From the smallest virus to the biggest gene: marching towards gene therapy for duchenne muscular dystrophy

Abstract

The gene encoding dystrophin, which is altered in Duchenne muscular dystrophy, is one of the largest in mammalian species. Adeno-associated virus (AAV) is the smallest viral gene delivery vector with a "cargo" capacity of 6 kb and is the most effective gene delivery vehicle to muscle cells. Overlapping and trans-splicing AAV vector approaches are devised to do the "impossible."

Figure 1

Schematic outline of the dystrophin protein and the strategies to deliver the micro- and mini-dystrophin genes by AAV. The N-terminus (N) of the dystrophin protein interacts with γ-actin. The body of the dystrophin body is consisted of 24 spectrin-like repeats and four hinges. Repeats 3, 20, and 24 are marked with numerical numbers. Hinge 3 (gray color) is different from other hinges and it contains a viral protease site. The cysteine-rich (CR) domain interacts with dystroglycan (DG). The C-terminus (C) of the dystrophin protein interacts with syntrophin (Syn) and dystrobrevin (Dbr). Syntrophin recruits nNOS to the sarcolemma. The 3.8 kb microgene is missing the regions from repeat 4 to repeat 23 as well as the C-terminal domain. This microgene can be delivered by a single intact AAV virion. The 6 kb minigene has a smaller deletion (from hinge 2 to repeat 19). The minigene can be efficiently expressed by the trans-splicing AAV vectors.

Figure 2

AAV gene therapy reduces dystrophic pathology in a mouse model of Duchenne muscular dystrophy. Representative photomicrographs of serial sections of an AAV treated muscle at two months after gene therapy. A, immunostaining for dystrophin (Dys). Dystrophin expression (green) is only seen in AAV infected myofibers. Nuclei are stained with DAPI (blue). B, Hematoxylin-eosin (HE) staining shows small degenerative myofibers in untreated areas (arrows). The treated muscle is protected from degeneration. C, Masson trichrome (MT) staining illustrates fibrosis (blue) in untreated area (arrow). D, Non-specific esterase (NE) staining reveals macrophage (small dark brown cells) infiltration in untreated areas (arrows). Yellow squares mark the same myofiber in serial sections. Scale bar, 50 μm.