Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophin-deficient dogs

Abstract

Duchenne (DMD) and golden retriever (GRMD) muscular dystrophy are caused by genetic mutations in the dystrophin gene and afflict striated muscles. We investigated systemic gene delivery in 4-day-old GRMD dogs given a single intravenous injection of an AAV9 vector (1.5 x 10(14) vector genomes/kg) carrying a human codon-optimized human mini-dystrophin gene under control of the cytomegalovirus (CMV) promoter. One of the three treated dogs was euthanized 9 days later due to pre-existing conditions. Scattered mini-dystrophin-positive myofibers were seen by immunofluorescent (IF) staining in numerous muscles. At the end of the 16-week study, the other two dogs showed generalized muscle expression of mini-dystrophin in ~15% to nearly 100% of myofibers. Western blot and vector DNA quantitative PCR results agreed with the IF data. Delayed growth and pelvic limb muscle atrophy and contractures were seen several weeks after vector delivery. T-2 weighted magnetic resonance imaging (MRI) at 8 weeks showed increased signal intensity compatible with inflammation in several pelvic limb muscles. This marked early inflammatory response raised concerns regarding methodology. Use of the ubiquitous CMV promoter, extra-high vector dose, and marked expression of a human protein in canine muscles may have contributed to the pathologic changes seen in the pelvic limbs.

Figure 1

Histopathological changes in muscles 16 weeks after AAV9-CMV-mini-dystrophin vector intravenous injection of golden retriever muscular dystrophy (GRMD) dogs Emerald (E) and Amethyst (A) at 4 days of age. (a) Semitendinosus (E), (b) psoas major (E), (c) peroneus longus (A), (d) diaphragm (E), (e) cranial tibial (A), and (f) vastus medialis (E). All muscles have changes typical of GRMD, including small group myofiber necrosis (arrows in b, d, and e) and regeneration (arrows in c). Muscle is otherwise relatively normal histologically in all but the vastus medialis (f) in which marked deposition of fat is evident. Hematoxylin and eosin; bar = 300 µm.

Figure 2

T2-weighted magnetic resonance images of pelvic limb muscles 8 weeks after AAV9-CMV-mini-dystrophin vector intravenous injection of golden retriever muscular dystrophy dogs Emerald and Amethyst at 4 days of age. Transverse (left) and sagittal (right) images of (a–d) Amethyst and (e–h) Emerald are seen. The images in c,d,g, and h have been segmented and color coded to outline individual muscles. Signal intense lesions are particularly pronounced in the vastus heads of the quadriceps and adductor muscles. These changes persisted with fat saturation suggesting that they most likely represent fluid due to inflammation or edema.

Figure 3

Body-wide extensive human mini-dystrophin expression 16 weeks after intravenous injection of AAV9-CMV-mini-dystrophin vector in golden retriever muscular dystrophy dogs (a) Emerald and (b) Amethyst at 4 days of age. Cryo-thin sections of the muscle and heart were stained by immunofluorescence with antibody against human mini-dystrophin (red color) and counterstained with DAPI to show cell nuclei (blue color). Variable and extensive gene expression in multiple skeletal muscles is readily visible. Photomicrographs were taken from the areas of best mini-dystrophin expression in each muscle. a,b, Bar = 250 µm.

Figure 4

Lack of immune infiltration and dystrophic lesions in muscles with uniform human mini-dystrophin expression 16 weeks after intravenous injection of AAV9-CMV-mini-dystrophin vector in golden retriever muscular dystrophy dog Emerald at 4 days of age. The long head of the triceps muscle of Emerald was cryo-thin-sectioned. Consecutive sections were subjected to H&E and fluorescent staining with antibodies against mini-dystrophin (DYS, green color), sarcoglycan-γ (SG-γ, red color), CD4 (red color), and CD8 (red color) T cells. Note the lack of T-cell infiltration and dystrophic lesions in the muscle. Bar = 150 µm. H&E, hematoxylin and eosin.

Figure 5

Analyses of mini-dystrophin expression by western blot in muscle and heart samples at necropsy 16 weeks after intravenous injection of AAV9-CMV-mini-dystrophin vector in golden retriever muscular dystrophy dogs Emerald and Amethyst at 4 days of age. Human mini-dystrophin and low levels of endogenous revertant dystrophin are seen in nearly all skeletal muscles. Low levels of human mini-dystrophin are seen in the left ventricle of the heart, whereas revertant dystrophin is undetectable. (a) Emerald and (b) Amethyst.

Figure 6

Analysis of AAV vector DNA by quantitative PCR in muscle and heart samples at necropsy 16 weeks after intravenous injection of AAV9-CMV-mini-dystrophin vector in golden retriever muscular dystrophy dogs Emerald and Amethyst at 4 days of age. Numbers are shown on a per nucleus (diploid genome) basis. The data were obtained by dividing total vector copy numbers by 2 × the total copy numbers of an endogenous single-copy gene glucagon in the PCR reactions of each muscle sample.