Hyaluronidase enhances recombinant adeno-associated virus (rAAV)-mediated gene transfer in the rat skeletal muscle

Skeletal muscle is a privileged target for long-term rAAV-mediated gene tra nsfer in mouse, rat dog and non-human primates. Intramuscular injections of rAAV encoding human factor IX in hemophilia B patients have been initiated based on promising results gathered in affected dogs. We found that intram uscular rAAV administration in rats resulted in restricted transduction ess entially along the myofibers axis with poor lateral diffusion. This suggest ed that the transduction rate might be limited by the ability of the virus to reach sites distant from the injection point We tested whether hyaluroni dase, an enzyme which dissociates the extracellular matrix, could enhance v ector diffusion when injected in the rat muscle before administration of rA AV encoding either nuclear-localized beta-galactosidase (rAAVCMVnlsLacZ) or the human alpha-1-antitrypsin (rAAVCMVhAAT) under the control of the cytom egalovirus immediate-early promoter (CMV). The results showed that pretreat ment of the rat anterior tibialis muscle with hyaluronidase resulted in: (I ) a larger diffusion of the virus indicated by an increase in the area cont aining LacZ-transduced fibers, and (2) a two- to three-fold increase of tra nsduction efficiency measured by the number of LacZ-positive fibers or by t he hAAT serum concentration. We also provide evidence that hyaluronidase wa s well tolerated and was not associated with short- or long-term toxicity e valuated by morphological studies. Finally, in our experimental conditions, hyaluronidase did not promote rAAV dissemination to other organs as assess ed by PCR to detect vector sequences. We conclude that pretreament of skele tal muscle by hyaluronidase, a clinically available reagent, was harmless a nd resulted in a consistent and significant increase in rAAV diffusion and transduction levels.