Evaluation of vascular delivery methodologies to enhance rAAV6-mediated gene transfer to canine striated musculature.

A growing body of research supports the development of recombinant adeno-associated viral (rAAV) vectors for delivery of gene expression cassettes to striated musculature as a method of treating severe neuromuscular conditions. However, it is unclear whether delivery protocols that achieve extensive gene transfer in mice can be adapted to produce similarly extensive gene transfer in larger mammals and ultimately patients. Consequently, we sought to investigate methodological modifications that would facilitate rAAV-mediated gene transfer to the striated musculature of canines. A simple procedure incorporating acute (i) occlusion of limb blood flow, (ii) exsanguination via compression bandage, and (iii) vector "dwell" time of <20 minutes, markedly enhanced the transduction of limb muscles, compared with a simple bolus limb infusion of vector. A complementary method whereby vector was infused into the jugular vein led to efficient transduction of cardiomyocytes and to a lesser degree the diaphragm. Together these methods can be used to achieve transgene expression in heart, diaphragm, and limb muscles of juvenile dogs using rAAV6 vectors. These results establish that rAAV-mediated gene delivery is a viable approach to achieving systemic transduction of striated musculature in mammals approaching the dimensions of newborn humans.

Macrophage fusion, giant cell formation, and the foreign body response require matrix metalloproteinase 9.

Macrophages undergo fusion to form multinucleated giant cells in several pathologic conditions, including the foreign body response (FBR). We detected high levels of matrix metalloproteinase (MMP)-9 during macrophage fusion in vitro and in foreign body giant cells (FBGCs) in vivo. Wild-type (WT) bone marrow-derived macrophages were induced to fuse with IL-4 in the presence of MMP-9 function-blocking antibodies and displayed reduced fusion. A similar defect, characterized by delayed shape change and abnormal morphology, was observed in MMP-9 null macrophages. Analysis of the FBR in MMP-9 null mice was then pursued to evaluate the significance of these findings. Specifically, mixed cellulose ester disks and polyvinyl alcohol sponges were implanted s.c. in MMP-9 null and WT mice and excised 2-4 weeks later. Histochemical and immunohistochemical analyses indicated equal macrophage recruitment between MMP-9 null and WT mice, but FBGC formation was compromised in the former. In addition, MMP-9 null mice displayed abnormalities in extracellular matrix assembly and angiogenesis. Consistent with a requirement for MMP-9 in fusion, we also observed reduced MMP-9 levels in MCP-1 null macrophages, previously shown to be defective in FBGC formation. Collectively, our studies show abnormalities in MMP-9 null mice during the FBR and suggest a role for MMP-9 in macrophage fusion.

Fluorophore-labeled myosin-specific antibodies simplify muscle-fiber phenotyping.

Skeletal muscles are frequently analyzed for composition of phenotypically distinct myofibers, as a functional determinant. We describe an improved myofiber phenotyping procedure, involving cryosection co-incubation with fluorophore-labeled myosin heavy-chain (MyHC)-isoform-specific antibodies. This technique identifies multiple fiber "types" on a single section, thereby reducing reagents and processing, and offers side-by-side comparison of samples from multiple species including mice. These advances are valuable for studying the physiological attributes of skeletal muscle in health and disease.