Revertant fibres and dystrophin traces in Duchenne muscular dystrophy: Implication for clinical trials

Arechavala‐Gomeza, Virginia; Kinali, Maria; Feng, Lucy; Guglieri, Michela; Edge, G.; Main, Marion; Hunt, David M.; Lehovsky, Jan; Straub, Volker; Bushby, Kate; Sewry, Caroline A.; Morgan, Jennifer E.; Muntoni, Francesco

doi:10.1016/j.nmd.2010.03.007

Cited by 80 publications

(66 citation statements)

References 40 publications

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“…The expression of very low levels of trace dystrophin in patients with DMD has been observed before [1], [2], but emerging methodologies on imaging and quantification of expression levels have shown that trace dystrophin is expressed more commonly in nearly all fibers in the majority of DMD muscle biopsies [3]–[7]. The incidence of revertant fibers that express relatively high levels of dystrophin is generally low (<5%) and appears to be dependent on the deletion and can reach 14% [6], [7]. Trace dystrophin and revertant fibers can be recognized and accounted for in immunofluorescence analysis (IFA) of muscle biopsy cross-sections.…”

Section: Introductionmentioning

confidence: 94%

“…One image analysis method determined the average dystrophin intensity from all fibers in an image that co-localized with a spectrin signal in a software-generated mask defining the sarcolemmal area [4]. Another method assessed dystrophin expression by manually placing circles on randomly selected areas of the sarcolemma of a selected number of fibers per biopsy and measured the maximum membrane intensity within that circle [5], [6]. It has been reported that the intensity of dystrophin may vary between fibers in a healthy control muscle [4], [5] and that this variability is even more pronounced in BMD and DMD muscle [2], [3], [5].…”

Section: Introductionmentioning

confidence: 99%

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A Sensitive, Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

et al. 2014

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Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.

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Section: Introductionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

A Sensitive, Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

et al. 2014

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“…In one of these (patient 2 in that report) the response consisted of CD4 + cells, whereas in the other (patient 4) it was mediated by CD8 + cells. It had been postulated that such priming was unlikely, due to the presence of revertant fibers in many patients (estimated, using different degrees of sensitivity in the analysis, as occurring between 0.01 and 47% of patients with DMD [Kissel et al, 1991;Fanin et al, 1992Fanin et al, , 1995Klein et al, 1992;Thanh et al, 1995;Uchino et al, 1995;Winnard et al, 1995;Arechavala-Gomeza et al, 2010]). In these fibers, dystrophin protein is expressed because of secondary mutations within the individual muscle fibers-most commonly, altered splicing that restores the reading frame and allows translation of exons downstream of the primary mutation.…”

Section: Introductionmentioning

confidence: 99%

Anti-Dystrophin T Cell Responses in Duchenne Muscular Dystrophy: Prevalence and a Glucocorticoid Treatment Effect

et al. 2013

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Duchenne muscular dystrophy (DMD) typically occurs as a result of truncating mutations in the DMD gene that result in a lack of expression of the dystrophin protein in muscle fibers. Various therapies under development are directed toward restoring dystrophin expression at the subsarcolemmal membrane, including gene transfer. In a trial of intramuscular adeno-associated virus (AAV)-mediated delivery of a therapeutic minidystrophin construct, we identified in two of six subjects the presence of a population of T cells that had been primed to recognize dystrophin epitopes before transgene delivery. As the presence of preexisting T cell immunity may have a significant effect on the success of therapeutic approaches for restoring dystrophin, we sought to determine the prevalence of such immunity within a DMD cohort from our Muscular Dystrophy Association clinic. Dystrophin-specific T cell immunity was evaluated in subjects with DMD who were either receiving the glucocorticoid steroid prednisone (n=24) or deflazacort (n=29), or who were not receiving steroids (n=17), as well as from normal age-matched control subjects (n=21). We demonstrate that increasing age correlates with an increased risk for the presence of anti-dystrophin T cell immunity, and that treatment with either corticosteroid decreases risk compared with no treatment, suggesting that steroid therapy in part may derive some of its benefit through modulation of T cell responses. The frequency of dystrophin-specific T cells detected by enzyme-linked immunospot assay was lower in subjects treated with deflazacort versus prednisone, despite similar overall corticosteroid exposure, suggesting that the effects of the two corticosteroids may not be identical in patients with DMD. T cells targeted epitopes upstream and downstream of the dystrophin gene mutation and involved the CD4⁺ helper and/or CD8⁺ cytotoxic subsets. Our data confirm the presence of preexisting circulating T cell immunity to dystrophin in a sizable proportion of patients with DMD, and emphasize the need to consider this in the design and interpretation of clinical gene therapy trials.

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“…It had been postulated that such priming was unlikely, due to the presence of revertant fibers in many patients (estimated, using different degrees of sensitivity in the analysis, as occurring between 0.01 and 47% of patients with DMD [Kissel et al, 1991;Fanin et al, 1992Fanin et al, , 1995Klein et al, 1992;Thanh et al, 1995;Uchino et al, 1995;Winnard et al, 1995;Arechavala-Gomeza et al, 2010]). In these fibers, dystrophin protein is expressed because of secondary mutations within the individual muscle fibers-most commonly, altered splicing that restores the reading frame and allows translation of exons downstream of the primary mutation.…”

mentioning

confidence: 99%

Anti-Dystrophin T Cell Responses in Duchenne Muscular Dystrophy: Prevalence and a Glucocorticoid Treatment Effect

Flanigan¹,

Campbell²,

Viollet³

et al. 2013

Human Gene Therapy Methods

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Duchenne muscular dystrophy (DMD) typically occurs as a result of truncating mutations in the DMD gene that result in a lack of expression of the dystrophin protein in muscle fibers. Various therapies under development are directed toward restoring dystrophin expression at the subsarcolemmal membrane, including gene transfer. In a trial of intramuscular adeno-associated virus (AAV)-mediated delivery of a therapeutic minidystrophin construct, we identified in two of six subjects the presence of a population of T cells that had been primed to recognize dystrophin epitopes before transgene delivery. As the presence of preexisting T cell immunity may have a significant effect on the success of therapeutic approaches for restoring dystrophin, we sought to determine the prevalence of such immunity within a DMD cohort from our Muscular Dystrophy Association clinic. Dystrophin-specific T cell immunity was evaluated in subjects with DMD who were either receiving the glucocorticoid steroid prednisone (n = 24) or deflazacort (n = 29), or who were not receiving steroids (n = 17), as well as from normal age-matched control subjects (n = 21). We demonstrate that increasing age correlates with an increased risk for the presence of anti-dystrophin T cell immunity, and that treatment with either corticosteroid decreases risk compared with no treatment, suggesting that steroid therapy in part may derive some of its benefit through modulation of T cell responses. The frequency of dystrophin-specific T cells detected by enzymelinked immunospot assay was lower in subjects treated with deflazacort versus prednisone, despite similar overall corticosteroid exposure, suggesting that the effects of the two corticosteroids may not be identical in patients with DMD. T cells targeted epitopes upstream and downstream of the dystrophin gene mutation and involved the CD4 + helper and/or CD8 + cytotoxic subsets. Our data confirm the presence of preexisting circulating T cell immunity to dystrophin in a sizable proportion of patients with DMD, and emphasize the need to consider this in the design and interpretation of clinical gene therapy trials.

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Revertant fibres and dystrophin traces in Duchenne muscular dystrophy: Implication for clinical trials

Cited by 80 publications

References 40 publications

A Sensitive, Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

A Sensitive, Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

Anti-Dystrophin T Cell Responses in Duchenne Muscular Dystrophy: Prevalence and a Glucocorticoid Treatment Effect

Anti-Dystrophin T Cell Responses in Duchenne Muscular Dystrophy: Prevalence and a Glucocorticoid Treatment Effect

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