Systemic AAV Micro-dystrophin Gene Therapy for Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin gene mutation. Conceptually, replacing the mutated gene with a normal one would cure the disease. However, this task has encountered significant challenges due to the enormous size of the gene and the distribution of muscle throughout the body. The former creates a hurdle for viral vector packaging and the latter begs for whole-body therapy. To address these obstacles, investigators have invented the highly abbreviated micro-dystrophin gene and developed body-wide systemic gene transfer with adeno-associated virus (AAV). Numerous microgene configurations and various AAV serotypes have been explored in animal models in many laboratories. Preclinical data suggests that intravascular AAV micro-dystrophin delivery can significantly ameliorate muscle pathology, enhance muscle force, and attenuate dystrophic cardiomyopathy in animals. Against this backdrop, several clinical trials have been initiated to test the safety and tolerability of this promising therapy in DMD patients. While these trials are not powered to reach a conclusion on clinical efficacy, findings will inform the field on the prospects of body-wide DMD therapy with a synthetic micro-dystrophin AAV vector. This review discusses the history, current status, and future directions of systemic AAV micro-dystrophin therapy.

Keywords: AAV; BMD; Becker muscular dystrophy; DMD; Duchenne muscular dystrophy; adeno-associated virus; clinical trial; dystrophin; micro-dystrophin; systemic gene therapy.

Figure 1

Historical Milestones in the Development of Systemic AAV Micro-dystrophin Gene Therapy

Figure 2

Adeno-associated Viral Vector (A) A representative electron microscopic image of the AAV vector. Arrowhead, a fully packaged AAV particle. Arrow, an empty AAV particle. (B) Examination of AAV purity with SDS-PAGE silver staining. Lanes 1, 2, and 3 show a gradual increase of the purity after one, two, and three rounds of purification. The highly pure AAV stock has three viral proteins (VPs) at the ratio of VP1:VP2:VP2 ≈ 1:1:10.

Figure 3

Full-Length Dystrophin and Representative Micro-dystrophins Full-length dystrophin contains an N-terminal domain (N), 24 spectrin-like repeats (R1 to R24), four hinges (H1 to H4), a cysteine-rich domain (CR), and a C-terminal domain (CT). ΔDysM3 is the first synthetic micro-dystrophin. Δ3990, ΔR4–23/ΔC and μDys5R are three micro-dystrophins currently in use in clinical trials. H2 is marked in orange to indicate that it compromises micro-dystrophin function in the mouse DMD model (see Banks et al. for details). R16 and R17 are marked in red to indicate that they are the nNOS-binding domain (see Lai et al., for details).

Figure 4

AAV Micro-dystrophin Gene Therapy Ameliorated Muscle Disease in the Murine and Canine DMD Models (A) Systemic AAV micro-dystrophin injection improved skeletal muscle function in mdx mice. Treatment significantly improved specific tetanic force and resistance to eccentric contraction-induced force drop in the extensor digitorum longus muscle (see Shin et al. for details). Error bar, mean ± SEM. (B) Systemic AAV micro-dystrophin injection improved cardiac hemodynamics in mdx mice (see Bostick et al. for details). (C) AAV micro-dystrophin therapy improved histology (left) and reduced pathological muscle calcification (right) in the extensor carp ulnaris muscle in affected dogs (see Shin et al. for details).

Figure 5

First AAV Micro-dystrophin Clinical Trial Direct injection of the AAV vector to the biceps of a patient by Dr. Jerry Mendell (asterisk). The injection was assisted by an interventional radiologist (triangle) and a neurologist (square). The radiologist and the neurologist guided and monitored the injection process with ultrasound and electromyography, respectively, to make sure AAV was delivered into viable muscle (see Mendell et al. for details).

Figure 6

Sarcolemmal nNOS Delocalization Contributes to DMD Pathogenesis In normal muscle, nNOS is localized at the sarcolemma. This allows immediate diffusion of nitric oxide (NO) to the vasculature and vasodilation in contracting muscle. In DMD, the loss of sarcolemmal nNOS compromises this process and leads to functional ischemia. The H&E-stained image illustrates focal ischemic lesions (arrow) as the first observable histological change in a 3-week-old affected dog. Despite the absence of dystrophin, histologically, the majority of myofibers appeared normal at this age.