Human Molecular Genetics, 2002, Vol. 11, No. 15 1719-1730
© 2002 Oxford University Press
Stable micro-dystrophin gene transfer using an integrating adeno-retroviral hybrid vector ameliorates the dystrophic pathology in mdx mouse muscle
1Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway – University of London, Egham, Surrey TW20 0EX, UK, 2Gene Targeting Unit, Department of Neuromuscular Diseases, Imperial College School of Medicine, Charing Cross Hospital, St Dunstan's Road, London W6 8RP, UK, 3Laboratoire de Thérapie Génique, CHU Hotel Dieu, Bâtiment Jean Monnet, 44000 Nantes, France, 4Department of Molecular Genetics, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-higashi, Kodaira, Tokyo 187, Japan and 5Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, Unité de Virologie Humaine, INSERM U412, Ecole Normale Supérieure de Lyon, 69364 Lyon, France
Received March 25, 2002; Accepted May 15, 2002
The ability to transfer the dystrophin gene stably to the skeletal muscle of DMD patients is a major confounding issue in establishing an effective gene therapy for this disease. To overcome this problem, we have examined the ability of muscle fibres from mdx mice to act as in situ factories of retroviral vector production. Tibialis anterior (TA) muscles from 4-week-old mdx mice were injected with an adenoviral vector expressing LacZ within a retroviral expression cassette (AdLZIN). Retroviral vector production was induced by the inclusion of two additional adenoviral vectors expressing retroviral gag–pol (AdGagPol) and 10A1 env genes (Ad10A1). Upon introduction of infected muscles into cell culture, colonies of ß-galactosidase-expressing myotubes formed only in cultures where the muscle was injected with AdLZIN, AdGagPol and Ad10A1, but not from muscle injected with AdLZIN only. Muscles from mdx/nude mice producing retroviral vector displayed a 4.6-fold increase in ß-galactosidase-positive myofibres after 1 month, compared with contralateral muscle in the same animal injected with AdLZIN and AdGagPol only. By constructing a hybrid adeno-retroviral vector expressing a truncated micro-dystrophin construct (AdµDyIN), we were able to partially correct the mdx dystrophic phenotype. AdµDyIN-mediated expression of micro-dystrophin in mdx TA muscle restored the formation of the dystrophin-associated glycoprotein complex and significantly reduced the level of muscle degeneration over uninjected controls. By stimulating in situ production of retroviral vector expressing micro-dystrophin, we achieved 92%±6% transduction of myofibres in the TA muscle by 4 weeks. Strikingly, by 3 months post injection, micro-dystrophin was still expressed to high levels in nearly all the myofibres of the TA muscle. By comparison, there was a pronounced drop in the levels of micro-dystrophin expressed by muscles injected with AdµDyIN only. Finally, using a novel PCR approach, we detected reverse-transcribed, integrated proviral sequences in TA muscle genomic DNA by 4 weeks post injection, the levels of which were found to increase after 3 months.
* To whom correspondence should be addressed: Tel: +44 1784443545; Fax: +44 1784434326; Email: g.dickson{at}rhul.ac.uk
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