Hyaluronidase Increases Electrogene Transfer Efficiency in Skeletal Muscle

Abstract

Electrogene transfer (EGT) of plasmid DNA into skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. We report here that preinjecting hyaluronidase (HYAse) significantly increases the gene transfer efficiency of muscle EGT. Three constructs encoding mouse erythropoietin (pCMV/mEPO), secreted alkaline phosphatase (pCMV/SeAP), and luciferase (pGGluc) were electroinjected intramuscularly in BALB/c mice and rabbits with and without HYAse pretreatment. Preinjection 1 or 4 hr before EGT increased EPO gene expression by about 5-fold in mice and maintained higher gene expression than plasmid EGT alone. A similar increment in gene expression was observed on pretreatment with HYAse and electroinjection of pCMV/mEPO into rabbit tibialis muscle. The increment of gene expression in rabbits reached 17-fold on injection of plasmid pCMV/SeAP and 24-fold with plasmid pGGluc. Injection of a plasmid encoding beta-galactosidase (pCMV/beta gal/NLS) and subsequent staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside indicated that HYAse increased the tissue area involved in gene expression. No irreversible tissue damage was observed on histological analysis of treated muscles. HYAse is used in a variety of clinical applications, and thus the combination of HYAse pretreatment and muscle EGT may constitute an efficient gene transfer method to achieve therapeutic levels of gene expression.

High-efficiency plasmid gene transfer into dystrophic muscle

“…It has previously been demonstrated that the pretreatment of skeletal muscle with bovine hyaluronidase, prior to plasmid electrotransfer, can significantly improve transfection efficiencies compared to electrotransfer alone, 12,14 and we have previously published data demonstrating that the pretreatment of muscle with hyaluronidase can reduced the damage associated with electrotransfer in nondystrophic muscle. 12 The improvements in transfection efficiency achieved with hyaluronidase and electrotransfer may be a result of improved accessibility of the plasmid DNA to the muscle fibre surface.…”

“…This difference in the method of entry of plasmid into muscle fibres may explain why hyaluronidase pretreatment improves electrotransfer, but not the direct injection, of plasmid DNA. 12,14 We speculate that the hyaluronidase pretreatment permeabilises the pericellular matrix of the muscle fibre, thus increasing the effective concentration of plasmid at the membrane leading to increased uptake.…”

“…Recently, further improvements have been reported by Vilquin et al, 11 who achieved 37% transfection following multiple site injections and electrotransfer. In addition, it has also been shown that pretreatment of the muscle with hyaluronidase followed by electrotransfer can result in significant improvements in plasmid gene transfer, 12,14 and that 50% levels of transfection in nondystrophic muscle can routinely be achieved in our hands. 12 The results presented here demonstrate that very efficient plasmid gene transfer can be achieved with a single injection into the dystrophic muscle of the mdx mouse mediated by hyaluronidase and electrotransfer, despite the presence of increased levels of connective tissue within the muscle.…”

Electro-gene therapy of collagen-induced arthritis by using an expression plasmid for the soluble p75 tumor necrosis factor receptor-Fc fusion protein

“…A total of 90 units of type VI-S hyaluronidase from bovine testes (Sigma, St. Louis, MO, USA) were resuspended in 50 ml of sterile saline solution and injected 10 min prior to electroporation as previously described. 42 Commercially available caliper electrodes (model 383, BTX, San Diego, CA, USA) were used for electroporation. The caliper electrodes were applied to the shaved skin on either side of the marked DNA injection point, and the calipers were closed to a gap of 5 mm, so that electrical contact with the skin was maximized.…”

Transferência gênica in vivo por meio de eletroporação em músculo sóleo de ratos wistar.

“…O pré-tratamento com hialuronidase, anterior a injeção de plasmídeo, pode aumentar significativamente a eficiência de transferência gênica, comparado a eletroporação sozinha (MCMAHON et al, 2001); (MENNUNI et al, 2002). Tem sido relatado um aumento de eficiência de transferência gênica, com valores próximos de 10% (VICAT et al, 2000) a 37% (VILQUIN et al, 2001) e até 50% em musculatura não distrófica, associando o tratamento prévio de hialuronidase ao método de eletroporação (GOLLINS et al, 2003); (MCMAHON et al, 2001).…”