 kb of the human MBNL1 locus. Green boxes correspond to exons and black lines to introns. Tested promoter regions are indicated as P1 and P2; a yellow circle denotes a predicted CpG island. (B) Schematic representation of H3K27Ac marks, typical of promoter regions, on seven human cell lines. (C) 5′-Ends of ESTs mapping to the MBNL1 locus support two potential transcription start sites for the gene. Data according to the UCSC Genome Browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093125#pone.0093125-Meyer1" target="_blank">[58]</a>. (E,F,H,I) Direct visualization of the eGFP reporter under the control of the ME enhancer. (D–F) Enhancer-less Hsa-P1 construct is a negative control. (G–I) Flies carrying the ME enhancer upstream of the human MBNL1 promoter (Hsa-P1) reproduce Muscleblind expression in the somatic musculature. (E,H) Lateral and (F,I) ventral views of late embryos. All micrographs were taken at 200× magnification. Anterior is to the left and dorsal up, unless otherwise stated.</p

NE reproduces <i>muscleblind</i> expression in the central nervous system.

<p>(A) Schematic representation of the reporter construct used to transform the <i>Drosophila</i> germline. Fluorescence confocal images of lateral (B–D) and ventral (E–J) views of late <i>Drosophila</i> embryos expressing construct (A) co-stained with anti-GFP (green) and anti-Mbl antibodies (red). NE drives expression in the CNS (arrows; C,I) overlapping Muscleblind expression (B,E,H; D,G,J shows the merge in yellow). No signal of the reporter was observed in tissues other than CNS. Endogenous Muscleblind expression in the muscles is in focus in (E,G,H,J). Micrographs were taken at 200× (B–G) and 400× magnification (H–J). Anterior is to the left and dorsal up unless otherwise stated.</p

ME reproduces <i>muscleblind</i> expression in the embryonic somatic musculature.

<p>(A) Localization of the putative <i>cis</i>-regulatory modules M1, M2, ML, H and M3, indicated as orange boxes, in the context of the <i>muscleblind</i> genomic locus. Fluorescence confocal images of lateral (B,E,H,) and ventral (C,F,I,) views of late <i>Drosophila</i> embryos. (D,G) Schematic representation of the reporter constructs used to transform the germline of <i>Drosophila</i>. In control <i>yw</i> flies (B,C) an anti-Mbl antibody detects robust expression in the somatic musculature and in the CNS (green). Direct visualization of the GFP reporter under the control of the ME enhancer in the pH-Stinger vector (E,F,H,I). Promoter-less ME constructs (D–F) do not activate GFP expression and serve as negative controls. Flies carrying the ME enhancer upstream of <i>Hsp70</i> (G–I) reproduce Muscleblind expression in the somatic musculature but not in the CNS. All micrographs were taken at 200× magnification. Anterior is to the left and dorsal up unless otherwise stated.</p

RbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences

Myotonic dystrophy type 1 and type 2 (DM1, DM2) are caused by expansions of CTG and CCTG repeats, respectively. RNAs containing expanded CUG or CCUG repeats interfere with the metabolism of other RNAs through titration of the Muscleblind-like (MBNL) RNA binding proteins. DM2 follows a more favorable clinical course than DM1, suggesting that specific modifiers may modulate DM severity. Here, we report that the rbFOX1 RNA binding protein binds to expanded CCUG RNA repeats, but not to expanded CUG RNA repeats. Interestingly, rbFOX1 competes with MBNL1 for binding to CCUG expanded repeats and overexpression of rbFOX1 partly releases MBNL1 from sequestration within CCUG RNA foci in DM2 muscle cells. Furthermore, expression of rbFOX1 corrects alternative splicing alterations and rescues muscle atrophy, climbing and flying defects caused by expression of expanded CCUG repeats in a Drosophila model of DM2