An Isolated Limb Infusion Method Allows for Broad Distribution of rAAVrh74.MCK.GALGT2 to Leg Skeletal Muscles in the Rhesus Macaque

Abstract

Recombinant adeno-associated virus (rAAV)rh74.MCK.GALGT2 is a muscle-specific gene therapy that is being developed to treat forms of muscular dystrophy. Here we report on an isolated limb infusion technique in a non-human primate model, where hindlimb blood flow is transiently isolated using balloon catheters to concentrate vector in targeted leg muscles. A bilateral dose of 2.5 × 1013 vector genomes (vg)/kg/limb was sufficient to induce GALGT2-induced glycosylation in 10%–60% of skeletal myofibers in all leg muscles examined. There was a 19-fold ± 6-fold average limb-wide increase in vector genomes per microgram genomic DNA at a bilateral dose of 2.5 × 1013 vg/kg/limb compared with a bilateral dose of 6 × 1012 vg/kg/limb. A unilateral dose of 6 × 1013 vg/kg/limb showed a 12- ± 3-fold increase in treated limb muscles compared to contralateral untreated limb muscles, which received vector only after release into the systemic circulation from the treated limb. Variability in AAV biodistribution between different segments of the same muscle was 125% ± 18% for any given dose, while variability between the same muscle for any given treatment dose was 45% ± 7%. These experiments demonstrate that treatment of muscles throughout the leg with rAAVrh74.MCK.GALGT2 can be accomplished safely using an isolated limb infusion technique, where balloon catheters transiently isolate the limb vasculature, but that intra- and inter-muscle transduction variability is a significant issue.

rAAVrh74.MCK.GALGT2 Protects against Loss of Hemodynamic Function in the Aging mdx Mouse Heart

“…49 Previous WFA and CT antibody-staining studies showed that both mouse (mdx and WT) and rhesus macaque (WT) cardiomyocytes have little, if any, endogenous expression of Galgt2-induced glycosylation and that the majority of such staining is confined to intercalated disks. 49,57 Delivering rAAVrh74.MCK.GALGT2 intravenously via the tail vein led to dose-dependent overexpression of the human GALGT2 transgene, with increased glycosylation evidenced by increased WFA staining ( Figures 1A-1C). WFA staining occurred along the entirety of the sarcolemmal membrane in mouse cardiomyocytes, which were marked by co-staining with an antibody to laminin a2 ( Figure 1B).…”

rAAVrh74.MCK.GALGT2 Demonstrates Safety and Widespread Muscle Glycosylation after Intravenous Delivery in C57BL/6J Mice

“…rAAVrh74.MCK. GALGT2 is a muscle-specific gene therapy designed to overexpress the human GALGT2 transgene (also called B4GALNT2 ) in heart and skeletal muscle, where it acts as a β1,4 N-acetylgalactosaminyltransferase to glycosylate α dystroglycan 1, 2, 3, 4, 5, 6, 7, 8, 9. In adult skeletal muscle, expression of GALGT2 is confined to the neuromuscular and myotendinous junction, and its expression is also very low in the heart 10, 11, 12.…”

Bridging from Intramuscular to Limb Perfusion Delivery of rAAV: Optimization in a Non-human Primate Study

“…We chose to compare two intra-vascular delivery protocols that have been previously published as efficacious routes of AAV administration to large animal models (Table S1). The first method involves intra-arterial delivery with a double balloon catheter to the pelvic extremity, a method that has been successfully used in non-human primates 7 . Variations of the intra-arterial technique have also been successfully used for AAV delivery in both non-human primates and canine models (Table S1).…”