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Developments in Gene Therapy for Muscular Dystrophy
in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618
of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618
3Center for Gene Therapy, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618
dystrophin; Duchenne; limb-girdle; vector; adenovirus; gutted; gutless; helperdependent; AAV; lentivirus
Gene therapy for muscular dystrophy (MD) presents significant challenges,
including the large amount of muscle tissue in the body, the large size of many genes defective in
different muscular dystrophies, and the possibility of a host immune response against the
therapeutic gene. Overcoming these challenges requires the development and delivery of suitable
gene transfer vectors. Encouraging progress has been made in modifying adenovirus (Ad) vectors to
reduce immune response and increase capacity. Recently developed gutted Ad vectors can deliver
full-length dystrophin cDNA expression vectors to muscle tissue. Using muscle-specific promoters to
drive dystrophin expression, a strong immune response has not been observed in mdx mice.
Adeno-associated virus (AAV) vectors can deliver small genes to muscle without provocation of a
significant immune response, which should allow long-term expression of several MD genes. AAV
vectors have also been used to deliver sarcoglycan genes to entire muscle groups. These advances
and others reviewed here suggest that barriers to gene therapy for MD are surmountable. Microsc.
Res. Tech. 48:223–238, 2000. r 2000 Wiley-Liss, Inc.
The muscular dystrophies (MDs) are a common set of
genetic disorders. Since none of the underlying biochemical defects were described until 10 years ago, treatment
for these disorders has been mostly palliative. In the
absence of a biological explanation for muscle weakness, little progress toward identifying rational treatments could be expected. This situation changed with
the identification of dystrophin in 1987 (Koenig et al.,
1987), which cleared the way for biochemical studies of
the defect underlying DMD and offered hope that other
dystrophies might be similarly understood.
The genetics of many types of MD have now been
delineated in considerable detail. In addition to mutations of dystrophin and the dystrophin-associated glycoproteins (Bonnemann et al., 1996; Ozawa et al., 1995),
mutations in genes including calpain (Richard et al.,
1995), caveolin-3 (Minetti et al., 1998), laminin-alpha2
(Helbling-Leclerc et al., 1995), fukutin (Kobayashi et
al., 1998), and emerin (Bione et al., 1994) have been
shown to cause forms of MD (Fig. 1). Each of these
molecules must play a functionally important role in
muscle and so offers an opportunity for understanding
the biology of muscle and the pathogenesis of dystrophy.
The potential of gene therapy for treatment of genetic
disease was understood long before the development of
positional techniques for identification of disease genes
(Freese, 1972). When the dystrophin gene was identified, many scientists hoped that gene therapy for DMD
would follow quickly. However, the difficulties of using
gene transfer to treat DMD soon became clear. The
major obstacles included the large size of the dystrophin gene (Table 1), the large mass of post-mitotic
muscle cells in the body, and the tendency of the
immune system to reject novel antigens. It is now clear
that efficient, long-term transfer of dystrophin required
significant new vector technologies beyond those available in 1987.
Today there is room for cautious optimism that DMD
and other muscular dystrophies will be treated with
gene therapy. Scientists have achieved a new understanding of the requirements for, and obstacles to,
successful gene transfer. There is greater respect for
both the capabilities and the limitations of available
viral vectors. By choosing the best vector for use in a
particular setting, significant success has already been
The earliest clinical gene therapy protocols targeted
cells of the hematopoietic system (Culver et al., 1991;
Rosenberg et al., 1990). There were at least two advantages to this choice. First, gene transduction could be
performed ex vivo, which allowed for in vitro selection
of genetically altered cells. Second, there are diseases of
the hematopoietic system in which corrected cells should
have a selective growth advantage over uncorrected
cells, allowing for successful therapy even when a
minority of target cells are transduced. In principle, it
can be argued that the muscular dystrophies should
share these advantages. Dystrophic myoblasts can be
isolated and manipulated ex vivo. Transplantation of
corrected myoblasts into dystrophic muscle could restore normal muscle function. Since myoblasts can
proliferate considerably before fusing into myofibers,
*Correspondence to: Jeffrey S. Chamberlain, Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan
48109-0618. E-mail:
Received 20 October 1999; accepted in revised form 21 October 1999
Fig. 1. The dystrophin-glycoprotein complex (DGC). The DGC
forms a structural link between the actin cytroskeleton and laminin-2
in the extracellular matrix. Integral components of this link include
dystrophin, the dystroglycans, the sarcoglycans (SGs), sarcospan (SP),
and various proteins associated with the C-terminus of dystrophin,
including syntrophin (SYN), dystrobrevin (Db), and MAST. Many
forms of muscular dystrophy (MD) are caused by mutations in genes
coding for DGC proteins. Shown are congenital MD, one form of which
is caused by mutations in a laminin subunit; limb-girdle MD, forms of
which are caused by mutations of each of the SGs; Duchenne MD,
caused by mutations in the dystrophin gene; and Bethlem myopathy,
caused by mutations in subunits of collagen type VI.
TABLE 1. DNA sequences used for gene therapy
of muscular dystrophy
At the present time, direct gene transfer to muscle is
a more promising approach for gene therapy of MDs, so
this review is focused on transduction of muscle tissue
in vivo. Muscle tissue has several advantageous characteristics for gene therapy. First, the bulk of skeletal
muscle is easily accessible for experimental manipulation. In addition, muscle tissue is efficiently transduced
by commonly used viral vectors, including adenovirus
and AAV (see Fig. 4; Kessler et al., 1996; Xiao et al.,
1996). Myofibers have long lifespans in vivo, which
should facilitate long-term gene transfer. Finally, few
alternative treatments are available for the muscular
dystrophies, which justifies the development of potentially expensive gene replacement therapies. On the
other hand, gene therapy of muscle presents at least
one daunting challenge: muscle tissue comprises over
40% of body mass. Most muscular dystrophies—with
the possible exception of merosin-deficient CMD (Vilquin et al., 1996)—are caused by cell-autonomous defects, which argues that the majority of muscle cells will
have to be treated individually (Phelps et al., 1995;
Rafael et al., 1994). Currently most gene delivery to
muscle is accomplished by intramuscular injection of
vector particles, an impractical approach for treatment
of all muscles in the body. In the future, vascular
delivery systems will have to be developed to make gene
replacement practical (see Fig. 5; Greelish et al., 1999).
The various forms of MD are not equally well suited
to the development of gene replacement therapy. Duchenne muscular dystrophy has received the most
attention because it is the most common type of dystrophy and its pathogenesis was the first to be elucidated
at the molecular level (Fig. 1). Replacement of a single
isoform of dystrophin in striated muscle is sufficient for
elimination of the major symptoms of the disease, an
important consideration given the widespread expression of dystrophin isoforms (Cox et al., 1993). Furthermore, low-level dystrophin expression in a simple majority of muscle fibers has been shown to suffice for
elimination of symptoms (Phelps et al., 1995). One of
the major difficulties with the development of gene
Conventional Ad
BMD minigene
⌬H2-R19 minigene
Gutted Ad
MCK (full length)
MCK (truncated)
MCK (synthetic)
Human skeletal ␣-actin
Sizea (approximate kb)
aFor viral vector systems, listed in bold, the approximate capacity for foreign
DNA is given. For potentially therapeutic genes, the length of the coding region of
the cDNA is given. The last five items in the table refer to frequently used
an individual myoblast or myogenic stem cell could
correct a large volume of muscle tissue (Engel and
Franzini-Armstrong, 1994). Also, corrected muscle fibers would likely display a strong selective advantage
over dystrophic fibers, which have a limited half-life
(Morgan et al., 1993). Unfortunately, the replicative
capacity of myoblasts from dystrophic humans is low,
which greatly limits the possibilities for culture, correction, and reinfusion of these cells (Blau et al., 1983).
Heterologous myoblast transfer, which could bring normal dystrophin genes into the body, has met with little
success (Mendell et al., 1995). This latter scenario has
been limited by immunologic rejection of donor myoblasts and by the loss of myogenic potential following
large-scale culturing in vitro (Gussoni et al., 1996;
Karpati et al., 1993). Nonetheless, recent advances in
identifying myogenic stem cells suggest that ex vivo
strategies might one day be developed for the muscular
dystrophies (Ferrari et al., 1998; Gussoni et al., 1999).
replacement therapy for DMD has proven to be the
large size of the dystrophin gene, which has required
development of entirely new vector systems for efficient
delivery (see Figs. 4, 6; Table 1).
The sarcoglycanopathies, as targets for development
of gene therapy, share some of the advantages of DMD
and are caused by defects in relatively small genes (Fig.
1; see Fig. 7; Table 1). Since alpha- and gammasarcoglycan are expressed primarily or exclusively in
striated muscle, gene transfer to striated muscle should
correct most symptoms of limb-girdle muscular dystrophy (LGMD) 2D and 2C (Bonnemann et al., 1995;
Coral-Vazquez et al., 1999; Jung et al., 1996; Lim et al.,
1995; McNally et al., 1994; Nigro et al., 1996; Noguchi
et al., 1995; Roberds et al., 1993, 1994; Straub et al.,
1999). Beta- and delta-sarcoglycan, however, are expressed in both smooth and striated muscle, so complete correction of LGMD2E and 2F may require more
widespread gene expression (Coral-Vazquez et al., 1999;
Straub et al., 1999). Though the levels of gene transfer
required for functional correction have not been systematically investigated, it is likely that correction of a
majority of fibers would be sufficient, given the fact that
the sarcoglycanopathies and DMD cause dystrophy
through an effect on the same protein complex (Fig. 1;
Straub and Campbell, 1997). Perhaps most importantly, the coding region of each sarcoglycan gene is less
than 2 kb, enabling delivery using an adeno-associated
virus (AAV) vector (Fig. 4; Table 1; Lim and Campbell,
1998). AAV has been shown to deliver neoantigens to
muscle without elicitation of an immune response-the
basic goal of all gene replacement therapies (Kessler et
al., 1996; Xiao et al., 1996). The major drawback to
sarcoglycanopathies as models for gene therapy of MD
is their relatively low incidence, which is probably less
than 5% of that of DMD (Ljunggren et al., 1995).
Other muscular dystrophies have received limited
attention as candidates for gene therapy. Merosindeficient CMD (MCMD), for example, appears to be
caused by a defect that is not entirely cell-autonomous,
which could allow phenotypic correction despite transduction of a lower proportion of fibers (Fig. 1; Vilquin et
al., 1996). Correction of MCMD has been investigated
in transgenic mouse models, where it was shown that
expression of laminin-alpha2 from a muscle promoter
corrected the muscle phenotype but failed to correct a
relatively minor neurological phenotype (Kuang et al.,
1998). Unfortunately, the laminin-alpha2 mRNA is
about 9.5 kb (Kuang et al., 1998), too long for delivery
using AAV or conventional Ad vectors (Table 1). EmeryDreifuss muscular dystrophy (EDMD) is caused by
deficiency of emerin, a widely expressed protein of the
inner nuclear membrane and intercalated disks (Bione
et al., 1994; Bonne et al., 1999; Cartegni et al., 1997;
Manilal et al., 1996; Nagano et al., 1996). Since the
most lethal features of EDMD are caused by absence of
the protein in heart tissue, rather than skeletal muscle
tissue, this disease offers the opportunity for effective
intervention through treatment of a relatively small
mass of tissue (Emery, 1987). In addition, the emerin
coding region is less than 1 kb, which allows for its
delivery in an AAV vector (Table 1). To date, development of gene therapy for EDMD has been hampered by
the lack of an animal model.
For a recessive genetic disease or a dominant disease
caused by haploinsufficiency, delivery of the disease
gene itself is an obvious therapeutic choice. Unfortunately, gene replacement will not be feasible in all
cases. First, some muscular dystrophies are dominantly inherited and not amenable to gene replacement. Second, gene replacement could lead to an immune response against the therapeutic protein in
patients who do not express any significant protein
from their disease locus. Finally, the size of the intact
disease gene may be too large for delivery with the viral
vector of choice (Table 1). To overcome these difficulties,
it may be necessary to deliver a minigene, a homologous
gene, or a modulatory gene instead of the disease gene.
The use of a minigene for therapy of DMD was
originally suggested by mutation analysis in patients
with mild forms of Becker muscular dystrophy (BMD;
Love et al., 1990). A number of these patients had large
deletions in their dystrophin genes, which nonetheless
led to the accumulation of small proteins with substantial functional capacity (Arahata et al., 1991; Bulman et
al., 1991; Monaco et al., 1988). Considerable attention
has been focused on a patient with a deletion of 46% of
the dystrophin coding sequence but very mild BMD
(BMD minigene in Table 1; England et al., 1990). This
allele functions well in transgenic animals (Figs. 2, 3;
Phelps et al., 1995). Similar minigenes have been
delivered to muscles using both adenoviral and retroviral vectors and some efficacy was observed in young or
immunosuppressed animals (Deconinck et al., 1996;
Dunckley et al., 1993; Ragot et al., 1994; Vincent et al.,
1993). The obvious problem with the use of such
minigenes is that the means of their identification,
study of patients with BMD, ensures that they are less
than completely effective (Figs. 2, 3). It is, therefore,
hoped that creation of recombinant dystrophin minigenes in the laboratory might yield more effective
minigenes. For example, we have observed that modification of some naturally occurring BMD minigenes to
restore an integral number of spectrin-like repeats
resulted in a smaller molecule with superior effectiveness (⌬H2-R19 in Table 1; Figs. 2, 3; Chamberlain et al.,
unpublished data). Several other promising alleles,
shown to restore the dystrophin-associated glycoprotein complex and small enough to be delivered using
AAV, have unfortunately failed to provide functional
correction in transgenic mice (Cox et al., 1994, Chamberlain et al., unpublished data).
Delivery of a homologous gene may also prove useful
in some circumstances. This approach could eliminate
concerns about an immune response triggered by delivery of a formerly missing protein. The best example is
delivery of utrophin for therapy of DMD. Utrophin is a
protein closely related to dystrophin, but which is
normally localized to the neuromuscular and myotendinous junctions (Helliwell et al., 1992, 1994). Expression
of utrophin uniformly on the sarcolemma using transgenic animal technologies prevented development of
dystrophic pathology in mdx mice (Tinsley et al., 1998).
Utrophin expression on the sarcolemma could in theory
be achieved either by delivery of utrophin in a viral
vector or by delivery of factors that stimulate transcrip-
Fig. 2. Histology of wild-type, mdx, and transgenic mdx muscle.
Top-left panel, marked ⌬H2-R19, shows muscle histology in an mdx
mouse made transgenic for a synthetic dystrophin minigene lacking
an integral number of spectrin-like repeats. The histology shown is
normal, as can be seen by comparison with the wild-type panel at
lower-right: variation in fiber size is minimal and myonuclei are
located at the periphery of their fibers. The top-right panel, marked
⌬17–48, shows muscle histology in an mdx mouse made transgenic for
a minigene isolated from a patient with mild Becker muscular
dystrophy. The histology of this muscle is close to normal, but an
occasional central nucleus is observed, indicating low-level muscle
regeneration. The bottom-left panel shows typical muscle histology in
mdx mice. Extreme variation in fiber size, frequent centrally nucleated fibers, and some fibrosis are observed. Prominent central nucleation and fiber size variation indicate ongoing muscle regeneration.
Fig. 3. Functional correction of dystrophy
in transgenic mdx mice. The specific force, or
force per unit cross-sectional area, produced
by dystrophic muscle (mdx) is reduced compared to that produced by healthy muscle
(C57). Transgenic mdx mice bearing a Becker
muscular dystrophy (BMD) dystrophin minigene, called ⌬17–48, display partial correction of this parameter. Transgenic mice bearing a synthetic minigene with an integral
number of spectrin-like repeats, called ⌬H2R19, are fully corrected.
tion of the endogenous gene (Gramolini et al., 1999;
Khurana et al., 1999). This approach might be advantageous because the utrophin coding region is almost as
large (10.3 kb) as that of dystrophin (Table 1; Tinsley et
al., 1992). The possibility of using homologous gene
products for therapy has also been raised in other forms
of muscular dystrophy, though there is no evidence yet
that this would work. For example, M-laminin or
merosin appears later in muscle development than does
an earlier isoform called A-laminin, which differs from
merosin only in the identity of its heavy chain (Fig. 1;
Leivo and Engvall, 1988; Sanes et al., 1990; Xu et al.,
1994). If the A-laminin heavy chain could be delivered
to muscle or expression of the endogenous locus could
be forced, perhaps this approach would be sufficient to
alleviate dystrophy. Similarly, epsilon-sarcoglycan is a
widely expressed gene with similarity to alpha-sarcoglycan, and it has been suggested that high-level expression of epsilon-sarcoglycan might allow for restoration
of the sarcoglycan complex in LGMD type 2D (Ettinger
et al., 1997).
Another approach to treating the muscular dystrophies would be to deliver a modulatory gene together
with, or instead of, a replacement for the mutant gene.
The MDs are characterized by constant cycles of muscle
fiber degeneration and regeneration that eventually
fail (Fig. 2; Blau et al., 1983; Emery, 1993; McArdle et
al., 1995). If these cycles could be slowed, or if regeneration could be made more robust, it is reasonable to
assume that the dystrophic process would be delayed or
alleviated. Genes that might achieve these goals include trophic factors that stimulate muscle hypertrophy, such as IGF-1 (Barton-Davis et al., 1998); regulatory molecules that can stimulate formation of new
myogenic precursors, such as MyoD (Lattanzi et al.,
1998; Megeney et al., 1996); or inhibitors of necrosis or
apoptosis, such as calpastatin or Bcl family members
(Tidball et al., 1995).
Most muscular dystrophies are caused by absence of
a protein in muscle cells. Although some dystrophies
have less obvious effects on the central nervous system,
dysfunction of heart or skeletal muscle leads to death.
For DMD and MCMD, it has been proven that gene
replacement in striated muscle alone can alleviate the
major features of the disease (Cox et al., 1993; Kuang et
al., 1998). For these reasons, gene delivery to muscle
cells is the most likely route to an effective treatment.
Achievement of this goal will require (1) a suitable
vector that can be mass-produced cheaply and (2) an
efficient means for delivery of the vector to the surface
of muscle fibers. Vector delivery to the muscle surface is
usually accomplished on a small scale by multiple
injections throughout the muscle; however, a systemic
vascular delivery system would be vastly superior if one
could be developed. Dystrophin-positive fibers appear
to have a survival advantage over negative fibers,
which might allow for use of a less efficient delivery
system in the long term, especially if this selective
advantage also applies to the other forms of dystrophy
(Morgan et al., 1993).
A vector that can achieve long-term persistence will
be required. Since many patients with MD are diagnosed early in life, and the goal of an ideal therapy is
extension of lifespan into the normal range, the ideal
therapy would retain its effectiveness for about 70
years. This goal could be achieved by multiple treatments throughout life, but each individual treatment
should retain effectiveness for at least several years.
Experience has shown that long-term effectiveness
depends on avoidance of the host immune response, so
an appropriate vector must evade the immune system
or be administered together with immune suppression.
A suitable vector must also be capable of carrying and
delivering the therapeutic gene(s). In some cases the
disease gene will be large, but even small genes may
need to be regulated using large, tissue-specific regulatory elements or might need to be delivered together
with a modulatory gene. Several approaches can be
taken to facilitate delivery of long stretches of DNA.
First, development of new viruses as gene delivery
vectors should be pursued. Development of a herpes
simplex virus (type 1) system, for example, is encouraging in this regard (Huard et al., 1995; Marconi et al.,
1999). Second, conventional vector systems could be
adapted so as to increase their capacity for foreign
DNA, as has been recently achieved with the development of gutted Ad vectors (Fig. 4; see Fig. 6; Fisher et
Fig. 4. Vectors for gene therapy. The genome of Wild-type Ad may
be divided into early and late regions. Early regions, designated by
gradient shading, are replaced by exogenous DNA in conventional Ad
vectors. 1st generation vectors contain deletions in the E1 and E3
regions. 2nd generation vectors contain deletions in additional early
regions-E2B in this example. Ad5␤dys is an example of a gutted Ad
vector, in which virtually the entire genome can be replaced by
exogenous DNA. AAV vectors also contain deletions of nearly the
entire viral genome, except for the viral ITRs, which are required for
replication and packaging of the vector. The ITRs are only 145 bp in
size and are not shown in this diagram. Lentiviral vectors contain an
initial short stretch of viral RNA required for export and packaging of
full-length transcripts in the packaging line. The remainder of the
genome may be replaced with exogenous sequences, except for a small
region near the polyadenylation signal that is required for reverse
al., 1996; Kochanek et al., 1996; Kumar-Singh and
Chamberlain, 1996). Use of these vectors eliminates
the host immune response against viral proteins and
may reduce the response against the therapeutic protein (Schiedner et al., 1998). These vectors have recently allowed our laboratory and others to deliver
dystrophin without elicitation of a major immune response (Fig. 4; see Fig. 6).
and production of immunogenic late proteins occurs
only if the early phase progresses through the onset of
viral DNA replication.
Conventional Ad vectors are created by the replacement of viral early genes with an exogenous expression
cassette (Fig. 4). The vectors can be propagated in
packaging cell lines that express the deleted early
genes, but are essentially replication-defective in noncomplementing cell lines and in vivo. First-generation
vectors lack E1, which is involved in transactivation of
other viral genes, so immunogenic viral proteins are
expressed at a greatly reduced level (Gaynor and Berk,
1983; Nevins, 1981; Yang et al., 1996b).
Adenoviral vectors have some advantageous properties for gene therapy. The serotypes used in creation of
gene delivery vectors usually cause only mild or subclinical disease in the wild (Brandt et al., 1969; Schmitz et
al., 1983). As a result, inadvertently generated, replication-competent virus is unlikely to harm an immunecompetent patient. Adenoviruses can be readily grown
to very high titer. Since Ad virions remain tightly
associated with lysed cells, within whose nucleus they
are packed into crystalline arrays, concentrated stocks
are easily prepared through low-speed centrifugation
and collection of lysed cells. Adenoviruses efficiently
infect most human cell types, including immature
muscle cells (see Fig. 7), though most lymphocytes are
relatively resistant. Finally, the popularity of Ad vec-
Adenoviral Vector Technology
Adenoviral replication is usually divided into two
stages, early and late, which are divided by the onset of
DNA replication. Only a subset of viral genes, called E1,
E2, E3, and E4, are expressed during the early phase
(Fig. 4). These genes prepare the host cell for viral
replication by stimulating production of necessary precursors and helping to prevent a host immune response. During the late phase genes for structural
components of the Ad virion are expressed. Obviously,
these structural proteins must produced at high levels;
perhaps because of this, they are also the principal
targets of the host immune response (Jooss et al.,
1998a). Expression of the late genes requires DNA
replication, though the mechanism behind late gene
activation is not entirely understood (Thomas and
Mathews, 1980). As a result, expression of late genes
tors has led to development of simple means for their
manipulation (Chartier et al., 1996; reviewed in Graham and Prevec, 1991).
Unfortunately, Ad vectors have drawbacks that limit
their usefulness for gene replacement therapy of muscular dystrophy. The cloning capacity of first-generation
vectors is only about 8 kb, which is the amount of space
made available by deletion of E1 and E3 (Fig. 4; Table 1;
Bett et al., 1993). Obviously, first-generation Ad vectors
cannot deliver full-length dystrophin, since the coding
region of dystrophin alone is 11 kb. These vectors can,
however, be used to deliver shortened forms of dystrophin that are generated in the laboratory. Ad vectors
can also be used to deliver smaller genes involved in
other forms of muscular dystrophy (see Fig. 7).
A more serious difficulty with first-generation Ad
vectors is leaky expression of immunogenic viral proteins in vivo. Despite deletion of E1, viral gene expression and even limited replication can occur in noncomplementing cells over a longer time scale. Expression
of viral proteins leads to a host immune response and
elimination of gene expression from transduced tissues
(Dai et al., 1995; Dong et al., 1996; Gaynor and Berk,
1983; Nevins, 1981; Van Ginkel et al., 1995; Yang et al.,
1994, 1996b; Yang and Wilson, 1995; Zsengeller et al.,
1995). The resulting inflammatory process can even
lead to muscle damage and exacerbation of weakness.
Immunosuppressive drugs can partially overcome this
problem (Lochmuller et al., 1996), but immunosuppression has its own risks and so development of an
improved vector would be preferable.
Second Generation Ad Vectors
Several groups created new Ad vectors lacking additional early genes in an effort to address problems with
first-generation vectors (Fig. 4; Amalfitano et al., 1998;
Armentano et al., 1995; Gorziglia et al., 1996; Schaack
et al., 1995; Wang et al., 1995; Weinberg and Ketner,
1983). The deletions target E2A, E2B, and E4, which
are the remaining early regions of the Ad genome. For
each deletion a corresponding packaging cell line must
be generated, which expresses the missing proteins in
trans. These second-generation vectors provide additional cloning capacity and should further attenuate
the virus. Further inhibition of viral replication is
desirable for two reasons. First, these highly modified
second-generation vectors are less likely to generate
replication-competent virus during large-scale, clinicalgrade vector preparation. Second, since expression of
viral late genes requires replication, complete inhibition of Ad genome replication should abolish late gene
expression, which would eliminate the host immune
response against late proteins (Amalfitano et al., 1998).
E2A, E2B, and E4 deletions introduced into Ad
vectors have provided about 1.4 kb, 1 kb, and 1.9 kb of
additional cloning capacity, respectively. Individually,
none of the deletions yields a dramatic increase in
cloning capacity; nonetheless, combining two or three of
these additional deletions might allow delivery of fulllength dystrophin via an Ad vector. However, since
many second-generation vectors grow to lower viral
titers than first-generation vectors or wild-type Ad
(Zhou et al., 1996), combining all these deletions into a
single vector may prove difficult.
Second-generation vectors have been shown to elicit
a reduced immune response and thereby allow prolonged transgene expression (Gao et al., 1996; Hu et al.,
1999; O’Neal et al., 1998; Wang et al., 1997). For
example, an E1-, E2B-, and E3-defective vector designed in our laboratory demonstrated prolonged transgene expression and persistence of vector genomes,
along with reduced toxicity, in the liver (Hu et al.,
1999). Similar experiments to test the properties of
E2B-defective vectors in muscle are underway. Secondgeneration vectors therefore offer significant promise in
some settings, but further investigation of their advantages for treatment of MD are required.
Gutted Adenoviral Vectors
Gutted, or helper-dependent, Ad vectors may overcome many drawbacks associated with conventional Ad
technology (Fig. 4). These deleted genomes were the
first Ad vectors to be developed (Solnick, 1981; Thummel et al., 1981), but interest in their use has expanded
recently with several demonstrations of their advantages for gene transfer. Growth and purification of
these viruses was extremely laborious until the recent
development of new techniques (Hardy et al., 1997;
Hartigan-O’Connor et al., 1999; Parks et al., 1996).
Further improvements will still be required before
routine, large-scale growth of clinical-grade gutted
virus is feasible.
Gutted vectors contain cis-acting DNA sequences
that direct adenoviral replication and packaging but do
not contain viral coding sequences (Fig. 4; Fisher et al.,
1996; Kochanek et al., 1996; Kumar-Singh and Chamberlain, 1996). Theoretically, the vectors can accommodate up to about 37 kb of exogenous DNA, though 28–30
kb is more typical (Fig. 4; Table 1). Since gutted vectors
do not contain any viral genes, expression of viral
proteins is not possible. Gutted vectors are defective
viruses produced by replication in the presence of a
helper virus, which provides all necessary viral proteins in trans. Like other defective viruses, gutted
viruses are normally prepared as a mixture with helper
The starting material for production of all gutted
viruses is plasmid DNA (Fisher et al., 1996; Kochanek
et al., 1996; Kumar-Singh and Chamberlain, 1996).
These plasmids contain the viral origins of replication
(ITRs), the packaging signal (psi), and therapeutic DNA
to be carried by the gutted virus. Co-transfection of this
plasmid and helper viral DNA into a packaging cell line
leads to replication of the helper virus and concomitant
replication of the gutted virus, as directed by the viral
ITRs contained in the starting plasmid. Robust helper
virus replication causes lysis of the transfected cells.
The resulting lysate contains a large number of helper
virions and a relatively small number of gutted virions.
To increase the number and proportion of gutted virions
in the lysate, the initial mixture must be serially
passaged. During serial passage, for unknown reasons,
gutted virus is amplified more quickly than helper virus
and eventually substantial enrichment occurs. Particles containing gutted viral genomes, rather than
helper genomes, must then be purified on the basis of
their lower density (Fisher et al., 1996; Kochanek et al.,
1996; Kumar-Singh and Chamberlain, 1996).
A gutted vector can accommodate not only the fulllength dystrophin cDNA but also expression cassettes
coding for marker or modulatory proteins. The gutted
vector Ad5␤dys, for example, contains the full-length
dystrophin cDNA driven by a 3.3 kb muscle creatine
kinase promoter as well as a lacZ gene controlled by the
CMV promoter for monitoring vector production in
vitro and gene delivery in vivo (Fig. 4; Kumar-Singh
and Chamberlain, 1996). We have developed other
vectors that contain dystrophin, either murine or human, and combinations of other regulatory, reporter
gene, and phenotypic modulatory sequences (see Fig.
6). The very large cloning capacity of gutted vectors
opens a new world of therapeutic possibilities that were
previously not possible.
Ad5␤dys delivers full-length dystrophin (and ␤-galactosidase) to the skeletal muscles of mdx mice with an
efficiency comparable to that of first-generation viruses
(Kumar-Singh and Chamberlain, 1996; Hauser et al.,
unpublished data). Gene expression persists for over 1
year in immune-deficient SCID/mdx mice, which indicates that the complete absence of viral gene expression
does not destabilize gutted vector genomes in vivo. In
immune-competent mdx mice, by contrast, expression
of dystrophin persists for less than 1 month, suggesting
that dystrophin expression may be eliminated by an
immune response. Removal of all viral coding sequences from Ad5␤dys was therefore insufficient, by
itself, to eliminate a host immune response against all
antigens. A newer version of this gutted vector in which
␤-galactosidase is expressed from an inducible promoter leads to dystrophin expression for at least several months in immune-competent mice (Hauser et al.,
unpublished data). Gutted vectors should be designed
to provide targeted expression of therapeutic proteins
in the affected tissue only, a principle that was not
followed in construction of Ad5␤dys. As is discussed
further below, limiting gene expression to the target
tissue may allow avoidance of the immune response
and long-term gene expression.
Adeno-Associated Virus (AAV) Vectors
AAV particles were first identified by electron microscopic examination of human and simian Ad preparations (Atchison et al., 1965; Melnick et al., 1965). The
particles were soon recognized as defective viruses that
normally replicate only in the presence of Ad (Hoggan
et al., 1966). It is now known that several other viruses
and a variety of genotoxic treatments can also provide
helper functions (Schlehofer et al., 1986; Yakinoglu et
al., 1988; Yakobson et al., 1987, 1989). In fact, no helper
virus genes are directly involved in replication of AAV
DNA; instead, the helper virus seems to maximize
synthesis of cellular proteins involved in AAV replication (Muzyczka, 1992).
AAV is a 4,680-bp parvovirus with a fascinating and
unusual life cycle. In the absence of helper virus, AAV
infection is nonproductive and no progeny AAV particles are produced. Instead, the ssDNA genome may be
converted to a double-stranded form and become covalently associated with cellular DNA at a specific locus
on chromosome 19 (Kotin et al., 1990). If a helper virus
subsequently infects the cell, the AAV genome is excised
and begins to undergo lytic growth. The growth of AAV
reduces production of helper virus particles and lysis of
the cell eventually produces more AAV particles than
Ad particles (Atchison et al., 1965).
Gene delivery vectors based on AAV are prepared by
replacement of all viral coding sequences with therapeutic DNA (Fig. 4; Muzyczka, 1992). The total amount of
exogenous DNA that can be carried by AAV vectors is
currently less than 5 kb, which unfortunately eliminates many muscular dystrophy genes from consideration (Table 1). The only remaining viral sequences are
the AAV ITRs, which direct replication and packaging
of the vector construct. Both AAV proteins and helper
virus proteins must be provided in trans, an arrangement that will be recognized as similar to that of gutted
adenovirus vectors. Originally, AAV vector stocks were
therefore produced as a mixture of vector particles and
helper virus particles (Hermonat and Muzyczka, 1984).
Within the last 2 years, however, helper-virus-free
packaging systems have been developed based on a
better understanding of the requirements for efficient
AAV replication (Matsushita et al., 1998; Salvetti et al.,
1998; Xiao et al., 1998). Using these systems, pure AAV
vectors can be produced at titers nearly equivalent to
those of wild-type Ad.
AAV was originally developed as a gene therapy
vector based on the hope that genomic integration
would allow long-term expression of transgenes. AAV
has, in fact, been a successful vector because in some
tissues it provides very long-term expression of transgenes in the absence of an immune response. For
example, Xiao et al. (1996) found that expression of
␤-galactosidase from an AAV vector was stable for more
than 8 months in the muscle of adult, immunecompetent animals. Though humoral responses against
the AAV particles were observed, no cellular immune
response was apparent. This result is astonishing given
the robust cellular response observed after transduction of the ␤-galactosidase gene using Ad vectors.
It has turned out that lack of a cellular immune
response, not genomic integration, is the most important factor in maintenance of gene expression in muscle
cells. Jooss et al. (1998b) showed that administration of
an Ad-lacZ vector can elicit a cellular immune response
that destroys muscle fibers previously transduced by
AAV-lacZ, which could otherwise survive and continue
to express ␤-galactosidase. These authors also demonstrated that AAV-lacZ vectors fail to transduce antigenpresenting cells (APCs) whereas Ad vectors efficiently
transduce such cells. These data suggest that AAV
vectors achieve immune evasion and persistent gene
expression through avoidance of antigen presentation
by professional APCs such as dendritic cells. Furthermore, it seems that most of the AAV genomes in muscle
tissue are present in the form of large circular multimers (Duan et al., 1998). Formation of such multimers
may play a role in the persistence of vector DNA, but Ad
genomes can also survive inside the nucleus for long
periods, so physical persistence of episomal DNA can be
achieved through several mechanisms.
The striking success of AAV vectors in long-term
transduction of muscle tissue has given rise to hope
that muscular dystrophies caused by defects in small
genes may soon be treated using this vector. Some of the
exciting results achieved so far are described below.
Just as importantly, our insight into the mechanisms
used by AAV to achieve persistent gene expression may
be applicable to other vector systems that have not yet
achieved comparable success.
Lentiviral Vectors
Murine retroviral vectors were used in some of the
earliest efforts at gene replacement for DMD (Dunckley
et al., 1992, 1993). As murine retroviral vectors are
unable to stably infect non-dividing cells, post-mitotic
muscle fibers were not targeted in these studies. Instead, investigators attempted to transduce proliferating myoblasts in vitro or in vivo. Although transduction
of myoblasts with retroviral vectors is effective and
does lead to gene transfer after myoblast transplantation, the significant technical hurdles to myoblast transplantation remain. Transduction of myoblasts in vivo
might be expected to be effective, since muscle regeneration is such a prominent feature of dystrophic pathology
(Fig. 2). Unfortunately, direct injection of a murine
retrovirus carrying minidystrophin resulted in transduction of only a few percent of fibers at the site of injection
(Dunckley et al., 1993). Injection of bupivicaine, which
induces muscle necrosis and regeneration, doubled the
number of transduced fibers. More recently, implantation of retroviral-producer cells resulted in transduction of greater than 10% of fibers (Fassati et al., 1997).
Such an approach is, however, limited by problems
associated with immunological rejection of the producer cells. Finally, the 7.8 kb cloning capacity of
retroviral vectors prevents delivery of full-length dystrophin cDNA clones. As a result, the use of the murine
retroviruses for gene replacement therapy in muscle
will require significant technical innovation before it
becomes a promising approach.
Recently, however, new retroviral vectors have been
introduced that can integrate into the genome of postmitotic cells (Carroll et al., 1994; Naldini et al., 1996;
Poeschla et al., 1998). These vectors are based on
human or feline lentiviruses, which infect nondividing
cells as part of their normal life cycle (Lewis et al., 1992;
Weinberg et al., 1991). Like the older murine vectors,
these vectors are produced by expression of a packageable vector construct in a cell line that expresses viral
proteins (Fig. 4). Vector particles bud from the surface
of such cells continuously and can be harvested from
the supernatant. Because the lentivirus life cycle is
nonlytic and because the enveloped vector particles are
not as stable as naked Ad or AAV particles, the vector
titers obtained are relatively low (about 109 per ml)
even after concentration. The size of the vector construct is limited to about 10 kb by constraints on
retroviral packaging, which again prohibits transfer of
the full-length dystrophin cDNA (Table 1).
Lentiviral vectors efficiently transduce post-mitotic
neurons, hepatocytes, and muscle fibers (Kafri et al.,
1997; Naldini et al., 1996). Injection of a lentiviral
vector expressing GFP into muscle tissue resulted in
transduction of about half the muscle fibers at the site
of injection (Kafri et al., 1997). Gene expression was
maintained for at least 2 months without elicitation of a
major immune response. It should be noted, however,
that the number of particles injected was several orders
of magnitude lower than the number of Ad or AAV
particles that are usually injected. Elicitation of a
significant cellular immune response may require attainment of a certain gene expression threshold, which
may not have been achieved in this case (Tripathy et al.,
1996). Alternatively, perhaps the VSV-G envelope protein, with which the vector was pseudotyped, does not
allow efficient infection of dendritic cells. Further development of this technology might someday allow for
effective treatment of muscular dystrophy.
A single injection into muscle tissue typically results
in transduction of cells within 0.5–1 cm of the injection
site. This limited vector diffusion means that saturation of a single muscle group would require multiple
injections. Saturation of many large human muscles,
including the heart and diaphragm, would require
hundreds of injections and is probably impractical on a
large scale. Therefore, if viral vectors can be proven
suitable for stable transduction of muscle on a small
scale, a means for vector delivery to a large mass of
muscle tissue needs to be developed.
Arterial delivery holds promise as a means to overcoming this difficulty (Fig. 5; Greelish et al., 1999;
Welling et al., 1996). Unfortunately, transport of viral
vectors across normal vascular endothelium is poor,
even when high hydrostatic pressure is applied (Greelish et al., 1999; Jejurikar et al., 1997). Greelish et al.
(1999) found that arterial instillation of Ad-lacZ under
elevated hydrostatic pressure led to efficient transduction of vasculature but resulted in limited muscle
infection (Fig. 5A–C). After vasodilation with papaverine and endothelial permeabilization with histamine,
however, widespread transduction of entire muscle
groups was achieved (Fig. 5D, G, H). Transduction was
strictly limited to muscle groups served by the perfused
arteries, confirming that gene delivery was accomplished through arterial delivery (Fig. 5E, F). The
treatment was effective in both hindlimb and heart
using either Ad or AAV vectors (Fig. 5I). If arterial
delivery can be performed safely and routinely, then
transduction of the majority of human muscle tissue for
gene replacement may be feasible.
Effective gene delivery depends not only on transport
of vector to target cells, but also on subsequent infection. Tropism of Ad vectors for muscle fibers has been
observed to decline with muscle maturation, during
which process myofibers down-regulate expression of
cellular receptors for adenovirus (Acsadi et al., 1994a;
Acsadi et al., 1994b; Nalbantoglu et al., 1999). As a
result, infection of immature or regenerating muscle by
Ad is more efficient than infection of mature muscle.
AAV, in contrast, seems to infect mature muscle as
efficiently as immature muscle (Snyder et al., 1997). It
is unknown whether the infectability of dystrophic
human muscle will present a serious impediment to
gene therapy of MD with Ad vectors, as little is known
about Ad tropism for human muscle. Some success has
been attained in modifying the tropism of adenovirus to
improve infection of macrophage, endothelial, smooth
muscle, fibroblast, and T cells (Douglas et al., 1996;
Stein et al., 1999; Wickham et al., 1997); however,
engineering of an adenovirus with dramatically increased tropism for skeletal muscle has not yet been
achieved. Combining a vascular delivery method with a
tropism-modified Ad vector could result in efficient gene
Fig. 5. Gene transfer across the endothelial barrier: histamine
and papaverine increase permeability to viral particles. A–C: Pattern
of gene transfer in the absence of inflammatory mediators demonstrates interference of microvascular barrier with adenovirus transport. A: ␤-galactosidase activity in whole-mount-stained leg of adult
rat 4 days after arterial infusion with AdCMVlacZ. X-gal shows that
virus uptake is limited to microvasculature; muscle fibers do not stain.
B,C: Staining is seen in most capillaries but absent in muscle fibers of
the tibialis anterior. D–I: Efficient gene transfer to adult skeletal
muscle fibers after forced exudation with histamine and papaverine.
D: Entire hindlimb from rat dissected before whole-mount-staining to
expose multiple cross-sections; universal fiber uptake is visible on
most cross-sections. E,F: Gross (E) and light microscopic (F) appearance of marker gene distribution in the quadriceps shows detail on
adjacent rectus femoris and vastus medialis. A tourniquet at the level
of the common femoral artery occluded blood supply to the rectus
femoris, preventing virus delivery and infection. G: Semimembranosus and adductor brevis with adjacent saphenous artery. H: Nomarski
micrograph of tibialis anterior shows unstained wall of arteriole (right
center) against a backdrop of uniformly stained muscle fibers. I:
Heterotopically transplanted heart after isolated perfusion with histamine and papaverine analogous to that used for isolated limb.
Reprinted from Greelish J P, Su L T, Lankford E B, Burkman J M,
Chen H, et al. 1999. Stable restoration of the sarcoglycan complex in
dystrophic muscle perfused with histamine and a recombinant adenoassociated viral vector. Nature Med 5:439–443, with permission of the
transfer to human muscle using a systemic delivery
Transgenic mice have proven the feasibility of gene
replacement in muscle as a means for treatment of
DMD (Figs. 2, 3; Corrado et al., 1996; Cox et al., 1993;
Phelps et al., 1995; Rafael et al., 1994; Wells et al.,
1995). Several investigators have created mdx mice
expressing a variety of levels of full-length or truncated
dystrophins in muscle tissue. More recently, similar
studies have been performed using utrophin vectors
(Tinsley et al., 1996, 1998). These animals have provided detailed information on the level of gene expression needed to prevent occurrence of dystrophy, the
functional capacity of different therapeutic molecules,
and the percentage of muscle fibers that must express
the transgene to prevent disease progression. After
generation of transgenic mice and confirmation that a
particular molecule is effective in preventing dystrophy,
transduction using viral vectors can be attempted.
Obviously, it has been more difficult to demonstrate
functional correction of dystrophy using viral vectors
than to demonstrate prevention of dystrophy with
transgenes that are uniformly expressed in all muscles.
Since most viral vectors are unable to carry a fulllength dystrophin, many studies have focused on dystrophin minigene vectors. Transgenic mdx mice have been
created that express a truncated dystrophin minigene
of 6.3 kb in length (Fig. 2; Phelps et al., 1995). This
molecule, missing about half of the coding region, was
based on the mutation discovered in a mildly affected
Becker muscular dystrophy patient with a deletion of
exons 17–48 (BMD minigene in Table 1). Few signs of
dystrophy were observed in mice expressing this minidystrophin, though low-level muscle regeneration was
detected on careful histological analysis (Phelps et al.,
1995). Muscles from these animals also display slight
deficits in their ability to generate force and resist
injury (Fig. 3). These data show that this minidystrophin does not completely prevent dystrophy; however,
the observed lack of fibrosis and almost normal force
development indicate that smaller proteins could be
useful in a clinical setting. In fact, a recombinant
adenovirus expressing this minidystrophin was shown
to prevent muscle pathology if injected within one week
of birth (Deconinck et al., 1996; Vincent et al., 1993).
Older mdx mice and dystrophic humans represent more
difficult challenges due to a functional immune system,
advanced pathology, and down-regulation of adenovirus receptors. Despite these difficulties, the same virus
was found to be reasonably effective when injected into
older mdx mice that had been immunosuppressed with
FK506 (Lochmuller et al., 1996; Yang et al., 1998).
These data offer hope that more effective minidystrophin molecules might be designed in the laboratory
using knowledge of dystrophin structure and function.
The BMD deletion described above results in the loss of
a non-integral number of spectrin-like repeats forming
the rod domain of dystrophin. Since this truncation
likely disrupts the structure of the dystrophin rod
domain, a variety of modified truncations have been
engineered in attempts to develop a highly functional
dystrophin encoded by a cDNA less than 6 kb in size
(Phelps et al., 1995; Rafael et al., 1994; Yuasa et al.,
1998; Hauser and Chamberlain, unpublished data).
Transgenic mdx mice expressing some of these modified
alleles have recently been observed to be more effective
than the exon 17–48 truncation in preventing muscular
dystrophy in mdx mice (Figs. 2, 3). Experiments to
assess the efficacy of delivery of these modified cDNAs
to dystrophic muscle using Ad and retroviral vectors
are currently in progress.
As described earlier, sarcolemmal expression of utrophin, a dystrophin-related protein normally localized to
Fig. 6. Delivery of full-length dystrophin using a gutted adenoviral
vector. Ghumdys is a gutted adenoviral vector containing the fulllength human dystrophin cDNA driven by a muscle creatine kinase
(MCK) promoter. The vector contains no reporter gene. Top: Expression of dystrophin in mdx mouse muscle 5 days after injection of the
vector. Note cytoplasmic accumulation of dystrophin, indicating highlevel expression. Bottom left: Marked DYS, shows immunofluores-
cence staining for dystrophin. Arrowheads have been placed within
two weakly positive fibers. Bottom right: Marked Merge, shows
accumulation of Evans blue dye in dystrophin-negative fibers. Evans
blue is a dye that permeates fiber with sarcolemmal disruption,
indicating severe injury. Note that the weakly dystrophin-positive
fibers do not take up dye, despite their proximity to damaged fibers.
neuromuscular and myotendinous junctions, prevents
dystrophy in transgenic mdx mice (Tinsley et al., 1998).
This observation is important because it is possible that
DMD patients with null alleles could mount an immune
response against dystrophin, which might be perceived
as a neoantigen. Even mdx mice, which express a low
level of dystrophin in revertant myofibers, have been
shown to mount a CTL response against dystrophin
expressed by transplanted C57/BL10 myoblasts (Ohtsuka et al., 1998). In contrast, no strong immune
response has been noted in experiments using gutted
vectors to deliver dystrophin to mdx mice, so concerns
about immune response in human patients may prove
unfounded. Considerable work has been done with a
synthetic utrophin minigene analogous to the common
exon 17–48 deleted minidystrophin (Deconinck et al.,
1997; Tinsley et al., 1996). In transgenic mdx mice, this
allele provides significant improvement in dystrophic
pathology based on histological and functional criteria;
in fact, the improvement in phenotype is roughly
similar to that obtained with the 6.3 kb minidystrophin
(Phelps et al., 1995).
Undoubtedly the most direct and efficacious route to
gene therapy of DMD would be delivery of full-length
dystrophin itself-true gene replacement (Fig. 6). Expression of even low levels of dystrophin in transgenic mdx
mice eliminated the major symptoms of dystrophy.
Dystrophin expression at levels between 20 and 5,000%
of wild-type prevented all signs of dystrophy without
deleterious side effects (Cox et al., 1993; Phelps et al.,
1995). Mouse and human dystrophin molecules were
found to be equally effective in these studies (Phelps et
al., 1995). Animals with expression levels below 20% of
wild-type level displayed intermediate signs of dystrophy, indicating that even low-level dystrophin expression may have therapeutic benefit. Some lines of transgenic mice produced dystrophin in a variable pattern
(Phelps et al., 1995; Rafael et al., 1994). These nonuniformly expressing lines provide data on the percentage of fibers that must be transduced to alleviate
symptoms. Comparison of the dystrophic pathology of
several such lines indicated that at least 50% of muscle
fibers must accumulate moderate amounts of dystrophin to prevent a severe dystrophy.
The major difficulty with delivery of full-length dystrophin is the fact that most gene delivery vectors
cannot accommodate the large size of the gene (Table
1). Until recently, full-length dystrophin could only be
delivered to muscle using myoblast transplantation or
naked DNA injection. However, as discussed above,
recent improvements in helper-dependent or ‘‘gutted’’
Ad vectors have allowed delivery of full-length dystrophin (Fig. 6; Kochanek et al., 1996; Kumar-Singh and
Chamberlain, 1996). We found that a gutted vector
containing both dystrophin and ␤-galactosidase expression cassettes, Ad5␤dys (Fig. 4; Kumar-Singh and
Chamberlain, 1996), efficiently delivered full-length
dystrophin to skeletal muscle, but expression persisted
for less than 1 month. The presence of a lymphocytic
infiltrate surrounding dystrophin- and ␤-galactosidasepositive fibers suggested that an immune response was
responsible for loss of expression.
Since Ad5␤dys does not contain any viral coding
sequences, it seemed likely that the immune response
was directed against either dystrophin or ␤-galactosidase, the two transgenes carried by the vector (Fig. 4).
␤-galactosidase, which plays no therapeutic role in the
vector, is known to elicit a potent T-cell-mediated
response when delivered to immune-competent mice
using conventional Ad vectors (Tripathy et al., 1996;
Yang et al., 1996a). To reduce expression of ␤-galactosidase, we created a new form of the gutted vector,
GE␤dys, in which the inducible ecdysone promoter
drives lacZ expression. Use of this promoter greatly
limits expression in mouse and human tissues, which
do not express the ecdysone receptor (EcdR), but allows
for titering of gutted vector preparations in modified
293 cells expressing EcdR. Since provocation of a strong
CTL response is thought to require gene expression in
antigen-presenting cells, attenuation of gene expression by use of such a promoter should reduce the
immune response. In fact, lymphocytic infiltration of
mdx muscles injected with GE␤dys was dramatically
reduced, indicating successful reduction of the CTL
response despite robust dystrophin expression in myofibers. Muscles injected with GE␤dys expressed dystrophin for at least 4 months post-injection, which was the
longest time point tested (Salvatori et al., unpublished
These data suggest that gutted vectors will allow
delivery and long-term expression of full-length dystrophin, although avoidance of immune response remains
a critical issue. Additional studies addressing the persistence and functional capacity of gutted vectors lacking
all reporter genes have yielded highly encouraging
results, suggesting that phase I clinical trials of gutted
vectors should proceed (Fig. 6).
The dystrophin-glycoprotein complex found in skeletal muscle includes four related proteins named sarcoglycans (Fig. 1; Campbell and Kahl, 1989; Ervasti et al.,
1990; Yoshida and Ozawa, 1990). Sarcoglycan deficiency is now known to cause four forms of autosomal
recessive LGMD-one for each sarcoglycan molecule
(Bonnemann et al., 1995; Jung et al., 1996; Lim et al.,
1995; Noguchi et al., 1995; Roberds et al., 1994).
Fig. 7. Recombinant ␦-SG adenovirus mediates high efficiency
gene transfer into BIO 14.6 hamster muscle. A: Age-matched quadriceps from F1B, BIO 14.6, and BIO 14.6 injected with 109 particles of
␣-SG adenovirus or ␦-SG adenovirus (tissue harvested 7 days postinjection) were analyzed by immunofluorescence using an antibody
specific for ␦-SG. Bar ⫽ 50 µm. B: ␦-SG adenovirus particles (109 ) were
injected into the quadriceps femoris muscle of BIO 14.6 hamster.
Seven days later, muscle cryosections were prepared and subjected to
immunofluorescence using antibodies specific for individual sarcoglycan proteins, as shown. Bar ⫽ 50 µm. Reprinted from Holt K H, Lim L
E, Straub V, Venzke D P, Duclos F, et al. 1998. Functional rescue of the
sarcoglycan complex in the BIO 14.6 hamster using delta-sarcoglycan
gene transfer. Mol Cell 1:841–848, with permission of the publisher.
Collectively these forms of muscular dystrophy are
called sarcoglycanopathies.
A naturally occurring hamster mutant, the BIO 14.6
cardiomyopathic hamster, is available for delta-sarcoglycan (delta-SG) deficiency, so gene replacement studies
have focused on this gene (Fig. 7; Nigro et al., 1997;
Sakamoto et al., 1997). It is likely that a gene replacement therapy effective in amelioration of delta-SG
deficiency could be directly adapted to the other sarcoglycanopathies. Holt et al. were the first to explore the
feasibility of gene replacement in the BIO 14.6 hamster
(Fig. 7; Holt et al., 1998). Due to the small size of the
sarcoglycan genes, these authors were able to deliver
delta-SG in a conventional E1, E3-deleted adenovirus
(Fig. 7A, Table 1). They found that injection of delta-SG
Ad, but not control alpha-SG Ad, restored all four
sarcoglycans to the sarcolemma (Fig. 7B). Injection of
young (3-week-old) hamsters with the Ad vector resulted in expression for at least several months, which
also occurs after injection of Ad into young, but not
mature, mice. Delta-SG Ad injection also prevented the
development of morphological abnormalities, as assessed by the percentage of muscle fibers with central
nucleation, and maintained sarcolemmal integrity.
These results are very similar to those achieved in
young mdx mice after delivery of full-length dystrophin
or dystrophin minigenes (Deconinck et al., 1996; Vincent et al., 1993).
Unlike the dystrophin gene, the sarcoglycan genes
are all small enough to be delivered using AAV (Table
1). Since AAV has proven capable of gene delivery to
adult animals without elicitation of an immune response, it should be possible to inject older BIO 14.6
hamsters and maintain expression for a long period.
For example, Greelish et al. (1999) injected 9-week-old
hamsters and evaluated injected muscle 13 weeks after
gene transfer, when stabilization of sarcolemmal integrity was demonstrated through exclusion of procion
orange dye. Such studies offer hope that sarcoglycan
deficiency could be corrected in adult, immune-competent humans. This hypothesis will be the focus of a
number of limited human clinical trials over the next
few years (Mendell and Wilson, personal communication).
There has been encouraging progress towards establishing gene replacement as a viable therapy for muscular dystrophy. Understanding the limitations of available gene transfer vectors led to the development of
new options including gutted Ad, AAV, and lentiviral
vectors. Gutted Ad vectors have allowed delivery of
full-length dystrophin to adult muscle for the first time.
Even in immune-competent animals, gene expression
can be maintained for at least several months. In the
future, these vectors may provide the capacity necessary for tightly regulated expression of therapeutic
genes or delivery of multiple expression cassettes.
Recent advances in the growth and purification of these
gutted vectors should allow evaluation of their usefulness in disparate settings. AAV vectors have had encouraging success in gene delivery to the muscles of immunecompetent animals. Even proteins that are known to be
very immunogenic, such as ␤-galactosidase, can be
expressed for long periods. It is hoped that expression of
small muscular dystrophy disease genes using AAV will
be comparatively easy.
Many challenges remain before gene replacement
therapy for MD can be declared a success. Production of
viral vectors at very large scale and high purity remains problematic, so further improvements will be
required before delivery of full-length dystrophin to
humans is practical. Long-term functional correction,
as opposed to prevention, of the dystrophic phenotype
in mouse models has not yet been demonstrated. The
immunologic consequences of delivering disease genes
to patients with null mutations are still unknown.
Finally, safe, consistent delivery of viral vectors to a
large mass of muscle tissue has not been shown to be
Stable gene transduction that provides functional
correction at the level of single cells has been demonstrated. In the future, we hope that viral vectors such
as gutted Ad, AAV, and others being developed will
allow meaningful correction of muscular dystrophy at
the level of whole muscles and human patients. Making
this leap in scale is the challenge of the years to come.
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