Preclinical development of an innovative drug for Myotonic Dystrophy
- Autores: Irene González Martínez
- Directores de la Tesis: Rubén Artero Allepuz (dir. tes.), Estefanía Cerro Herreros (dir. tes.), Federico Pallardo Calatayud (tut. tes.)
- Lectura: En la Universitat de València ( España ) en 2025
- Idioma: inglés
- Número de páginas: 308
- Tribunal Calificador de la Tesis: Alejandro Garanto Iglesias (presid.), Arturo López Castel (secret.), Gisela Nogales-Gadea (voc.)
- Programa de doctorado: Programa de Doctorado en Fisiología por la Universitat de València (Estudi General)
- Menciones:
- Internacional
- Calificación: Sobresaliente Cum Laude
- Materias:
- Enlaces
- Resumen
Myotonic dystrophy type 1 (DM1) is a rare, progressive, and multisystemic disease caused by the expansion of CTG triplets in the untranslated region (3'-UTR) of the dystrophia myotonica protein kinase (DMPK) gene. This alteration produces mutant RNA that accumulates in the cell nucleus, where it sequesters proteins from the Muscleblind-like family of splicing factors, specifically MBNL1 and MBNL2, which are key regulators of alternative splicing with partially redundant functions. The functional loss of these proteins leads to a widespread disruption of molecular processes that contribute to the onset and progression of clinical symptoms, such as myotonia (difficulty relaxing muscles after contraction) and muscle atrophy. Specifically, restoring MBNL1 function has been proposed as an effective therapeutic strategy, as its overexpression reverses multiple pathological phenotypes in both cell and animal models. This thesis focuses on the development and optimization of antisense oligonucleotides (ASOs), specifically antimiRs targeting microRNAs miR-23b and miR-218, whose activity naturally represses the production of MBNL1/2 and whose expression is increased in the disease. The inhibition of these microRNAs allows for the upregulation of endogenous MBNL levels, thereby helping to correct the molecular and functional defects characteristic of DM1.
The work is structured into three main sections. First, a platform based on the Quantitative Dot Blot (QDB) technique was optimized and validated. This method enables the quantitative detection of MBNL1 with high sensitivity, reproducibility, and high-throughput capacity. This tool facilitated the systematic screening of multiple antimiR variants in both cell and murine models.
Second, phosphorodiamidate morpholino oligomer (PMO)-based antimiRs were developed and conjugated to cell-penetrating peptides (CPPs), designed to enhance their delivery to skeletal muscle. These compounds demonstrated high efficacy in both in vitro and in vivo models, achieving the restoration of MBNL levels, correction of aberrant splicing events, improvement of myotonia, and better muscle histology. Furthermore, they displayed a favorable safety profile, with no evidence of toxicity or immune activation, although their accumulation in the kidney relative to target tissues, such as muscle, remained quite high.
Third, an alternative optimization strategy was explored through the lipid conjugation of antimiRs with oleic acid. This modification improved biodistribution and prolonged therapeutic effects following a single low-concentration dose (6 mg/kg).
Oleic-conjugated compounds preferentially reached skeletal muscle and maintained their effects up to 15 days post-treatment, demonstrating sustained efficacy in the murine model. Moreover, studies in human, porcine, and rat cell cultures confirmed that the mechanism of action is not affected by genetic background and is conserved across species.
Taken together, this work provides strong evidence supporting the therapeutic value of optimized antimiRs targeting miR-23b and miR-218 and proposes scalable, transferable methodologies, such as QDB, for this and other similar preclinical developments.
The results support the potential of this approach to advance toward new oligonucleotide-based therapies for the treatment of neuromuscular diseases such as DM1.
