Nanotherapy for Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a lethal X-linked childhood muscle wasting disease caused by mutations in the dystrophin gene. Nanobiotechnology-based therapies (such as synthetic nanoparticles and naturally existing viral and nonviral nanoparticles) hold great promise to replace and repair the mutated dystrophin gene and significantly change the disease course. While a majority of DMD nanotherapies are still in early preclinical development, several [such as adeno-associated virus (AAV)-mediated systemic micro-dystrophin gene therapy] are advancing for phase I clinical trials. Recent regulatory approval of Ataluren (a nonsense mutation read-through chemical) in Europe and Exondys51 (an exon-skipping antisense oligonucleotide drug) in the United States shall offer critical insight in how to move DMD nanotherapy to human patients. Progress in novel, optimized nano-delivery systems may further improve emerging molecular therapeutic modalities for DMD. Despite these progresses, DMD nanotherapy faces a number of unique challenges. Specifically, the dystrophin gene is one of the largest genes in the genome while nanoparticles have an inherent size limitation per definition. Furthermore, muscle is the largest tissue in the body and accounts for 40% of the body mass. How to achieve efficient bodywide muscle targeting in human patients with nanomedication remains a significant translational hurdle. New creative approaches in the design of the miniature micro-dystrophin gene, engineering of muscle-specific synthetic AAV capsids, and novel nanoparticle-mediated exon-skipping are likely to result in major breakthroughs in DMD therapy. WIREs Nanomed Nanobiotechnol 2018, 10:e1472. doi: 10.1002/wnan.1472 This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies.

FIGURE 1

Histopathology and dystrophin immunostaining. Top panels show representative photomicrographs of hematoxylin–eosin (HE)-stained skeletal muscle cross-sections from a normal and an affected mouse. Bottom panels are serial sections immunostained with a monoclonal antibody that recognizes dystrophin. In normal muscle, dystrophin is localized at the muscle cell membrane. Lack of dystrophin leads to muscle degeneration/regeneration, necrosis, inflammatory cell infiltration, and replacement of deceased muscle by fat and fibrotic tissues. The presence of centrally located nuclei in dystrophic muscle fibers indicates recent fiber regeneration. Asterisks represent the same myofiber in serial muscle sections. The interstitial fluorescence signals seen in the immunostaining image of the affected mouse muscle is due to cross-reaction of the secondary antibody (Alex 594-conjugated anti-mouse antibody) to the inflamed mouse muscle.

FIGURE 2

Systemic AAV-9 micro-dystrophin gene transfer results in bodywide dystrophin restoration in striated muscles in the mouse model of Duchenne muscular dystrophy. (a) Representative dystrophin immunostaining photomicrographs from skeletal muscles of normal, untreated, and adeno-associated virus (AAV)-treated mdx mice. Normal muscle shows sarcolemmal expression of dystrophin, which is enriched at the neuromuscular junction (arrows). Dystrophic muscle has no dystrophin expression at the sarcolemma. Cross-reaction of the immunostaining antibody with inflamed/fibrotic interstitial tissues illustrates an irregular pattern of dystrophic muscle with extremely large and small myofibers. Systemic AAV-9 micro-dystrophin therapy restores dystrophin expression in the quadriceps (Quadro), extensor digitorum longus (EDL), tibialis anterior (TA), and diaphragm (Diaph). (b) Representative dystrophin immunostaining photomicrographs from the heart of normal, affected, and AAV micro-dystrophin-treated mice. Scale bar in panel (a) applies to all images.

FIGURE 3

A single intravenous injection of the adeno-associated virus (AAV)-9 micro-dystrophin vector leads to bodywide muscle transduction in the canine model of Duchenne muscular dystrophy. (a) Top panels: representative dystrophin immunostaining photomicrographs from skeletal muscles of normal and affected dogs. Bottom panels: representative dystrophin immunostaining photomicrographs from different skeletal muscle of an AAV-treated dog. Tem, temporalis; Ster, sternohyoid; TM, teres major; SP, superficial pectoral; Tri, triceps; ECU, extensor carpi ulnaris; FCU, flexor carpi ulnaris; LD, latissimus dorsi; LT, longissimus thoracis; VL, vastus lateralis; BF, biceps femoris; Ton, tongue. (b) Representative dystrophin immunostaining photomicrographs from the heart of normal, affected, and AAV mciro-dystrophin-treated dogs.