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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 Wakefield
1
, JM Tinsley
1
, MJA Wood
1
, R Gilbert
1
, G Karpati
2
and KE Davies
1
1
Department of Human Anatomy and Genetics, University of Oxford, Oxford, UK; and
2
Neuromuscular Research Group, Montreal
Neurological Institute, McGill University, Montreal, Quebec, Canada
Duchenne muscular dystrophy (DMD) is a progressive mus-
cle wasting disorder caused by the lack of a subsarcolemmal
protein, dystrophin. We have previously shown that the dys-
trophin-related protein, utrophin is able to compensate for
the lack of dystrophin in the mdx mouse, the mouse model
for DMD. Here, we explore whether utrophin delivered to the
limb muscle of dystrophin/utrophin-deficient double knockout
(dko) neonatal mice can protect the muscle from subsequent
dystrophic damage. Utrophin delivery may avoid the poten-
tial problems of an immune response associated with the
delivery of dystrophin to a previously dystrophin-deficient
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
progressive muscle wasting disease that affects one in
3500 boys every year. It is caused by a mutation within
the dystrophin gene which encodes a large cytoskeletal
subsarcolemmal protein, dystrophin.
1
This protein pro-
vides a crucial link between the actin cytoskeleton and a
group of proteins anchored in the cell membrane – the
dystrophin protein complex (DPC).
2
In turn, the DPC
maintains an interaction with the laminin component of
the extracellular matrix. Dystrophin is thus thought to
play a role in stabilising the membrane during cycles of
muscle contraction and relaxation.
3
DMD is characterised
by repeated cycles of muscle necrosis and regeneration
leading to the eventual replacement of muscle fibres by
connective and adipose tissue.
4
Regenerated fibres are
recognised by the presence of centrally located nuclei as
opposed to the peripherally located nuclei seen in
normal fibres.
Gene therapy strategies using viruses to deliver
replacement dystrophin are currently being tested in dys-
trophin-deficient mdx mice.
5,6
One problem encountered
by this approach is an immune response caused not only
by the viral vector proteins but also by the delivery of
dystrophin to a previously dystrophin-deficient host. The
immunogenic nature of viral vectors is now being
Correspondence: KE Davies, Department of Human Anatomy and Gen-
etics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK
Received 16 June 1999; accepted 13 September 1999
generation recombinant adenovirus containing a utrophin
minigene. Up to 95% of the fibres continued expressing the
minigene 30 days after injection. Expression of utrophin
caused a marked reduction from 80% centrally nucleated
fibres (CNFs) in the uninjected dko TA to 12% in the injected
dko TA. Within the region of the TA expressing the utrophin
minigene, a significant decrease in the prevelance of
necrosis was noted. These results demonstrate that the utro-
phin minigene delivered using an adenoviral vector is able
to afford protection to the dystrophin/utrophin-deficient mus-
cle of the dko mouse.
Gene Therapy (2000) 7, 201–204.
addressed in the form of gutted adenoviruses, viable
viruses that only contain the inverted terminal repeats
and a packaging signal of the adenoviral genome and
consequently do not produce any viral proteins.
7
How-
ever, delivery of dystrophin still could pose a problem,
possibly requiring the use of long-term immunosuppres-
sive regimens.
8
Utrophin is a ubiquitously expressed protein similar in
amino acid sequence to dystrophin.
9,10
In adult skeletal
muscle, however, it is restricted to the neuromuscular
and myotendinous junctions and is thought to play a role
in the maintenance of junctional folds.
11
Studies in vitro
have shown that utrophin is able to bind both to actin
and to the DPC.
12
Furthermore, it is known that in normal
fetal muscle, utrophin is localised to the extrajunctional
sarcolemma before being replaced by increasing levels of
dystrophin during development.
13
In regenerating mus-
cle fibres, utrophin is also localised at the extrajunctional
sarcolemma. Thus it is proposed that utrophin could
offer a replacement role for dystrophin.
10
We have shown
that up-regulation of utrophin in transgenic mdx mice
results in its localisation to the sarcolemma and correc-
tion of the dystrophic phenotype.
14,15
As utrophin is already present in DMD patients, there
will be no immune problems with the delivery of a utro-
phin gene. Using a first generation adenovirus, we
decided to explore the delivery of utrophin to dystrophic
mouse muscle. However, due to the limited 8 kb insert
capacity of this adenovirus, a truncated utrophin mini-
gene was employed. This has been constructed through
Utrophin minigene protects dystrophin/utrophin-deficient muscle
PM Wakefield
et al
202
Gene Therapy
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 ×10
11
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 m
m
Tris, 150 m
m
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-
gene that lacks a proportion of the rod domain, as
ascertained from a mildly affected Becker muscular
dystrophy patient.
14
Previous experiments for the delivery of a utrophin
containing adenovirus to dystrophic muscle have utilised
the mdx mouse.
16
Although the mdx mouse does show an
underlying dystrophic pathology and hence has proved
a valuable model for DMD, it does not show the severe
and progressive muscle wasting observed in a DMD
patient (other than in the diaphragm), and has a normal
life span. Furthermore, the mdx mouse shows significant
levels of utrophin localised to the sarcolemma of regener-
ating fibres thus leading to difficulty when trying to
assess the delivery of a therapeutic utrophin gene. We
therefore used the severely affected dystrophin/
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 fol-
lowing 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 ×10
11
p.f.u. (5 l). The con-
tralateral 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 dur-
ing 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
utrophin
14
revealed that 95% of the fibres at the site of
the injection were now expressing the recombinant utro-
phin at the muscle fibre surface. Staining was not restric-
ted 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 utro-
phin expression, which is difficult to distinguish from the
delivered protein. Sections from the uninjected contralat-
eral 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
utrophin had the ability to restore the DPC, antibodies
against ␣-dystrobrevin, ␣-sarcoglycan and -dystrogly-
can were used (Figure 1). In all cases, the presence of
sarcolemma-bound utrophin coincided with a restoration
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 per-
iod 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 macro-
phage 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
from mice that were injected before necrosis had com-
menced, showed a similar level of macrophage infil-
Gene Therapy
Figure 2 Haemotoxylin and eosin stained sections to show a reduction in
the numbers of centrally nucleated fibres (CNFs) when dko dystrophic
muscle is injected with a utrophin containing adenovirus. Macrophage
infiltration (MP) and necrosis is markedly decreased and the majority of
fibres contain peripherally located nuclei (PNF).
Figure 3 Injection of a utrophin containing adenovirus into the TA of six
dko mice results in a marked reduction in the number of centrally
nucleated fibres in comparison with the uninjected contralateral control.
Utrophin minigene protects dystrophin/utrophin-deficient muscle
PM Wakefield
et al
204
Gene Therapy
tration in the injected leg with none being observed in
the control leg (data not shown).
Thus we have demonstrated successful delivery of a
utrophin minigene to dystrophic limb muscle of the
dystrophin/utrophin dko mouse. Using an adenovirus
vector it was possible to transduce up to 95% of the mus-
cle fibres around the site of the injection. A significant
decrease in the number of CNFs from 80% in the unin-
jected control TA to just 12% in the injected TA and a
marked decrease in the level of macrophage infiltration
shows that the delivered utrophin is able to protect the
muscle from necrosis.
It has been suggested that functional correction of mus-
cle groups would require a minimum of 20% of the fibres
to be expressing dystrophin.
19
Assuming that utrophin
levels would need to be similar, infection of 95% of the
fibres would provide for a normalised phenotype.
Observing sections at the other end of the muscle from
where the injection was carried out showed levels of
between 50 and 60% utrophin expression (data not
shown). This indicates that the adenovirus is able to
spread throughout the small murine TA muscle and
maintain a level of infectivity that would still be thera-
peutic. However, it must be considered that the adeno-
virus would not show much impressive spread through-
out larger muscles. Analysis of the soleus muscle of an
injected leg shows that the virus is unable to spread from
one muscle to the next, an observation that has been
alluded to previously using -galactosidase expressing
adenoviruses.
20
This work demonstrates that it is possible to deliver a
therapeutic utrophin gene to dystrophic muscle of the
dko mouse and afford substantial protection against
muscle necrosis and a dystrophic phenotype.
Acknowledgements
We are most grateful to the Muscular Dystrophy Group
of Great Britain and Northern Ireland, the Muscular Dys-
trophy Association (USA) and the MRC (UK & Canada)
for their support of this work. We would also like to
thank Dr Jill Rafael and Dr Derek Blake for helpful dis-
cussions and advice on the antibodies.
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