Fig 1.
Biodistribution of AAV genomes in muscles and non-muscle organs after rAAVrh74.MHCK7.GALGT2 treatment of GRMD dogs.
rAAV vector genomes were measured in skeletal muscles (A), heart (B), and non-muscle organs (C) by qPCR. Four GRMD dogs were dosed with 2x1014vg/kg (2E+14), with average transduction levels shown (lighter bars), while one GRMD dog was dosed with 6x1014vg/kg (black bars, 6E+14). Abbreviations: Diaph (Diaphragm), Lat (Lateral) Head Tri (Triceps), Pect (Deep Pectoral), Bic Brach (Biceps Brachii), SDF (Superficial Digital Flexor), Mid Glut (Middle Gluteal), RF (Rectus Femoris), VL (Vastus Lateralis), Cran Sart (Cranial Sartorius), BF (Biceps Femoris), ST (Semitendinosus), SM (Semimembranosus), MG (Medial head, Gastrocnemius), LG (Lateral head, Gastrocnemius), Cran Tib (Cranial Tibialis), LDE (Long Digital Extensor), R (Right), L (Left), IVS (Interventricular Septum). Errors are SD for n = 4 for 2x1014vg/kg group.
Fig 2.
GALGT2-induced glycosylation in heart and skeletal muscles after rAAVrh74.MHCK7.GALGT2 treatment of GRMD dogs.
WFA staining was scored in each cardiomyocyte or skeletal myofiber in muscle sections taken from four GRMD dogs dosed with 2x1014vg/kg (lighter bars 2E+14) or one GRMD dog with 6x1014vg/kg (black bars, 6E+14). Errors are SD for n = 4 for the 2x1014vg/kg group.
Fig 3.
WFA staining after treatment of GRMD dogs with rAAVh74.MHCK7.GALGT2.
Muscle sections from 6-month-old GRMD dogs treated (or untreated) at 3 months of age with 2x1014vg/kg of rAAVrh74.MHCK7.GALGT2 were assayed for GALGT2-induced glycosylation. Sections were stained with WFA (green), to identify βGalNAc made by GALGT2, and with antibody to laminin α2 (Lama2, red), to identify sarcolemmal membranes. Control shows merged red, green and blue fluorescence signal without WFA and with only secondary antibody, with DAPI (blue) added as a nuclear co-stain. Sections were stained from heart (left ventricle, LV), diaphragm, cranial sartorius (CS), and vastus lateralis (VL). All exposures are time matched. Bar is 100μm for all panels.
Table 1.
Functional data from GRMD dogs.
Fig 4.
Skeletal muscle pathology in rAAVrh74.MHCK7.GALGT2-treated GRMD dogs.
(A) Cranial sartorius, middle gluteus, and vastus lateralis muscles were cut in cross-section from 6-month-old untreated normal GR, untreated GRMD, and treated GRMD (treated with low dose, 2x1014vg/kg rAAVrh74.MHCK7.GALGT2 at 3 months) dogs. Sections were stained with H&E. Bar is 200μm. (B) Measures of fibrotic index for skeletal muscles from wild type golden retriever (GR) and golden retriever muscular dystrophy (GRMD) dogs, either untreated or treated (pooled low dose [2x1014vg/kg] plus high dose [6x1014vg/kg]) with rAAVrh74.MHCK7.GALGT2. Fibrotic index (percentage non-muscle area in muscle) measures were based on the following values: 0, no evident fibrosis, 1, 0<x≤5% fibrosis, 2, 5%<x≤15% fibrosis, 3, 15%<x≤25% fibrosis, 4, 25%<x≤50% fibrosis, and 5, x>50% fibrosis. Errors are SEM for n = 9 (GR), 26 (GRMD Untreated), 54 (GRMD Treated Low and High Dose) different muscle biopsies.
Fig 5.
Immune responses to rAAVrh74.MHCK7.GALGT2 treatment of GRMD dogs.
(A) Highest reciprocal dilution at which serum antibodies to rAAVrh74 capsid protein could be detected in one untreated GRMD dog (0 dose), four different GRMD dogs treated with low dose of rAAVrh74.MHCK7.GALGT2 (2x1014vg/kg), and in one dog treated with high dose of rAAVrh74.MHCK7.GALGT2 (6x1014vg/kg). (B) Highest reciprocal dilution at which serum antibodies could be identified to rAAV1, rAAV2, rAAV6, rAAV8, or rAAV9 capsid protein after treatment with rAAVrh74.MHCK7.GALGT2. (C) Interferon gamma (IFN-g) expressing cells were quantified in ELISpot assays of peripheral blood mononuclear cells (PBMCs) taken from GRMD dogs at 3 months of age (baseline, pretreatment) and at 45 or 90 days after treatment with rAAVrh74.MHCK7.GALGT2. Positive ELISpots to identify activated T cells were measured in response to 1 of 3 overlapping peptide pools made against the rAAVrh74 capsid protein and to 1 of 2 overlapping peptide pools made against the GALGT2 protein. ELISpot scores of 50 or more (line) are considered positive.
Table 2.
Serum chemistry tests.
Fig 6.
Expression of glycosylated α-dystroglycan and utrophin in response to rAAVrh74.MHCK7.GALGT2 treatment of GRMD dogs.
(A) Lectin precipitations of non-ionic detergent whole protein lysates were completed using different amounts of cellular protein ranging from 0.1- to 3.0mg. Precipitates were solubilized in SDS denaturation buffer, separated by SDS-PAGE, and immunoblotted for α dystroglycan using the IIH6 monoclonal antibody, which recognizes functionally glycosylated αDG, or with antiserum made to the CORE DG polypeptide. The 160kDa region of the gene is shown for IIH6 and the 100-160kDa region is shown for CORE. WFA was used to precipitate α-dystroglycan containing βGalNAc, which is made by GALGT2, while WGA was used to precipitate glycosylated α dystroglycan not containing βGalNAc. (B) Muscle sections taken from the cranial sartorius muscle of normal untreated wild type golden retriever (GR), untreated golden retriever muscular dystrophy (GRMD), or GRMD dogs treated with 2x1014vg/kg rAAVrh74.MHCK7.GALGT2. Time-matched exposures are shown comparing immunostaining for dystrophin surrogates plectin 1 and utrophin as well as dystroglycan (DG). Bar is 200μm.