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

Gene Therapy (2000) 7, 1417–1420  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt VIRAL TRANSFER TECHNOLOGY BRIEF COMMUNICATION Hyaluronidase enhances recombinant adenoassociated virus (rAAV)-mediated gene transfer in the rat skeletal muscle D Favre1, Y Cherel2, N Provost1, V Blouin1, N Ferry1, P Moullier1 and A Salvetti1 Laboratoire de Thérapie Génique, CHU Hôtel-Dieu, Nantes; and 2UMR INRA-Ecole Vétérinaire, Nantes, France Skeletal muscle is a privileged target for long-term rAAVmediated gene transfer 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 intramuscular rAAV administration in rats resulted in restricted transduction essentially along the myofibers axis with poor lateral diffusion. This suggested that the transduction rate might be limited by the ability of the virus to reach sites distant from the injection point. We tested whether hyaluronidase, an enzyme which dissociates the extracellular matrix, could enhance vector diffusion when injected in the rat muscle before administration of rAAV encoding either nuclear-localized ␤-galactosidase (rAAVCMVnlsLacZ) or the human ␣-1-antitrypsin (rAAVCMVhAAT) under the control of the cytomegalovirus immediate–early promoter (CMV). The results showed that pretreatment of the rat anterior tibialis muscle with hyaluronidase resulted in: (1) a larger diffusion of the virus indicated by an increase in the area containing LacZ-transduced fibers, and (2) a two- to three-fold increase of transduction efficiency measured by the number of LacZpositive fibers or by the hAAT serum concentration. We also provide evidence that hyaluronidase was well tolerated and was not associated with short- or long-term toxicity evaluated by morphological studies. Finally, in our experimental conditions, hyaluronidase did not promote rAAV dissemination to other organs as assessed by PCR to detect vector sequences. We conclude that pretreament of skeletal muscle by hyaluronidase, a clinically available reagent, was harmless and resulted in a consistent and significant increase in rAAV diffusion and transduction levels. Gene Therapy (2000) 7, 1417–1420. Keywords: skeletal muscle; recombinant adeno-associated virus; hyaluronidase Recombinant adeno-associated viral vectors (rAAV) have emerged as an attractive alternative to retroviral and adenoviral vectors to transduce nondividing cells in vivo.1 Recombinant AAV vectors are derived from the ‘wildtype’ virus by deleting the rep and cap genes and replacing them with the transgene and transcription control elements. Usually, rAAV stocks are purified from 293 cells which are cotransfected with the AAV vector and a rep-cap expressing plasmid and subsequently infected with helper adenovirus. Alternatively, the adenoviral helper functions required for the production of rAAV can be provided by a nonreplicative plasmid resulting in rAAV stocks free of detectable adenovirus.2 Despite the broad host range of rAAV in vitro, three preferential targets have been identified in vivo: the retina, skeletal muscle, and the central nervous system, where rAAV efficiently transduced post-mitotic and differentiated cells such as photoreceptors, myofibers and neurons.3–5 Skeletal muscle is an easily accessible target for gene delivery and is highly vascularized. Efficient rAAV transduction of myofibers can be used to achieve the sustained in situ expression of reporter genes or the Correspondence: P Moullier, Laboratoire de Thérapie Génique, CHU Hotel-Dieu, 44035 Nantes, France Received 18 February 2000; accepted 3 May 2000 systemic delivery into the circulation of therapeutic proteins. The mouse has been used most commonly for rAAV evaluation in vivo and the anterior tibialis muscle remains the preferred site of vector administration. The small size of the target combined with large volumes of injected rAAV (30–100 ␮l) allow diffuse transduction of the muscle reaching up to 40% of myofibers.6,7 In the present study rAAV-mediated transduction of the rat anterior tibialis muscle was analyzed. Intramuscular injection of rAAVCMVnlsLacZ into 250 g Wistar rats resulted in transduction essentially along the axis of myofibers with little lateral diffusion (data not shown). On average, 1% myofibers were routinely transduced using 7.8 × 109 rAAVCMVnlsLacZ particles (2.5 × 107 transducing particles as measured by LacZ staining of adenovirus co-infected 293 cells) in a total volume of 250 ␮l saline, injected perpendicularly at three different sites at equal distances along the longitudinal axis. Increasing the total volume of injection to 500 ␮l did not improve the level of transduction, nor did the longitudinal administration of the rAAV vector (data not shown). Actually, increasing the volume above 250 ␮l resulted in obvious leakage outside the muscle despite making a cutaneous incision and exposing the muscle before rAAV injection. Overall, these observations indicated that rAAV had a restricted capacity to diffuse from the injection site and suggested that the confined diffusion of the vector was limiting the transduction rate of muscle fibers. Hyaluronidase enhances rAAV muscle transduction D Favre et al 1418 To improve rAAV diffusion and transduction we tested the effect of injecting hylauronidase into the muscle before rAAV administration. Hyaluronidase (Choay, Sanofi, France), is a mucolytic enzyme involved in the depolymerization and hydrolysis of hyaluronic acid, a major constituent of the extracellular matrix. One hundred units of the enzyme in 250 ␮l saline were injected into the muscle of 12 rats 3 h before the administration of 2.5 × 107 rAAVnlsLacZ transducing particles (group B). The control group (group A) contained 12 animals receiving 250 ␮l of saline instead of hyaluronidase and, 3 h later, the same dose of rAAVCMVnlsLacZ particles as group B. Four weeks later, the animals were killed and muscles were excised, paraformaldehyde-fixed and stained with X-gal to detect ␤-galactosidase activity. The results presented in Figure 1 indicate that pretreatment of the skeletal muscle with hyaluronidase increased rAAV diffusion compared with untreated muscle. To determine if hyaluronidase treatment also affected the rAAV trans- a b duction rate, the number of ␤-galactosidase-expressing myofibers was counted for each animal in both groups (Figure 2). The number of positive fibers was found, on average, to be two-fold higher in hyaluronidase-treated animals than in the mock-injected controls (231 ± 118.3 versus 114.9 ± 61.7, respectively). Altogether, these data indicate that pretreament of skeletal muscle with hyaluronidase improved the diffusion of the rAAV vector in the tissue and resulted in a two-fold increase in the transduction events. To validate this observation further, this protocol was re (...truncated)