Prevention of pathology in mdx mice by expression of utrophin: analysis using an inducible transgenic expression system

Abstract

Duchenne muscular dystrophy results from the absence of dystrophin, a cytoskeletal protein. Previously, we have shown in a transgenic mouse model of the disease (mdx) that high levels of expression of the dystrophin-related protein, utrophin can prevent pathology. We developed a new transgenic mouse model where muscle specific utrophin expression was conditioned by addition of tetracycline in water. Transgene expression was turned on at different time points: in utero, at birth, 10 and 30 days after birth. We obtained moderate levels of expression, variable from fibre to fibre (mosaicism) but sufficient to induce a correct localization of the dystro-sarcoglycan complex. Histology revealed a reduction of necrotic foci and of the percentage of centronucleated fibres, which remained still largely above the normal level. Isometric force was not improved but the resistance to eccentric contractions was significantly stronger. When utrophin expression was activated 30 days after birth, improvements were marginal, suggesting that the age at which utrophin therapy is initiated could be an important factor. Our results also provide an unexpected insight into the pathogenesis of the dystrophinopathies. We observed a complete normalization of the characteristics of the mechano-sensitive/voltage-independent Ca(2+) channels (occurrence, open probabilities and Ca(2+) currents), while the classical markers of dystrophy were still abnormal. These observations question the role of increased Ca(2+) channel activity in initiating the dystrophic process. The new model shows that utrophin therapy, initiated after birth, can be effective, but the extent of correction of the various symptoms of dystrophinopathy critically depends on the amount of utrophin expressed.

Soluble TNF-α receptor secretion from healthy or dystrophic mice after AAV6-mediated muscle gene transfer

“…Alternatively, such an approach has to be associated with a second treatment, which avoids muscle degeneration. This latter result may be obtained using different strategies, including a gene therapy approach allowing for dystrophin, mini-or microdystrophin expression; 23 gene transfer-mediated overexpression of utrophin; 28 antisense approaches consisting of the delivery of oligonucleotides (or rAAVs allowing their production) targeted to splicing elements of the dystrophin pre-mRNA that can induce exon skipping and thereby restore the open reading frame and the production of a functional dystrophin; 29 ribosomal readthrough of premature termination codons by compounds such as PTC124; 30 or pharmacological treatment to increase levels of utrophin. 31 …”

Guérir la myopathie de Duchenne par l’utrophine ?

“…Les approches développées pour stimuler l'expression d'utrophine chez la souris mdx adulte ont, tout au plus, permis d'augmenter celle-ci de 2 à 3 fois, nettement moins que dans la lignée transgénique Fiona, où aucun signe de dystrophie n'était plus détectable. Les résultats obtenus avec la transgenèse conditionnelle [25] indiquent que la quantité-seuil requise pour corriger un paramètre déficitaire diffère selon le paramètre considéré. La dystrophie ne pourra être vaincue que lorsque la quantité d'utrophine nécessaire à la correction du(des) paramètre(s) responsable(s) aura été obtenue.…”

“…Ce contrôle est réversible et offre la possibilité de déclen-cher ou de réprimer l'expression du transgène à n'importe quel moment de la vie de l'animal. Ce type de construction génétique a été développé pour l'expression conditionnelle de l'utrophine dans les muscles de souris mdx à différents moments (avant et après la naissance), suivie d'une analyse de l'état dystrophique des muscles à l'âge adulte (3 mois) [25]. Quantitativement, la surexpression par rapport à l'état mdx est modérée, bien inférieure à celle obtenue dans la lignée transgénique Fiona.…”

“…Quantitativement, la surexpression par rapport à l'état mdx est modérée, bien inférieure à celle obtenue dans la lignée transgénique Fiona. Elle est cependant suffisante pour obtenir une relocalisation des DAG, une réduction du pourcentage de fibres centronucléées et une récupération de 40% pour la résistance aux contractions excentriques [25]. Un certain niveau de guérison a donc pu être obtenu.…”

Utrophin suppresses low frequency oscillations and coupled gating of mechanosensitive ion channels in dystrophic skeletal muscle

“…28,29 In support of this idea, transgenic mice lacking both dystrophin and utrophin have severe muscle disease that more closely resembles Duchenne dystrophy 30,31 and increased transgenic expression of utrophin reduces the muscle pathology in the mdx mouse. 32,34 In a preceding paper, we showed that genetic depletion of utrophin in muscle fibers from mdx mice produces a gene dosedependent increase in the opening probability of single MS channels in recordings from membrane patches. 35 The increase in MS channel opening in the utrophin mutants lasted seconds rather than milliseconds.…”