Delineation of Duchenne muscular dystrophy gene therapy using genetically engineered mice

Duchenne muscular dystrophy (DMD) is a genetically inherited debilitating muscle disorder affecting young boys due to the loss of dystrophin protein in muscle and the heart. Affected individuals lose their mobility and become confined to a wheel chair in early teens. They develop heart disease towards the end stage of the disease. Heart failure or breathing complications leads to death. Currently there is no cure for DMD. Gene therapy has shown great promise to restore the lost dystrophin protein in DMD. During my PhD, I have addressed several important aspects of dystrophin gene therapy. The findings from these studies will benefit development of an effective gene therapy for DMD and will open the door for some important future studies. Here I briefly describe the findings in my research. In the first study I addressed whether muscle-only rescue affects the heart. I used mice genetically engineered (alias transgenic) to carry dystrophin in skeletal muscle but not in the heart. To more closely mimic the human heart condition, I aged these mice to 23-months, an aged dystrophin-null mice show heart failure similar to that of DMD patients. Evaluation of heart pathology, ECG and pump function revealed that the heart in transgenic mice does not notably differ from the dystrophin deficient mice. In other words, selective muscle only treatment did not improve or heighten heart disease in DMD. In the next study I evaluated whether continuous therapeutic dystrophin expression is essential for muscle and heart health. To evaluate this in a mouse model, I created two mouse models carrying therapeutic dystrophin in the heart or muscle. These mice were engineered in a way that permits intentional removal of the therapeutic gene from the heart or muscle using viral mediated enzyme delivery. I delivered the viral mediated enzyme to muscle or the heart of the respective mouse strain in adult mice. After 12-15 months of therapeutic dystrophin removal, I evaluated muscle and the heart from individual strain. The removal of muscle therapeutic dystrophin resulted muscle deterioration, reduced muscle weight, size and force. The heart pump function was noticeably weakened after removal of cardiac therapeutic dystrophin. These findings indicated that uninterrupted therapeutic dystrophin expression is essential to maintain muscle and heart health. In the third study, I proposed, based on patient data, that dystrophin may contain a heart protection region between repeats 16-19 (R16-19) of dystrophin. I studied this by comparing the heart pathology, ECG and pump function in two transgenic mouse models that express dystrophin in the heart with or without R16-19. In support of my hypothesis, addition of R16-19 completely rescued ECG and corrected an important heart function parameter that were not rescued in mice that lack R16-19. Lastly, I looked at whether very low levels of dystrophin can benefit DMD heart. To test this, I used a mouse model referred to as mdx3cv that was genetically modified using chemical induced mutations. These mice expressed about 3.3% of dystrophin compared to normal mice. The heart of mdx3cv mice showed similar level of damage as dystrophin deficient mice. Surprisingly, ECG and hemodynamic function were improved in mdx3cv mice. These results suggest that marginal level dystrophin expression can still help the heart but it is far from sufficient for full recovery

The FVB Background Does Not Dramatically Alter the Dystrophic Phenotype of Mdx Mice

The mdx mouse is the most frequently used animal model for Duchenne muscular dystrophy (DMD), a fatal muscle disease caused by the loss of dystrophin. Mdx mice are naturally occurring dystrophin-null mice on the C57BL/10 (BL10) background. We crossed black mdx to the white FVB background and generated mdx/FVB mice. Compared to that of age- and sex-matched FVB mice, mdx/FVB mice showed characteristic limb muscle pathology similar to that of original mdx mice. Further, the forelimb grip strength and limb muscle (tibialis anterior and extensor digitorum longus) specific force of mdx/FVB mice were significantly lower than that of wild type FVB mice. Consistent with what has been reported in original mdx mice, mdx/FVB mice also showed increased susceptibility to eccentric contraction-induced force loss and elevated serum creatine kinase. Our results suggest that the FVB background does not dramatically alter the dystrophic phenotype of mdx mice

100-fold but not 50-fold dystrophin overexpression aggravates electrocardiographic defects in the mdx model of Duchenne muscular dystrophy

Dystrophin gene replacement holds the promise of treating Duchenne muscular dystrophy. Supraphysiological expression is a concern for all gene therapy studies. In the case of Duchenne muscular dystrophy, Chamberlain and colleagues found that 50-fold overexpression did not cause deleterious side effect in skeletal muscle. 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 line with 50-fold overexpression, minidystrophin showed sarcolemmal localization and electrocardiogram abnormalities were corrected. 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. Excessive minidystrophin expression did not correct tachycardia, a characteristic feature of Duchenne muscular dystrophy. Importantly, several electrocardiogram parameters (QT interval, QRS duration and the cardiomyopathy index) became worse than that of mdx mice. Our data suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity