DMD Gene therapy Review

gene translation in DMD.

In contrast, viruses are ideal

vehicles for therapeutic gene transfer, having evolved to in-
fect specific cell populations with high efficiency. The choice
of virus is guided by factors including the target cell, im-
munogenicity, and required duration of transgene expres-
sion. Adenovirus was the early favorite for gene transfer
to muscle and other tissues including lung (cystic fibro-
sis) and liver (infectious, neoplastic, and metabolic disor-
ders). Disabling regions for replication (E1 and E2) and
regulatory functions (E3 and E4) resulted in recombinant
adenoviruses with presumed reduced ability to stimulate
immune responses while achieving sustained transgene ex-
pression. An extreme modification of adenovirus was the
deletion of all viral genes (“gutted”), enabling insertion of
as much as 35 kilobases (kb) of DNA and, thus, more than
enough for the entire dystrophin complementary DNA. This
approach gained momentum

despite several drawbacks

including challenges in vector production and concerns
about the emergence of replication-competent virus.

17,18

However, substantial uneasiness about the use of this vec-
tor was the outgrowth of the unexpected death of Jesse
Gelsinger in 1999 after transfer of the ornithine carbamy-
lase gene by adenovirus.

Within hours of intrahepatic ad-

ministration, Gelsinger experienced severe complications
and died 2 days later. The exact cause of his death has never
been fully delineated, but the clinical findings overlapped
with features of toxic shock syndrome.

After the death of Gelsinger, a member of the parvovi-

rus family, adeno-associated virus (AAV), a small single-
stranded DNA virus not associated with human disease,
took center stage for viral gene delivery. Adeno-asso-
ciated virus, a member of the Parvoviridae family, is a De-
pendovirus, requiring helper functions from other sources,
usually viruses, to complete its life cycle. The AAV ge-
nome is composed of approximately 4.7 kb containing the
replication and encapsidation genes. This viral genome is
flanked by two 145–base pair inverted terminal repeats.
Most often, adenoviral genes provide the helper func-
tions to activate and express the replication and encapsi-
dation genes required for rescue, replication, and pack-
aging. Herpesvirus can have a similar role in activating the
helper genes. A further attraction of the parvovirus fam-
ily is the recognition of the broad diversity of AAV sero-
types and genomic variants. Well over 120 AAV variants

have been isolated from various species including non-
human primates.

20,21

As of 2006, 11 AAV serotypes (AAV1-

11) had been described as candidates for gene therapy. Ad-
vantages of each are under intense investigation to establish
specific tropism relevant to the target tissue. The most
promising serotypes for transducing skeletal muscle are
AAV1, AAV2, AAV6, and AAV8.

22-25

Adeno-associated virus vectors have a relatively spotless

safety profile. An early concern was site-specific integration
on chromosome 19. While this attribute is characteristic
of wild-type AAV, it is a rare event with the recombinant
form of the virus (rAAV).

This was thought to be a po-

tential disadvantage, forcing transgene expression from an
episomal location. However, since the observed compli-
cations of insertional mutagenesis in patients with severe
combined immunodeficiency syndrome, genome integra-
tion with the potential to disrupt nearby genes is consid-
ered less desirable.

Loss of this attribute has not dimin-

ished the potential for long duration of transgene expression
best explained by the stability of the rAAV vector genome
to form stable episomal concatemers.

IMMUNOGENICITY OF AAV AND

POTENTIAL IMPACT ON GENE TRANSFER

A key issue evolving with the transfer of any gene deliv-
ery vehicle is the potential for an immune response. While
low immunogenicity was considered a major strength sup-
porting the use of rAAV in clinical trials, a number of ob-
servations have recently provided a more realistic view.
An obvious barrier to AAV transduction is the presence
of circulating neutralizing antibodies that preclude bind-
ing of the virion to its cellular receptor.

28,29

This potential

threat can be reduced by prescreening patients for AAV
serotype-specific neutralizing antibodies or through the
application of therapeutic maneuvers such as plasmapher-
esis before gene transfer. Alternatively, delivering the vi-
rus through an intravascular balloon catheter imparts a pro-
tected environment enhanced by the removal of blood from
the gene-targeted area, providing a hospitable environ-
ment for transduction.

Another challenge recently un-

covered is the development of a cell-mediated cytotoxic
T-cell (CT) response to AAV capsid peptides, limiting trans-
gene expression. In the human factor IX gene therapy trial

Cysteine-rich

N-terminal

R24

R23

R22

R21

R20

R19

R18

R17

R16

R15

R14

R13

R12

R11

R10

24-Spectrinlike repeats plus 4 hinges in the rod domain

Figure 1. Structure of dystrophin protein. The N-terminal contains the actin-binding domain. H indicates hinge; CR, cysteine-rich region; CT, C-terminal.

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in which rAAV was delivered to liver, only short-term trans-
gene expression was achieved and levels of therapeutic pro-
tein declined to baseline levels 10 weeks after vector in-
fusion.

This was accompanied by signs of hepatocellular

damage (elevation of serum transaminase enzyme val-
ues) and a CT response as measured by release of inter-
feron-

␥ directed toward specific AAV capsid peptides.

In this setting, AAV capsid sequences were displayed on
the surface of transduced liver cells by major histocom-
patibility complex class I molecules, leading to invasion
by activated CD8-positive CTs. To overcome this re-
sponse, transient immunosuppression may be required;
at least until AAV capsids are completely cleared.

Although data from the hemophilia B trial raise con-

cerns that cannot be ignored, additional findings suggest
that T-cell activation requires capsid binding to the hepa-
ran sulfate proteoglycan (HSPG) receptor, permitting vi-
rion shuttling into a dendritic cell pathway (referred to as
cross-presentation).

This is typical of the AAV2 (used in

the clinical factor IX gene therapy trial) but not for AAV
serotypes independent of HSPG binding. Further studies
are under way to confirm the privileged immune status

of virions deficient in HSPG binding. This is not a trivial
issue because gene transfer through a vascular approach
with HSPG-deficient AAV serotypes, such as AAV8 (see
“Gene Therapy by Vascular Delivery” section), seems prom-
ising. If AAV8 falls below the window of detection of the
immune system, its potential as a safe and effective gene
transfer vehicle is substantially enhanced.

MODIFYING DYSTROPHIN DNA

FOR GENE TRANSFER

The dystrophin protein has 4 major domains
(

Figure 1

2,33,34

as follow: the N-terminal, which binds

to cytoskeletal F-actin; a central rod domain composed of
24 spectrinlike repeats; the cysteine-rich domain, which
binds to the transmembrane protein

␣-dystroglycan, link-

ing noncovalently to

␤-dystroglycan, which together rep-

resent a vital component of the dystrophin glycoprotein com-
plex linking the extracellular matrix to the actin cytoskeleton
(

Figure 2

); and the C-terminal, which is not a prerequi-

site for maintaining stability of the muscle membrane. The
full-length dystrophin complementary DNA is 14 kb, pos-

Cytoplasm

Membrane

Actin

Dystrophin

Actin

F
A
K

Extracellular matrix

Sarcoglycan complex

Integrin-associated proteins

Laminin

Dystroglycan complex

Integrin

α7 β1

Syntrophins

Laminin

Figure 2. Illustration shows the major components of the dystrophin glycoprotein complex. Within the cytoplasm of the muscle fiber, the N-terminal of dystrophin
links to the actin cytoskeleton. The cysteine-rich C-terminal domain of dystrophin links to the membrane via the dystroglycan complex. Dystroglycan consists of

␤-dystroglycan, a transmembrane protein, and ␣-dystroglycan, a highly glycosylated extracellular membrane protein. Several G domains of laminin-2 bind

␣-dystroglycan to link the complex from the extracellular matrix through the membrane to the actin cytoskeleton.

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ing a substantial hurdle to the small packaging capacity of
AAV. The concept of small dystrophins that easily fit into
rAAV vectors is potentially useful and is based on clinical
observations in patients with large gene deletions of the rod
domain accompanied by a mild phenotype. The seminal
example involves a 61-year-old ambulatory patient with
Becker dystrophy with a rod domain deletion (exons 17-
48), equivalent to almost half (48%) of the coding region
of dystrophin.

Such findings encouraged a systematic study

in dystrophin-deficient (mdx) mice to find the optimal
complementary DNA that would both stabilize the sarco-
lemmal membrane and fit the restrictive capacity of AAV.

A consensus construct has emerged that permits deletion
of the C-terminal, and a rod domain consisting of either
4 (microdystrophin) or 5 or more (minidystrophin) spec-
trin repeats, combined with removal of the 5

⬘ and 3⬘ un-

translated regions.

35-37

An rAAV encoding minidystrophin

with these features

is undergoing clinical testing in a phase

1 gene therapy study by intramuscular injection in the
biceps muscle of boys with DMD at Columbus Children’s
Hospital and Research Institute, Columbus, Ohio.

GENE THERAPY BY VASCULAR DELIVERY

One challenge with gene therapy to the muscle is
achieving widespread delivery to multiple muscle
groups. We and others have been working on vascular
delivery methods to transfer therapeutic genes with the
intent of attaining clinically meaningful results. Several
AAV serotypes are more suitable for vascular delivery,
particularly AAV6 and AAV8. These serotypes demon-
strate a preferential capability for crossing the endothe-
lial vascular barrier and transducing muscle with a high
degree of efficiency, obviating the use of permeability-
enhancing agents or methods (

Figure 3

). There are

several important implications from such findings.
Using AAV serotypes with selective capacity to traverse
the endothelium for a clinical study avoids drugs (eg,
vascular endothelial growth factor, histamine, or pa-
paverine), high infusion pressures, or excessive viral
dosing, any of which adds potential for an adverse
event. A further implication of selective serotype transit
across the vascular barrier is that AAV8 does not rely on
HSPG receptor binding, potentially mitigating the need
for immunosuppression. This point is currently under
additional study and has important inferences for the
planning of clinical trials.

USE OF SURROGATE GENES TO MODIFY

THE DYSTROPHIC PHENOTYPE

Evidence from experimental gene replacement to the mdx
mouse shows that despite substantial correction of the
membrane defect by morphologic criteria (eg, protec-
tion against Evans blue dye permeability) and reversal
of the phenotype (reduced central nucleation) in ani-
mals treated at an early age, there remains a gap in full
restoration of contractile properties (protection against
eccentric contraction) using minidystrophins and mi-
crodystrophins.

39,40

This raises concerns that small dys-

trophin delivery may not restore the phenotype to nor-
mal in clinical studies. The extent of correction remains
to be established in clinical trials. Other approaches un-
der development may augment the therapeutic effect of
the small dystrophins.

The field is beginning to test “booster” genes as a thera-

peutic strategy. Such approaches include combinations
of small dystrophins with overexpression of insulin growth
factor-1, in particular, the muscle isoform (mIgf-1),

inhibition of the negative muscle growth regulatory fac-

Figure 3. Focal vascular delivery of recombinant adeno-associated virus serotype 8–green fluorescent protein (r-AAV8-GFP). Both heads of the gastrocnemius
muscle in 6-week-old beagles were treated by focal vascular delivery of rAAV8-GFP through the femoral artery using a fluoroscopy guided catheter. A, At 6 weeks
after transfer, more than 80% of the muscle fibers of the gastrocnemius muscle exhibited GFP expression. This section is from the central region of the medial
head of the gastrocnemius muscle. B, The hamstring muscle from the same limb that was not targeted by focal vascular delivery shows no GFP expression.

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tor myostatin. This approach has recently been illus-
trated in the mdx mouse, in which coinjection of the
rAAV-microdystrophin and rAAV–mIgf-1 vectors re-
sulted in a degree of protection against contraction-
induced injury not possible without dual gene therapy.

Similarly, the inhibition of myostatin results in larger and
stronger muscles with conservation of benefits, from mice
to humans. In addition to inhibiting myostatin with spe-
cific antibodies (as applied in a current clinical trial for
muscular dystrophies), follistatin is a potent myostatin
antagonist. Adeno-associated virus can deliver the gene
for follistatin and, because it is a secretory peptide, very
high serum levels can be reached, resulting in muscle hy-
pertrophy at regional and remote sites. When virus is in-
jected into the gastrocnemius and hamstring muscles in
animals, substantial increases in muscle mass are noted
at the site of injection and at remote sites (

Figure 4

We are exploring AAV-mediated gene therapy as a
form of combinational treatment of muscular dystrophy
that would also be applicable to other forms of muscle
disease.

Another novel surrogate gene strategy for muscular

dystrophy uses the enzyme CT GalNAc transferase, pref-
erably called Galgt2, normally restricted to the neuro-
muscular junction. At this site, Galgt2 is responsible for
glycosylating (adding a sugar moiety, the CT carbohy-
drate)

␣-dystroglycan. The CT carbohydrate (a name de-

rived from a common epitope shared with activated CTs)
bolsters

␣-dystroglycan in its linkage to the laminins of

the extracellular matrix, further ensuring membrane sta-
bility. Overexpression of Galgt2 in transgenic mice

by gene transfer using AAV results in the ectopic expres-
sion of glycosylated proteins (eg, CT carbohydrate–

␣-

dystroglycan), laminins usually confined to the neuro-
muscular junction (laminin

␣4 and laminin ␣5) and

utrophin along the entire myofiber. The net result is mem-
brane stability by the substitution of utrophin for dys-
trophin, creating a utrophin-glycoprotein complex. Stud-
ies by colleagues in our Gene Therapy Center

are laying

the groundwork for a clinical trial using the GALGT2 gene.

Another advantage accruing to this approach is that over-
expression of this normal gene averts the potential for
transgene immunogenicity.

VECTOR PRODUCTION METHODS

Extending gene therapy studies beyond small phase 1
safety trials is an important challenge for vector produc-
tion. To illustrate this point, if a single human dose is
determined to be 10

particles for a given application and

1000 patients with DMD are candidates for treatment,
10

particles would be required. Based on current vec-

tor production standards, each cell produces approxi-
mately 10

AAV particles, necessitating 10

cells to meet

the needs for a clinical trial. In practical terms, this would
translate to approximately 100 000 flasks or 1000 L of
cells in suspension, emphasizing both the limitations and
importance of scalable production methods.

Genetically engineered cell lines can be adapted to over-

come this potential barrier to clinical applications.

43-48

Three essential components are required for rAAV pro-
duction: the rAAV vector genome, the AAV trans-helper
genes (replication and encapsidation), and adenovirus
or herpesvirus helper genes delivered in plasmid DNA
form or via virus infection. Based on our experience, HeLa
cells yield the highest titers of rAAV per cell that we have
observed to date.

CONCLUSIONS

Multiple strategies are being developed for gene therapy
for muscular dystrophy. Clinically meaningful results are
anticipated through optimization of a vascular delivery
route using replacement, surrogate, or booster genes. Ad-
ditional studies are required to further define the immu-
nogenicity of AAV serotypes and transgenes and the po-
tential need for immunosuppression. Successful gene
therapy as a treatment for DMD is clearly on the hori-
zon, with the potential to improve the lives of patients
with this devastating disease.

Accepted for Publication: February 26, 2007.
Correspondence: Jerry R. Mendell, MD, Center for Gene
Therapy, Columbus Children’s Research Institute, 700 Chil-
dren’s Dr, Columbus, OH 43205 (mendellj@ccri.net).
Author Contributions: Study concept and design: Rodino-
Klapac, Chicoine, Kaspar, and Mendell. Acquisition of data:
Rodino-Klapac, Chicoine, Kaspar, and Mendell. Analysis
and interpretation of data: Rodino-Klapac, Chicoine, Kaspar,
and Mendell. Drafting of the manuscript: Rodino-Klapac,
Chicoine, Kaspar, and Mendell. Critical revision of the manu-
script for important intellectual content: Rodino-Klapac, Chi-
coine, Kaspar, and Mendell. Obtained funding: Kaspar and
Mendell. Administrative, technical, and material support:
Rodino-Klapac, Chicoine, Kaspar, and Mendell. Study su-
pervision: Chicoine, Kaspar, and Mendell.
Financial Disclosure: None reported.
Funding/Support: This study was supported by grant 5
U54 AR050733 from the Senator Paul D. Wellstone Mus-
cular Dystrophy Cooperative Research Centers, Na-
tional Institutes of Health.

Hindlimbs

Forelimbs
and torso

AAV1-GFP

AAV1-Follistatin

Figure 4. Muscle mass increases after recombinant adeno-associated virus
serotype 1 (rAAV1)–follistatin delivery.

␣-Sarcoglycan knockout animals

were injected with 1

⫻10

vector genomes of rAAV1-follistatin bilaterally

into the hamstring and gastrocnemius muscles at 3 weeks of age and
analyzed 13 weeks later. For control, AAV1-GFP (green fluorescent protein)
was used. Scale bars represent 10 cm.

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