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

Abstract

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 (μDys) are currently in clinical trials. They all carry H1 and H4. In this study, we investigated whether these two hinges are essential for μDy function in murine DMD models. Three otherwise identical μDys were engineered to contain H1 and/or H4 and were named H1/H4 (with both H1 and H4), ΔH1 (without H1), and ΔH4 (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 μDys expression in total muscle lysate. However, only H1/H4 and ΔH1 showed correct sarcolemmal localization. ΔH4 mainly existed as sarcoplasmic aggregates. H1/H4 and ΔH1, but not ΔH4, 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 ΔH1, but not by ΔH4. Next, we compared H1/H4 and ΔH1 in 6-week-old male mdx mice by intravenous injection (1E13 vector genome particles/mouse). Four months postinjection, H1/H4 significantly outperformed ΔH1 in extensor digitorum longus muscle force measurements but two constructs yielded comparable electrocardiography improvements. We conclude that H4 is essential for μDys function and H1 facilitates force production. Our findings will help develop next-generation μDys gene therapy.

Keywords: Duchenne muscular dystrophy; dystrophin associated glycoprotein complex; hinges; mdx; micro-dystrophin; muscle function.

Figure 1.

Absence of H4 but not H1 affected μDys localization. (A) Cartoon illustrations of three μDys constructs used in the study. Deleted regions are marked as dotted boxes. (B) Capillary western blot (visualized as virtual blot) detection of dystrophin and vinculin in the TA muscle following intramuscular injection. (C) Quantification of dystrophin expression in whole muscle lysate of the TA muscle following intramuscular injection. (D) Representative dystrophin immunofluorescence staining and quantification of dystrophin-positive myofibers in the TA muscle following intramuscular injection. Asterisk, p < 0.05. μDys, micro-dystrophin; AU, artificial unit; TA, tibialis anterior.

Figure 2.

H4 was essential for dystrophin-associated glycoprotein complex assembly. (A) Representative immunofluorescence staining photomicrographs for Dys, beta-DG, beta-SG, Dbr, Syn, and nNOS. Asterisk, same myofiber in serial sections in the same column. (B) Representative total muscle lysate western blot for dys, beta-DG, beta-SG, Dbr, Syn, and nNOS. Vinculin was used as the loading control. beta-DG, beta-dystroglycan; beta-SG, beta-sarcoglycan; Dbr, dystrobrevin; nNOS, neuronal nitric oxide synthase; Syn, syntrophin.

Figure 3.

TA muscle function was differently impacted by H1 deletion and H4 deletion. (A) Representative H&E staining photomicrographs and quantification of myofibers with centrally localized myonuclei. (B) Side-by-side comparison of the specific muscle force among WT (C57/BL6), mdx4cv (4cv), ΔH1 μDys-injected, and H1/H4 μDys-injected groups. Data from each muscle were plotted. (C) Side-by-side comparison of the eccentric contraction profile among WT, mdx4cv, ΔH1 μDys-injected, and H1/H4 μDys-injected groups. (D) Side-by-side comparison of the specific muscle force among WT, mdx4cv, ΔH4 μDys-injected, and H1/H4 μDys-injected groups. Data from each muscle were plotted. (E) Side-by-side comparison of the eccentric contraction profile among WT, mdx4cv, ΔH4 μDys-injected, and H1/H4 μDys-injected groups. Asterisk in (A, B, D): significantly different (p < 0.05) between indicated groups. Asterisk in (C, E): WT and H1/H4 μDys-injected groups are significantly higher than mdx4cv, ΔH1 μDys-injected, and ΔH4 μDys-injected groups. Cross (+) in (C): the WT group is significantly higher than mdx4cv and ΔH1 μDys-injected groups. Double cross (++) in (C): the H1/H4 μDys-injected group is significantly higher than the mdx4cv group. Pound sign (#) in (C, E): the wild-type group is significantly higher than the remaining three groups. Triple cross (+++) in (E): the H1/H4 μDys-injected group is significantly higher than mdx4cv and ΔH4 μDys-injected groups. Dollar sign ($) in (E): the H1/H4 μDys-injected group is significantly higher than the ΔH4 μDys-injected group. H&E, Hematoxylin and Eosin; WT, wild-type.

Figure 4.

H1 deletion compromised force recovery in the EDL muscle. (A) Representative dystrophin immunofluorescence staining photomicrographs. (B) Capillary western blot (visualized as virtual blot) detection of dystrophin and vinculin in the EDL muscle following systemic injection. (C) Quantification of dystrophin expression in whole muscle lysate of the EDL muscle following systemic injection. (D) Side-by-side comparison of the specific force of the EDL muscle among WT (C57/BL10), mdx, ΔH1 μDys-injected, and H1/H4 μDys-injected groups following systemic injection. Data from each mouse were plotted. (E) Side-by-side comparison of the eccentric contraction profile of the EDL muscle among WT, mdx, ΔH1 μDys-injected, and H1/H4 μDys-injected groups following systemic injection. Asterisk in (C, D): significantly different from the indicated groups. Asterisk in (E): significantly different from the remaining three groups. Cross (+) in (E): the H1/H4 μDys-injected group is significantly higher than the mdx group. Double cross (++) in (E): the H1/H4 μDys-injected group is significantly higher than mdx and ΔH1 μDys-injected groups. Pound sign (#) in (E): the mdx group is significantly different from ΔH1 μDys-injected and H1/H4 μDys-injected groups. EDL, extensor digitorum longus.

Figure 5.

ΔH1 μDys and H1/H4 μDys were equally effective in improving ECG. (A) Representative heart dystrophin immunofluorescence staining photomicrographs. (B) Side-by-side comparison of ECG parameters among WT (C57/BL10), mdx, ΔH1 μDys-injected, and H1/H4 μDys-injected groups. Asterisk: significantly different from the indicated groups. ECG, electrocardiography.