Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model

Abstract

Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder, caused by recessive mutations in the dystrophin gene. One of every 3,500 males suffers from DMD, yet no treatment is currently available. Genetic therapeutic approaches, using primarily myoblast transplantation and adenovirus-mediated gene transfer, have met with limited success. Adeno-associated virus (AAV) vectors, although proven superior for muscle gene transfer, are too small (5 kb) to package the 14-kb dystrophin cDNA. Here we have created a series of minidystrophin genes (<4.2 kb) under the control of a muscle-specific promoter that readily package into AAV vectors. When injected into the muscle of mdx mice (a DMD model), two of the minigenes resulted in efficient and stable expression in a majority of the myofibers, restoring the missing dystrophin and dystrophin-associated protein complexes onto the plasma membrane. More importantly, this AAV treatment ameliorated dystrophic pathology in mdx muscle and led to normal myofiber morphology, histology, and cell membrane integrity. Thus, we have defined minimal functional dystrophin units and demonstrated the effectiveness of using AAV to deliver the minigenes in vivo, offering a promising avenue for DMD gene therapy.

Figure 1

Construction of highly truncated minidystrophin genes. Dystrophin has four major domains: the N-terminal domain (N), the CR domain, the CT domain, and the central rod domain, which contains 24 rod repeats (R) and four hinges (H). The minidystrophin genes were constructed by deleting a large portion of the central rods and hinges and nearly the entire CT domain (except the last 3 aa). The minidystrophin genes subsequently were cloned between an MCK promoter (or a CMV promoter) and a poly(A) sequence in AAV vectors.

Figure 2

IF analysis of the dystrophin and DAP complexes in gastrocnemius muscle. (a) Cryosections of mdx muscle, at 3 months after treatment with construct AAV-MCK-Δ3849 or AAV-MCK-Δ3990, were IF-stained with an antibody against dystrophin (green) and then counterstained for cell nuclei with DAPI (blue). Photos were taken with a ×4 microscope lens. Note the widespread minidystrophin expression and peripheral nucleation in a majority of the myofibers. Also note the extensive central nucleation in minidystrophin-negative areas. (b) Cryosections of muscles from 15-week-old normal C57/B10 mice, from mdx mice treated either with vector AAV-MCK-Δ3849, AAV-MCK-Δ3990, or AAV-MCK-Δ4173, or from untreated mdx mice were IF-stained with antibodies for dystrophin (green) and counterstained with DAPI (blue) for nuclei (DYS + DAPI). Note the lack of central myonuclei. The consecutive sections also were stained with antibodies for α-sarcoglycan (α-SG), β-sarcoglycan (β-SG), and γ-sarcoglycan (γ-SG). Photographs were taken with a ×20 lens.

Figure 3

Long-term minidystrophin expression in mdx mice treated at a young age (a) or as adults (c–h) with AAV vectors containing different minigenes under the control of different promoters. IF staining of minidystrophin (green) and myonuclei counterstaining with DAPI (blue) were performed on gastrocnemius muscles isolated from (a) MCK-Δ3849-treated 10-day-old mdx for 6 months, (b) untreated 6-month-old mdx, (c) MCK-Δ3849-treated adult mdx for 2 months, (d) MCK-Δ3990-treated adult mdx for 2 months, (e) MCK-Δ3849-treated adult mdx for 4 months, (f) MCK-Δ3990-treated adult mdx for 4 months, (g) CMV-Δ3849-treated adult mdx for 6 months, and (h) CMV-Δ3990-treated adult mdx for 6 months.

Figure 4

Protection of muscle plasma membrane integrity by minidystrophin genes in mdx mice treated either at 10 days of age (a) or as adults (b). (a) Three months after AAV vector injection, either the treated mdx mice or the age-matched controls (normal C57/B10 and untreated mdx mice) were i.v. injected with Evans Blue dye. The gastrocnemius muscles then were collected and cryosectioned either from normal C57/B10 mice; from mdx mice treated at 10 days of age with AAV-MCK vectors Δ3849, Δ3990, or Δ4173; or from the untreated mdx mice. Normal dystrophin and minidystrophin expression was visualized by IF staining (Left, green). The leaky myofibers were visualized by the uptake of Evans Blue dye (Center, red fluorescence). Note the mutual exclusivity between dystrophin expression and Evans Blue dye uptake as shown by the merged images (Right). Photographs were taken with a ×10 lens. (b) Adult mdx gastrocnemius muscles were treated with AAV vectors containing Δ3990 minigene. Evans Blue dye tests were performed at 2 months after AAV-MCK-Δ3990 treatment (Left) or at 6 months after AAV-CMV-Δ3990 treatment (Right). Note the widespread minidystrophin expression (green) and the leaky myofibers (red), which were negative for minidystrophin staining.