Asterisk, significantly different from the other group.įigure 2 100-fold overexpression of the therapeutic ΔH2-R19 minidystrophin gene in the heart of transgenic line 26 worsened electrocardiogram (ECG) defects seen in mdx mice. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue color).

The cytosolic dystrophin inclusion bodies appear as vesicles. The right panel of line 26 images is an enlarged view of the boxed region of the middle panel and it was taken with a much reduced exposure time. Excessively expressed dystrophin forms inclusion body inside cardiomyocytes. The middle panel of line 26 images shows a short-exposure, high-power photomicrograph. The left panel (low-power images of line 29 heart) and the second to the left panel (low-power images of line 26 heart) had the identical exposure conditions. ( e) Representative dystrophin immunofluorescence staining from the heart of transgenic lines 26 and 29.

The level of expression was normalized to the loading control and BL10 control. ( d) Quantification of minidystrophin expression. ( c) Two representative dystrophin western blots of the heart of BL10, mdx and transgenic mice. ( b) Quantification of the minidystrophin gene copy number in transgenic mice. Arrow, the 3.4 kb diagnostic band for transgenic mice. ( a) A representative Southern blot photomicrograph. From the safety standpoint, it is essential to determine whether supraphysiological expression of a therapeutic mini/micro dystrophin gene can lead to deleterious side effects.įigure 1 Transgenic overexpression of a therapeutic minidystrophin gene in the heart of mdx mice. A likely consequence of this approach is unchecked expression and the production of excessive amount of dystrophin. To overcome these hurdles, investigators are forced to express a synthetic mini/micro dystrophin gene from a constitutive promoter (either ubiquitous or muscle-specific). This not only excludes the use of the full-length gene or cDNA as the therapeutic gene but also excludes the use of the endogenous dystrophin promoter in the expression cassette. It greatly exceeds the packaging limit of most viral gene delivery vectors. Unfortunately, the dystrophin gene is one of the largest genes in the mammalian genome. This will allow for spatially and temporally regulated expression of a full-length protein to meet developmental and physiological needs of muscle. Ideally, for gene therapy one would like to express the full-length gene or the full-length cDNA from the endogenous promoter. Introducing a functional dystrophin gene back to muscle by gene therapy holds a great promise to treat DMD. This X-linked disease mainly affects boys and young men. Our data suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity.ĭuchenne muscular dystrophy (DMD) is the most common childhood lethal muscle disease caused by dystrophin deficiency. Importantly, several electrocardiogram parameters (QT interval, QRS duration and the cardiomyopathy index) became worse than that of mdx mice. Excessive minidystrophin expression did not correct tachycardia, a characteristic feature of Duchenne muscular dystrophy. However, in the line with 100-fold overexpression, we not only detected sarcolemmal minidystrophin expression but also observed accumulation of minidystrophin vesicles in the sarcoplasm. In the line with 50-fold overexpression, minidystrophin showed sarcolemmal localization and electrocardiogram abnormalities were corrected. To determine whether excessive dystrophin expression in the heart is safe, we studied two lines of transgenic mdx mice that selectively expressed a therapeutic minidystrophin gene in the heart at 50-fold and 100-fold of the normal levels. In the case of Duchenne muscular dystrophy, Chamberlain and colleagues found that 50-fold overexpression did not cause deleterious side effect in skeletal muscle. Supraphysiological expression is a concern for all gene therapy studies. Gene Editing: Technology & Applicationsĭystrophin gene replacement holds the promise of treating Duchenne muscular dystrophy.

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