Duchenne muscular dystrophy (DMD) is a progressive disease caused by the loss of function of the protein dystrophin. This protein contributes to the stabilisation of striated cells during contraction, as it anchors the cytoskeleton with components of the extracellular matrix through the dystrophin-associated protein complex (DAPC). Moreover, absence of the functional protein affects the expression and function of proteins within the DAPC, leading to molecular events responsible for myofibre damage, muscle weakening, disability and, eventually, premature death. Presently, there is no cure for DMD, but different treatments help manage some of the symptoms. Advances in genetic and exon-skipping therapies are the most promising intervention, the safety and efficiency of which are tested in animal models. In addition to in vivo functional tests, ex vivo molecular evaluation aids assess to what extent the therapy has contributed to the regenerative process. In this regard, the later advances in microscopy and image acquisition systems and the current expansion of antibodies for immunohistological evaluation together with the development of different spectrum fluorescent dyes have made histology a crucial tool. Nevertheless, the complexity of the molecular events that take place in dystrophic muscles, together with the rise of a multitude of markers for each of the phases of the process, makes the histological assessment a challenging task. Therefore, here, we summarise and explain the rationale behind different histological techniques used in the literature to assess degeneration and regeneration in the field of dystrophinopathies, focusing especially on those related to DMD.

https://www.mdpi.com/1422-0067/23/24/16080/pdf?version=1671530316

Histological Methods to Assess Skeletal Muscle Degeneration and Regeneration in Duchenne Muscular Dystrophy

Disease-causing genes have been identified for many severe muscular and neurological genetic disorders. Advances in the gene therapy field offer promising solutions for drug development to treat these life-threatening conditions. Depending on how the mutation affects the function of the gene product, different gene therapy approaches may be beneficial. Gene replacement therapy is appropriate for diseases caused by mutations that result in the deficiency of the functional protein. Gene suppression strategy is suggested for disorders caused by the toxic product of the mutant gene. Splicing modulators, genome editing, and base editing techniques can be applied to disorders with different types of underlying mutations. Testing potential drugs in animal models of human diseases is an indispensable step of development. Given the specific gene therapy approach, appropriate animal models can be generated using a variety of technologies ranging from transgenesis to precise genome editing. In this review, we discuss technologies used to generate small and large animal models of the most common muscular and neurological genetic disorders. We specifically focus on animal models that were used to test gene therapies based on adeno-associated vectors and antisense nucleotides.

﻿Genetically modified animal models of hereditary diseases for testing of gene-directed therapy

La dystrophine est une proteine cytoplasmique qui lie physiquement le cytosquelette a la matrice extracellulaire par le biais du complexe dystrophine-proteines associees (DAPC), assurant ainsi la stabilite du sarcolemme. Des mutations dans le gene DMD codant pour la dystrophine, conduisant a l’absence de la proteine, sont a l’origine de la dystrophie musculaire de Duchenne qui est une maladie liee au chromosome X. Pour mes travaux de these, j’ai genere et caracterise un nouveau modele de souris transgeniques rapportrices, denomme DmdEGFP, qui exprime une proteine dystrophine endogene fusionnee avec la proteine fluorescente EGFP. La proteine dystrophine est liee dans sa region C-terminale qui est presente dans la majorite des isoformes. Dans le modele, une expression forte et naturelle de l’EGFP etait observee dans les muscles squelettiques, lisses, le cœur, le cerveau et l’œil, ce qui suggere un etiquetage correct de tous les isoformes de la dystrophine. La fluorescence de l’EGFP co-localisait exactement avec la dystrophine dans tous les sites. Dans le muscle squelettique, la dystrophine ainsi que d’autres proteines de la DAPC etaient exprimees dans des quantites normales et dans la bonne localisation subsarcolemmale. L’architecture du tissu musculaire squelettique etait normale, suggerant que la fonction de la proteine de fusion etait maintenue. In vitro, l’EGFP est egalement exprimee dans les fibres musculaires isolees, ainsi que dans les myotubes derives des cellules satellites. Par consequent, cette nouvelle souris rapportrice de la dystrophine devient un outil important pour la visualisation directe et in vivo de l’expression de la dystrophine.

Generation and Characterization of a DmdEGFP Reporter Mouse as a Tool to Investigate Dystrophin Expression

Duchenne muscular dystrophy is a complex and severe orphan disease. It develops when the organism lacks the expression of dystrophin - a large structural protein. Dystrophin is transcribed from the largest gene in the human genome. At the moment, there is no cure available. Dozens of groups all over the world search for cure. Animal models are an important component of both the fundamental research and therapy development. Many animal models reproducing the features of disease were created and actively used since the late 80’s until present. The species diversity spans from invertebrates to primates and the genetic diversity of these models spans from single mutations to full gene deletions. The models are often non-interchangeable; while one model may be used for particular drug design it may be useless for another. Here we describe existing models, discuss their advantages and disadvantages and potential applications for research and therapy development.

/pdf/duchenne-muscular-dystrophy-animal-models-4cpgcro41c.pdf

Yulia V. Ivanova

Papers

Duchenne Muscular Dystrophy Animal Models