Prevention of the Dystrophic Phenotype in Dystrophin/Utrophin-Deﬁcient Muscle Following Adenovirus-Mediated Transfer of a Utrophin Minigene

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Gene Therapy (2000) 7, 201–204  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt INHERITED DISEASE BRIEF COMMUNICATION Prevention of the dystrophic phenotype in dystrophin/utrophin-deﬁcient muscle following adenovirus-mediated transfer of a utrophin minigene

PM Wakeﬁeld1, JM Tinsley1, MJA Wood1, R Gilbert1, G Karpati2 and KE Davies1 1Department of Human Anatomy and Genetics, University of Oxford, Oxford, UK; and 2Neuromuscular Research Group, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada

Duchenne muscular dystrophy (DMD) is a progressive mus- generation recombinant adenovirus containing a utrophin cle wasting disorder caused by the lack of a subsarcolemmal minigene. Up to 95% of the fibres continued expressing the protein, dystrophin. We have previously shown that the dys- minigene 30 days after injection. Expression of utrophin trophin-related protein, utrophin is able to compensate for caused a marked reduction from 80% centrally nucleated the lack of dystrophin in the mdx mouse, the mouse model fibres (CNFs) in the uninjected dko TA to 12% in the injected for DMD. Here, we explore whether utrophin delivered to the dko TA. Within the region of the TA expressing the utrophin limb muscle of dystrophin/utrophin-deficient double knockout minigene, a significant decrease in the prevelance of (dko) neonatal mice can protect the muscle from subsequent necrosis was noted. These results demonstrate that the utro- dystrophic damage. Utrophin delivery may avoid the poten- phin minigene delivered using an adenoviral vector is able tial problems of an immune response associated with the to afford protection to the dystrophin/utrophin-deficient mus- delivery of dystrophin to a previously dystrophin-deficient cle of the dko mouse. Gene Therapy (2000) 7, 201–204. host. Dko muscle (tibialis anterior) was injected with a first

Keywords: Duchenne muscular dystrophy; adenovirus; utrophin; dystrophin/utrophin deﬁcient double knockout mice

Duchenne muscular dystrophy (DMD) is a severe and addressed in the form of gutted adenoviruses, viable progressive muscle wasting disease that affects one in viruses that only contain the inverted terminal repeats 3500 boys every year. It is caused by a mutation within and a packaging signal of the adenoviral genome and the dystrophin gene which encodes a large cytoskeletal consequently do not produce any viral proteins.7 How- subsarcolemmal protein, dystrophin.1 This protein pro- ever, delivery of dystrophin still could pose a problem, vides a crucial link between the actin cytoskeleton and a possibly requiring the use of long-term immunosuppres- group of proteins anchored in the cell membrane – the sive regimens.8 dystrophin protein complex (DPC).2 In turn, the DPC Utrophin is a ubiquitously expressed protein similar in maintains an interaction with the laminin component of amino acid sequence to dystrophin.9,10 In adult skeletal the extracellular matrix. Dystrophin is thus thought to muscle, however, it is restricted to the neuromuscular play a role in stabilising the membrane during cycles of and myotendinous junctions and is thought to play a role muscle contraction and relaxation.3 DMD is characterised in the maintenance of junctional folds.11 Studies in vitro by repeated cycles of muscle necrosis and regeneration have shown that utrophin is able to bind both to actin leading to the eventual replacement of muscle fibres by and to the DPC.12 Furthermore, it is known that in normal connective and adipose tissue.4 Regenerated fibres are fetal muscle, utrophin is localised to the extrajunctional recognised by the presence of centrally located nuclei as sarcolemma before being replaced by increasing levels of opposed to the peripherally located nuclei seen in dystrophin during development.13 In regenerating mus- normal fibres. cle fibres, utrophin is also localised at the extrajunctional Gene therapy strategies using viruses to deliver sarcolemma. Thus it is proposed that utrophin could replacement dystrophin are currently being tested in dys- offer a replacement role for dystrophin.10 We have shown trophin-deficient mdx mice.5,6 One problem encountered that up-regulation of utrophin in transgenic mdx mice by this approach is an immune response caused not only results in its localisation to the sarcolemma and correc- by the viral vector proteins but also by the delivery of tion of the dystrophic phenotype.14,15 dystrophin to a previously dystrophin-deficient host. The As utrophin is already present in DMD patients, there immunogenic nature of viral vectors is now being will be no immune problems with the delivery of a utrophin gene. Using a first generation adenovirus, we decided to explore the delivery of utrophin to dystrophic Correspondence: KE Davies, Department of Human Anatomy and Gen- mouse muscle. However, due to the limited 8 kb insert etics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK capacity of this adenovirus, a truncated utrophin mini- Received 16 June 1999; accepted 13 September 1999 gene was employed. This has been constructed through Utrophin minigene protects dystrophin/utrophin-deficient muscle PM Wakefield et al 202

Figure 1 Adenoviral delivery of utrophin to dystrophin/utrophin-deficient dystrophic muscle and subsequent restoration of the dystrophin-associated protein complex. Using a micromanipulator and glass syringe, the exposed tibialis anterior (TA) of 5-day-old dko mice were injected with 5 ␮lof AdCMV-Utr (kindly donated by Dr G Karpati, Montreal Neurological Institute) at a titre of 1.7 × 1011 virus particles/ml. The same muscle on the contralateral side acted as an uninjected negative control. The mice were killed after an expression period of 30 days and 8 ␮m sections taken for staining with haemotoxylin and eosin and immunostaining. For immunostaining, sections of 8 ␮m in thickness were blocked for 30 min in 10% heat inactivated foetal calf serum in 50 mm Tris, 150 mm NaCl pH 7.5 (TBS). Primary rabbit polyclonal antibodies against either utrophin (G3 courtesy of Dr CA Sewry, RPMS, London, UK), ␣-sarcoglycan, ␤-dystroglycan (courtesy of Dr JA Rafael, Oxford), or ␣-dystrobrevin (courtesy of Dr DJ Blake, Oxford) diluted in TBS were then added and incubated for 1 h at room temperature. Sections were washed three times in TBS for 5 min each then incubated for 1 h at room temperature with Cy3-conjugated affinity purified anti-rabbit IgG (Jackson Laboratories, Bar Harbor, ME, USA). Sections were then washed with TBS as before, mounted in Vectashield (Vector Laboratories, Peterborough, UK) and viewed using a Leica (Milton Keynes, UK) DMRBE microscope.

sequence comparison with a truncated dystrophin mini- a valuable model for DMD, it does not show the severe gene that lacks a proportion of the rod domain, as and progressive muscle wasting observed in a DMD ascertained from a mildly affected Becker muscular patient (other than in the diaphragm), and has a normal dystrophy patient.14 life span. Furthermore, the mdx mouse shows significant Previous experiments for the delivery of a utrophin levels of utrophin localised to the sarcolemma of regener- containing adenovirus to dystrophic muscle have utilised ating fibres thus leading to difficulty when trying to the mdx mouse.16 Although the mdx mouse does show an assess the delivery of a therapeutic utrophin gene. We underlying dystrophic pathology and hence has proved therefore used the severely affected dystrophin/

Gene Therapy Utrophin minigene protects dystrophin/utrophin-deficient muscle PM Wakefield et al 203 utrophin-deficient double knockout (dko) mice.17,18 In contrast to mdx mice, dko mice show many more clinical signs of DMD, which include a marked weight loss following weaning with an onset of joint contractures and kyphosis leading to premature death by 20 weeks of age. These mice show progressive muscular dystrophy and therefore provide an excellent model to observe an improvement in the clinical features of DMD. We have used an E1 + E3-deleted adenovirus vector containing the truncated utrophin minigene under the control of a murine cytomegalovirus promoter/enhancer. This was injected into the tibialis anterior (TA) of 5-day- old dko mice at a titre of 1.7 × 1011 p.f.u. (5 ␮l). The contralateral TA muscle was left uninjected to serve as a negative control for the immunostaining and also to observe the extent of damage caused to the muscle during the injection procedure. The mice were killed after 30 days following which the TA muscles were removed for immunostaining to detect the presence of utrophin and components of the DPC, and staining with haemotoxylin and eosin to observe muscle pathology. Staining with antibodies to the C-terminal region of utrophin14 revealed that 95% of the fibres at the site of the injection were now expressing the recombinant utrophin at the muscle fibre surface. Staining was not restricted to the neuromuscular junctions but was observed to be uniformly distributed along the sarcolemma (Figure 1). In many fibres, expression of utrophin was so high that cytoplasmic staining was also observed. Unlike with mdx mice, there was no spontaneously up-regulated utrophin expression, which is difficult to distinguish from the delivered protein. Sections from the uninjected contralateral TA used as a control did not stain with the utrophin antibodies (Figure 1). In common with DMD patients, lack of dystrophin in both mdx and dko mice results in a reduction of the DPC at the sarcolemma. In order to show that the delivered Figure 2 Haemotoxylin and eosin stained sections to show a reduction in utrophin had the ability to restore the DPC, antibodies the numbers of centrally nucleated fibres (CNFs) when dko dystrophic against ␣-dystrobrevin, ␣-sarcoglycan and ␤-dystrogly- muscle is injected with a utrophin containing adenovirus. Macrophage can were used (Figure 1). In all cases, the presence of infiltration (MP) and necrosis is markedly decreased and the majority of sarcolemma-bound utrophin coincided with a restoration fibres contain peripherally located nuclei (PNF). of members of the DPC. No restoration of the DPC was observed in the contralateral control muscle (Figure 1). The delivered utrophin affords a protective function to the muscle fibre by preventing necrosis and regeneration thus remains low (Figure 2). In the dko mice a major period of necrosis occurs at around 20 days of age following which the fibres retain centralised nuclei but degener- ation occurs at a much slower rate. It is interesting to note that this period of necrosis occurs about a week later in mdx mice suggesting that the low levels of sarco- lemmal-bound endogenous utrophin may be beneficial. There is a significant prevention in the formation of CNFs from an average of 80% in the TA of the uninjected leg to an average of 12% in the injected TA (Figure 3). Also, the fibres are more uniform in their size, again indicating a reversion to a normal phenotype. The level of macrophage infiltration is significantly lower in the injected TA, with the levels present being accounted either by the actual injection technique or through an immune reaction against adenoviral proteins (Figure 2). The morphology of the muscle as determined by analysis of H&E sections Figure 3 Injection of a utrophin containing adenovirus into the TA of six from mice that were injected before necrosis had com- dko mice results in a marked reduction in the number of centrally menced, showed a similar level of macrophage infil- nucleated fibres in comparison with the uninjected contralateral control.

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Oxford jected control TA to just 12% in the injected TA and a Monographs on Medical Genetics, Vol. 24. Oxford Medical Pub- marked decrease in the level of macrophage infiltration lications: Oxford, 1993. shows that the delivered utrophin is able to protect the 5 Quantin B et al. Adenovirus as an expression vector in muscle muscle from necrosis. cells in vivo. Proc Natl Acad Sci USA 1992; 89: 2581–2584. It has been suggested that functional correction of mus- 6 Acsadi G et al. Dystrophin expression in muscles of mdx mice cle groups would require a minimum of 20% of the fibres after adenovirus-mediated in vivo gene transfer. Hum Gene Ther 19 1996; 7: 129–140. to be expressing dystrophin. Assuming that utrophin 7 Clemens PR et al. In vivo muscle gene transfer of full-length dys- levels would need to be similar, infection of 95% of the trophin with an adenoviral vector that lacks all viral genes. Gene fibres would provide for a normalised phenotype. Therapy 1996; 3: 965–972. 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