The Implication of Hinge 1 and Hinge 4 in Micro-Dystrophin Gene Therapy for Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by dystrophin deficiency. Dystrophin consists of the amino terminus, central rod domain with 24 spectrin-like repeats and four hinges (H), cysteine-rich domain, and carboxyl terminus. Several highly abbreviated micro-dystrophins (mu Dys) are currently in clinical trials. They all carry H1 and H4. In this study, we investigated whether these two hinges are essential for mu Dy function in murine DMD models. Three otherwise identical mu Dys were engineered to contain H1 and/or H4 and were named H1/H4 (with both H1 and H4), Delta H1 (without H1), and Delta H4 (without H4). These constructs were packaged in adeno-associated virus serotype-9 and delivered to the tibialis anterior muscle of 3-month-old male mdx4cv mice (1E12 vector genome particles/muscle). Three months later, we detected equivalent mu Dys expression in total muscle lysate. However, only H1/H4 and Delta H1 showed correct sarcolemmal localization. Delta H4 mainly existed as sarcoplasmic aggregates. H1/H4 and Delta H1, but not Delta H4, fully restored the dystrophin-associated protein complex and significantly improved the specific muscle force. Eccentric contraction-induced force decline was best protected by H1/H4, followed by Delta H1, but not by Delta H4. Next, we compared H1/H4 and Delta H1 in 6-week-old male mdx mice by intravenous injection (1E13 vector genome particles/mouse). Four months postinjection, H1/H4 significantly outperformed Delta H1 in extensor digitorum longus muscle force measurements but two constructs yielded comparable electrocardiography improvements. We conclude that H4 is essential for mu Dys function and H1 facilitates force production. Our findings will help develop next-generation mu Dys gene therapy.

Automated summary

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by dystrophin deficiency. In this study, it was found that mu Dys with both H1 and H4 effectively localized to the muscle membrane, restored the dystrophin-associated protein complex, and improved muscle force. On the other hand, mu Dys with only H1 or without H4 did not have the same effects. Therefore, H4 is essential for mu Dys function and H1 facilitates force production. These findings will contribute to the development of next-generation mu Dys gene therapy.