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Reviews
MOLECULAR MEDICINE TODAY, JUNE 2000 (VOL. 6)
Towards a molecular therapy for
glycogen storage disease type II
(Pompe disease)
Yuan-Tsong Chen and Andrea Amalfitano
Glycogen storage disease type II (GSD-II), also known as Pompe disease, is a fatal genetic muscle
disorder caused by a deficiency of acid a-glucosidase, a glycogen-degrading lysosomal enzyme.
Currently, there is no treatment for this fatal disorder. However, several lines of research suggest the
possibility of future treatment. Enzyme replacement strategies hold the greatest hope for patients
currently affected by GSD-II, but future strategies could include in vivo or ex vivo gene therapy
approaches and/or mesenchymal stem cell or bone-marrow transplantation approaches. Each of the
approaches might eventually be combined to further improve the overall clinical efficacy of any one
treatment regimen. The lessons learned from GSD-II research will also benefit a great number of
individuals affected by other genetic disorders.
GLYCOGEN storage disease type II (GSD-II), also known as Pompe
disease or acid maltase deficiency (AMD), is an autosomal recessive
genetic disorder. GSD-II is heterogeneous in nature, with various
molecular defects in the lysosomal acid a-glucosidase (GAA) gene
resulting in partial or complete deficiency of GAA activity1–3. This enzyme defect results in lysosomal glycogen accumulation in almost all
tissues of the body, with cardiac and skeletal muscle the most seriously
affected.
GSD-II encompasses a range of clinical phenotypes, but myopathy
is common to all. The clinical phenotypes differ as to age of onset,
organ involvement and clinical severity. The most severe phenotype is
the infantile-onset form of GSD-II, originally described by J.C.
Pompe: affected infants can appear normal at birth, but soon develop
generalized muscle weakness with a ‘floppy baby’ appearance, feeding
difficulties, macroglossia, hepatomegaly and congestive heart failure
Yuan-Tsong Chen* MD, PhD
Professor and Chief
Andrea Amalfitano DO, PhD
Assistant Professor
Division of Medical Genetics, Department of Pediatrics, Duke
University Medical Center, Durham, NC 27710, USA.
Tel: 11 919 684 2036
Fax: 11 919 684 8944
*e-mail: [email protected]
caused by a rapidly progressive hypertrophic cardiomyopathy.
Death usually results from cardiorespiratory failure before the age of
one year.
The juvenile form of GSD-II typically presents as a delayed
achievement of motor milestones (if age of onset is early enough) or as
difficulty in walking in childhood. These physical signs are usually
followed by swallowing difficulties, proximal muscle weakness and
respiratory muscle involvement. Death from respiratory failure can
occur before the end of the second decade. Cardiomegaly is sometimes
observed, but overt cardiac failure does not usually occur.
The adult form of GSD-II presents as a slowly progressive myopathy, without cardiac involvement, and has its onset between the
second and seventh decades. The clinical picture is dominated by
slowly progressive proximal muscle weakness along with truncal involvement and greater involvement of the lower limbs. The most seriously affected muscle groups include the pelvic girdle, paraspinal and
diaphragm muscles. The initial symptoms in some patients can be
clinical signs of respiratory insufficiency, manifested by somnolence,
morning headache, orthopnea and exertional dyspnea.
In general, each of the GSD-II clinical phenotypes correlates with
residual GAA enzyme activities; the infantile form having less than
1%, juvenile 1–10%, and adult 10–40% of normal levels of GAA activity. The true incidence of all three forms of GSD-II has not been
thoroughly investigated, but estimates are in the range of 1:40 000–
100 000 live births4,5.
Previous attempts to treat GSD-II
At present, there is no effective treatment for GSD-II (reviewed in Ref.
1). Drugs such as epinephrine and glucagon, which enhance cytosolic
glycogen breakdown, have been tried without therapeutic effect6. This is
1357-4310/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1357-4310(00)01694-4
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MOLECULAR MEDICINE TODAY, JUNE 2000 (VOL. 6)
Plasma membrane
Endoplasmic reticulum
Glc I,
GlcII
ER
α-man
Golgi
α-Glucosidase
α1,2-Man
GnPT
Pathway
2
P
UCE
α1
,2
M
P
an
Lysosome
Pathway
1
GnT I
P
Man II
P
Acidified compartment
P
GnT II GalT/ST
P
pH
P
NH4Cl
P
P
P
Secretory
proteins
Secretory
granules
P
M-6-P
P
P
mediates appropriate muscle tissue-targeting
of GAA, and an insufficient supply of highly
purified enzyme. In one of the earliest clinical
applications of this method, GAA purified
from the fungus Aspergillus niger was administered to a patient with GSD-II (Ref. 6). After
three weeks of treatment, liver GAA activity
increased, and clearance of hepatic glycogen
storage vacuoles was demonstrated, but no effects were noted in the heart or skeletal muscles. Treatment was discontinued after four
months because of the development of an immune nephritis, and the patient died of cardiorespiratory insufficiency shortly afterwards.
Similar attempts at enzyme replacement therapy with GAA obtained from human placenta
have also been unsuccessful8. These early trials probably failed because mannose 6-phosphate (M-6-P) groups were not present in these
GAA preparations (see below).
Recent therapeutic approaches
Two approaches to the treatment of GSD-II
are currently being pursued: enzyme replacement and gene therapy. These are described
below.
Enzyme replacement therapy for
GSD-II
General considerations
The discovery of cell-surface receptors that
can mediate the delivery of lysosomal enzymes to target tissues renews the possibility
α1,2-Glc
α1,3-Man
β1,2-GlcNAc
of enzyme replacement for the treatment of
α1,3-Glc
α1,6-Man
β1,4-Gal
lysosomal storage diseases. We will start by
α1,2-Man
Manβ1,4-GlcNAc
α2,6-NeuNAc
reviewing the current understanding of lysoβ1,4-GlcNAc
somal-enzyme trafficking and receptor-mediated endocytosis.
Molecular Medicine Today
Lysosomal enzymes are synthesized in the
rough
endoplasmic reticulum, where a preFigure 1. Trafficking of lysosomal enzymes. The newly synthesized acid a-glucosidase that contains a highformed oligosaccharide is transferred from
mannose oligosaccharide is transferred from the endoplasmic reticulum to the Golgi network where it acquires
dolichol-phosphate to specific asparagines in
the mannose 6-phosphate (M-6-P) marker by the sequential action of N-acetylglucosaminylphosphotransferase (GnPT) and N-acetylglucosamine-1-phosphodiester a-N-acetylglucosaminidase, an uncovering enthe protein sequence (Fig. 1; reviewed in Refs
zyme (UCE) (pathway 2). The enzyme containing M-6-P residues then binds to M-6-P receptors and is trans9,10). The enzymes are then transferred to the
ported to the lysosome. Alternatively, it undergoes trimming of mannose, addition of sugars and formation of
Golgi apparatus where addition of M-6-P
a complex oligosaccharide and is secreted (pathway 1).
occurs via the sequential action of N-acetylglucosaminylphosphotransferase and N-acetylprobably because glycogen accumulation in the GSD-II patient is glucosamine-1-phosphodiester a-N-acetylglucosaminidase, the uncovprimarily located within lysosomes, whereas these drugs work in the ering enzyme. The modified enzyme that contains M-6-P then binds to
cytosol. A high-protein diet has also been tested, based on previous ev- M-6-P receptors on the Golgi network and is transported to the lysoidence that increased muscle catabolism occurs in GSD-II (Ref. 7). some. In addition, plasma membrane M-6-P receptors can also bind exThe high-protein diet seems to have some transient beneficial effects tracellular lysosomal enzymes and mediate their internalization and
in patients with juvenile or adult forms, but these interventions have localization to the lysosomes.
no effect in infantile patients1. Bone-marrow transplantation has also
It should be noted that not all lysosomal enzymes target lysosomes
been performed in GSD-II patients in an attempt to provide GAA via M-6-P receptors. For example, both GAA and b-glucocerebrosienzyme via secretion from donor cells. However, to date, these dase (the enzyme deficient in Gaucher disease) have M-6-P receptorinterventions have not been successful1.
independent pathways for lysosome targeting. Each of these enzymes
Previous attempts at enzyme-replacement for GSD-II were also can still be found in a number of organs, including liver, kidney, brain
unsuccessful, owing to an inadequate understanding of the route that and fibroblasts, in patients who are affected by I-cell disease, a genetic
P
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MOLECULAR MEDICINE TODAY, JUNE 2000 (VOL. 6)
condition resulting from a lack of effective phosphorylation of mannose
residues9,11. Furthermore, the plasma membrane is known to contain
carbohydrate receptors other than the M-6-P receptor such as the
asialoglycoprotein receptor (which can internalize glycoproteins containing galactose or N-acetylgalactosamine) and mannose receptors
(which bind mannose-exposed glycoproteins). The mannose receptor
(present in macrophages) is believed to be significantly involved
in the uptake of mannose-terminated b-glucocerebrosidase by the
macrophages of Gaucher disease patients12. These observations suggest that, in lysosomal storage diseases, the compatibility between the
carbohydrate chains of the administered enzyme and the type of receptors expressed on the plasma membrane of the target cells is critical for therapeutic success. Along these lines of reasoning, an effective
therapy is now available for Gaucher disease12,13, and some success
has been demonstrated in several types of mucopolysaccharidoses14,15,
as well as other lysosomal storage diseases, such as Fabry disease and
Niemann–Pick disease16,17.
The feasibility of using receptor-mediated endocytosis of GAA for
the replacement therapy of GSD-II was first demonstrated in pre-clinical studies18. When normal mice were treated with GAA containing M6-P (purified from bovine testis), the GAA was taken up by the heart
and skeletal muscles. Uptake of GAA was less evident with placentaderived GAA, a form of the enzyme that lacks the M-6-P residues.
Although these studies demonstrated the feasibility of using appropriately glycosylated GAA to exploit receptor-mediated uptake into target
tissues, it is clear that interspecies antigenicity of bovine GAA precludes the use of this source of enzyme in human therapy. A nonantigenic, human form of enzyme is required.
Recombinant enzyme therapy
Recombinant human acid a-glucosidase (rhGAA) has recently been
produced in its precursor form in Chinese hamster ovary (CHO) cell
lines19,20, and in transgenic mouse and rabbit milk21. Incubation of the
precursor form of rhGAA with primary fibroblasts derived from patients with infantile-onset GSD-II resulted in the uptake of the enzyme
and normalization of GAA activity and glycogen levels in the fibroblasts19,20. This uptake is inhibited by M-6-P, suggesting mediation of
uptake by M-6-P receptors. Injection of purified rhGAA into guinea
pigs resulted in increased GAA activity levels in liver, heart and skeletal muscle19. To study in vivo efficacy, however, an appropriate animal
model of GSD-II was required.
Naturally occurring GAA deficiency has been reported in Shorthorn
cattle, Brahman cattle and a strain of Japanese quail. Recently, mouse
models generated by targeted gene disruption have also become available22,23. The Japanese quail with acid-maltase deficiency (referred to
as the AMD quail) is a pertinent and practical animal model for the
study of GSD-II because of a readily demonstrable clinical phenotype
(Fig. 2). The disease in the AMD quail resembles the juvenile and
adult-onset forms of human GSD-II with its later onset, the presence of
residual GAA activity and characteristic histological changes noted in
affected muscles24.
AMD quail exhibit a progressive myopathy and cannot fly, lift their
wings or right themselves from the supine position (Fig. 2). Treatment
of symptomatic quail at four weeks of age with intravenous rhGAA
(14 mg kg21) every two to three days for 18 days (a total of seven injections) resulted in the ability of the quail to both right themselves
and flap their wings25. GAA activity also increased in most tissues examined. In heart and liver, glycogen levels and histopathology were
restored to normal (Fig. 3). In the pectoralis muscle, morphology was
Reviews
essentially normal except for above-normal numbers of glycogen
granules. In contrast, the muscles of control-treated AMD quail had
markedly increased glycogen granules, multivesicular autophagosomes, as well as interfasicular and intrafasicular fatty infiltrations
(Fig. 3). AMD quail that were treated with a lower dose of rhGAA (4.2
mg kg21) also demonstrated biochemical and histopathological improvements, but these were less dramatic than those noted in their
high-dose counterparts, indicating a dose response to rhGAA. An intermediate dose used in an extended treatment experiment (45 days)
also halted the progression of the disease. At present, we do not know
the exact mechanism by which exogenous rhGAA improves clinical
symptoms and histopathology. It is likely from the in vitro fibroblast
study that rhGAA is taken up by muscle cells via M-6-P receptor-mediated endocytosis. However, it is also possible that uptake of rhGAA
by muscle is mediated by other carbohydrate receptors.
Similarly, rhGAA purified from the milk of transgenic mice and
rabbits has been shown to correct GAA deficiency and reduce glycogen storage in heart and skeletal muscle of GSD-II knockout mice21,26.
The dose of the milk enzyme required to correct the enzyme deficiency
as well as the glycogen storage in the mouse model was larger than the
dose of the CHO-derived enzyme that had been used in the quail
model. The duration of the treatment required to see a therapeutic effect with the milk enzyme was also longer than with the CHO enzyme26. Furthermore, no clinical improvement was seen in the mouse
model following treatment with the milk-derived enzyme.
The observation that rhGAA improves muscle strength (in the quail
model) and histopathology and biochemical parameters (in both quail
and mouse models) suggests that rhGAA is a promising enzyme replacement therapy for human GSD-II. Based on these results, two
clinical trials using different enzyme sources have recently been initiated to investigate the potential of rhGAA to safely treat GSD-II patients. A Phase II study is being conducted in the Netherlands (Sophia’s
Children Hospital, Rotterdam) using GAA purified from the milk of
transgenic rabbits, and a Phase I/II study using rhGAA purified from
CHO cells is being conducted in the USA (Duke University Medical
Center, Durham, NC).
Although rhGAA could offer a promising therapy for GSD-II, two
potential problems can be foreseen. First, will the correction of the
Figure 2. Flip test. When placed on their backs, the normal quail (left) rights
itself immediately, whereas a quail with GSD-II (right) cannot right itself and
remains in the supine position. Courtesy of Dr Tateki Kikuchi.
247
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MOLECULAR MEDICINE TODAY, JUNE 2000 (VOL. 6)
Gene therapy for GSD-II
General considerations
Figure 3. Pectoralis muscle (a–c), liver (d–f) and heart (g–i) sections stained
with periodic acid-Schiff (PAS) from normal (a, d and g), AMD-control treated
(b, e and h) and AMD high-dose rhGAA treated (c, f and i) quail. Scale bar 5
50 mm. Reproduced with permission from Ref. 25.
muscle symptoms of GSD-II (over time) unmask neurologic symptoms in treated patients? This possibility exists because intravenously
administered rhGAA is unlikely to cross the blood–brain barrier, and
excessive glycogen is known to be stored in the nervous tissues,
particularly in infantile patients. Second, will antibodies develop
against the rhGAA and interfere with the effectiveness of the therapy?
The immune response is more likely to develop in the infantile patients as many of them carry a null mutation. In contrast, missense
mutations resulting in residual enzyme activity in juvenile and adult
patients might render these late-onset patients more immunologically
tolerant.
Glossary
Dyspnea – Shortness of breath.
Gaucher disease – A lysosomal storage disease resulting from a
deficiency of glucocerebrosidase, an enzyme that breakdowns cellular glycolipids.
Hepatomegaly – Enlargement of liver.
Hypertrophic cardiomyopathy – Enlargement of heart due to increased thickness of the heart wall.
Macroglossia – Enlargement of tongue.
Mannose 6-phosphate receptor – A cell receptor that recognizes
molecules containing mannose 6-phosphate.
Orthopnea – Discomfort of breathing that is relieved by reverting
to a sitting or standing position.
248
The specific cause of GSD-II is mutations within the GAA gene that
result in either lack of adequate transcription of the gene, or the expression and translation of a significantly altered GAA protein. Lack
of adequate GAA activity results in the abnormal accumulation of
glycogen, and its detrimental effects in muscle cells. The ultimate cure
for GSD-II would be to correct the responsible mutations in all of the
cells of an affected individual. However, this form of genetic therapy
is currently unachievable, and alternative modes of gene correction are
being investigated not only for GSD-II specifically, but also for genetic
diseases in general. Recently, it has been demonstrated that utilization
of RNA–DNA chimeric oligonucleotides can specifically target and
repair (via DNA mismatch repair mechanisms) specific one or two
base pair alterations in the genomes of mammalian cells. The most dramatic results have been achieved in hepatocytes (both in vitro and in
vivo), although confirmation of the results by other laboratories at this
time is still lacking27. Direct genetic correction of DNA mutations
causing GSD-II has not been investigated. It is also not clear whether
the primary target cells for such an intervention in GSD-II patients
(skeletal and cardiac muscle cells) are amenable to the mismatch repair
mechanisms noted in hepatocytes.
Gene therapy and ‘augmenting’ GAA activity
As the direct genetic correction of mutant DNA is still in its infancy, the
next level of genetic therapy would be the addition of normal GAA encoding sequences into cells that are deficient for GAA activity. This
‘augmentative’ form of gene therapy holds the most pragmatic view at
present for potential genetic therapies of GSD-II. In order to ‘add
back’ the genetic information encoding GAA to cells that lack GAA
activity, a vector is required to deliver the necessary information.
Ex vivo approaches
A number of intriguing studies have been performed to investigate several potential modalities for the gene therapy of GSD-II. In one example,
a viral vector might be used to transfer the GAA gene into fibroblasts and
myoblasts isolated directly from individuals affected by GSD-II. It is
hoped that, in this form of gene therapy, myoblasts from GSD-II patients
could be infected with the vector (transduced) ex vivo, then implanted
back into the muscles of the affected individuals. The corrected myoblasts would then either directly fuse with neighboring cells (supplying
them with GAA enzyme) and/or secrete the enzyme for uptake by other
cells not in direct contact with the genetically corrected cells.
Zaretsky and colleagues demonstrated that myoblasts transduced
with a GAA-encoding retrovirus were capable of expressing large
amounts of GAA; 30-fold higher than the levels found in myoblasts
from unaffected individuals28. Furthermore, the retrovirally encoded
GAA protein was capable of targeting lysosomes and reducing the
amount of accumulated glycogen in treated cells. The retrovirally
transduced cells were also capable of secreting the GAA enzyme,
allowing cross-correction of non-transduced GSD-II myoblasts in
tissue-culture systems28. Nicolino and colleagues further demonstrated
that a first-generation adenovirus (Ad) vector was also capable of
transducing the GAA gene into fibroblasts and muscle cells isolated
from affected patients29. The adenovirus-transduced cells were phenotypically corrected, and were also capable of secreting the recombinant GAA enzyme. However, further studies addressing the efficacy of
ex vivo myoblast therapy in animal models of GSD-II have yet to be
demonstrated.
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MOLECULAR MEDICINE TODAY, JUNE 2000 (VOL. 6)
Both studies highlight the potential of ex vivo gene therapy for GSDII, but other studies have also demonstrated the known limitations of
myoblast transfer for use in humans. These limitations include: (1) the
inherent difficulties of growing large quantities of cultured myoblasts;
(2) the decreased ability of cultured human myoblasts to fuse after implantation in vivo; and (3) whether uncertainty over enzyme production
from successfully implanted myoblasts would be capable of cross-correcting all of the muscles in an affected individual. Clinical studies of
myoblast transfer in patients affected by Duchenne muscular dystrophy
demonstrated that in vivo fusion of previously cultured human myoblasts can occur, but at a very low efficiency30. Ex vivo genetic correction of affected myoblasts is a promising approach, but further studies
will be required before myoblast transfer achieves clinical reality.
In vivo approaches
An optimum gene-transfer vector would be capable of transducing the
GAA gene into all of the affected cells of a patient after a simple direct
administration, as well as allowing for long-term expression of the
GAA protein either to prevent worsening of disease, or to effect a cure.
At present, no one vector can achieve all of these lofty goals; however,
several classes of vector have shown promise. DNA transfer mediated
by non-viral vectors in vivo is generally an inefficient process, although
skeletal muscles appear to be capable of uptake and expression of DNA
after a simple intramuscular injection31. Unfortunately, gene expression
is limited only to those muscle fibers close to the injection site. It has yet
to be demonstrated whether direct DNA injection of the GAA gene
would result in adequate expression (and secretion) of the GAA protein
to allow for cross-correction of non-injected muscle groups.
Direct intramuscular injection of viral vectors encoding GAA has
also been investigated. Pauly and colleagues demonstrated that direct intramuscular injection of a first-generation adenoviral vector (encoding
human GAA) into the cardiac or hind-limb muscles of normal neonatal
rats resulted in over-expression of GAA activity in each of the injected
tissues32. However, systemic secretion of GAA was not demonstrated.
Tsujino et al. confirmed that adenoviral vectors were also capable
of transducing the human GAA gene after direct injection into the
pectoralis muscles of the AMD quail model of GSD-II (Ref. 33).
Importantly, the latter studies confirmed that the in vivo delivery of the
GAA gene could result in adequate enzyme expression, as well as reduce
glycogen accumulation in the treated muscle groups33. However, crosscorrection of other muscle groups was not achieved in either study.
These important investigations demonstrated the potential of in vivo
approaches, but also highlighted their limitations, especially the inability to correct a significant number of muscle cells beyond the
vicinity of the local injection site. Surgically invasive techniques such
as the vascular isolation of a limb, followed by the re-circulation of a
vector-containing solution, allow for more dispersed transduction of
muscle cells by viral vectors34. The latter techniques are highly experimental, but with further investigation might allow an increased number of muscle cells to be transduced with hGAA-encoding vectors.
Furthermore, the targeting of specific muscle groups (for example the
intra-coronary delivery of a vector for cardiac transduction) might improve the function of the muscles primarily causing morbidity in some
forms of GSD-II (Ref. 35).
Hepatic gene therapy for a systemic muscle disorder
In light of the previously discussed limitations, our group is developing alternative strategies for the genetic therapy of a systemic muscle
disorder such as GSD-II. The conceptualization of one such approach
is outlined in Fig. 4. This approach capitalizes on the known propensity of adenoviral vectors to efficiently transduce hepatocytes after a
simple intravenous administration, rather than targeting muscle cells directly36. As the liver is a secretory organ, we hypothesized that the synthetic machinery of the liver could be harnessed to produce and secrete
large amounts of the GAA protein into the bloodstream of affected
individuals. Once secreted, the enzyme could be systemically distributed, resulting in the simultaneous cross-correction of major muscle
groups, without having to directly inject the gene transfer vector into
each of the respective muscles.
We recently confirmed that intravenous administration of a new
class of modified adenoviral vector allowed for extremely efficient
transduction of the hepatocytes of mice, including GAA-knockout
(GAA-KO) mice36,37. Specifically, the livers of adenovirally treated
GAA-KO mice expressed nearly 100-fold more GAA enzyme than is
normally detected in the tissues of wild-type mice. Furthermore, the
overexpression resulted in secretion of the precursor form of the enzyme into the bloodstreams of these mice, which was then subsequently taken up and targeted to the lysosomes of multiple muscle
groups. Within 12 days of a single injection, the heart, diaphragm,
quadriceps and gastrocnemius (calf) muscles were all essentially
cleared of their pathologic glycogen accumulations, as compared with
untreated, age-matched control animals37. Recently, other studies in
animal models of Fabry disease have also confirmed that lysosomal
storage disorders (other than GSD-II) can also benefit from adenovirus-mediated transduction of the liver, with subsequent secretion and
targeting of lysosomal enzymes to other tissues38.
Future directions
At present, enzyme replacement strategies hold the greatest hope for
patients currently affected by GSD-II, but future strategies might include in vivo or ex vivo gene therapy, or mesenchymal-stem-cell bonemarrow transplantation approaches. Currently, there are two ongoing
clinical trials of rhGAA replacement therapy in GSD-II (see above). No
efficacy data are yet available, but the pre-clinical data suggest that enzyme replacement therapy will be successful. However, there remain
challenges, particularly for the infantile patients. Extensive muscle
damage at the time of diagnosis might be beyond repair, and rapid progression of the disease (patients die, on average, three months after
diagnosis) might not allow sufficient time for muscle regeneration. A
successful treatment might unmask the underlying neurologic problem. Furthermore, immune responses could limit the efficacy of the
treatments. These potential problems might also be encountered with
gene therapy approaches.
For gene therapy, the liver secretion of lysosomal enzymes such as
the GAA protein for GSD-II offers much potential for the therapy of
GSD-II disease specifically, and a number of other lysosomal disorders in general. Critical questions that have yet to be addressed include
whether or not removal of glycogen actually results in improved muscle function in the treated animals, and how long the therapeutic effects
can persist. The latter point is a complex matter that depends not only
on duration of expression and secretion of the GAA gene from the liver,
but also on the lysosomal half-life of GAA in muscle cells, and the kinetics of re-accumulation of glycogen in muscle cells. In addition, immune responses (specifically, neutralizing antibody to the secreted
GAA, and/or cytotoxic T-cell mediated elimination of adenovirus infected hepatocytes) might limit the overall efficacy of this approach. The
use of modified vectors and/or other gene-transfer vectors (adenoassociated virus or lentivirus based vectors) might avoid these problems.
249
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MOLECULAR MEDICINE TODAY, JUNE 2000 (VOL. 6)
The outstanding questions
Muscle
cell
AdhGAA
Dpol
mechanisms are involved in muscle targeting of
• What
hGAA enzyme by either enzyme replacement therapy or
Hepatocyte
(a)
Lysosome
•
(b)
Nucleus
(c)
(e)
(e)
Lysosome
(d)
(f)
(g)
= hGAA receptor/transporter
(c)
Nucleus
(d)
= Processed (active) form of hGAA
= Precursor form of hGAA
Molecular Medicine Today
Figure 4. Hepatocyte production of hGAA in vivo. The diagram illustrates the important steps required to achieve systemic distribution and uptake of hepatically
produced GAA enzyme in hepatocytes or muscle cells. (a) Adenovirus entry into
the hepatocyte after intravenous administration. (b) Human GAA mRNA transcription/translation. (c) Binding of precursor GAA by mannose-6-phosphate
receptor. (d) Receptor mediated targeting of hGAA to lysosomal compartment.
(e) Cleavage of precursor form of hGAA upon entry into lysosome, generating
enzymatically active GAA. (f) Secretion of precursor hGAA from hepatocyte.
(g) Systemic distribution of precursor hGAA via the bloodstream.
The usefulness of alternative vectors will, however, be dictated by
whether or not the vector can transduce enough cells, or secrete
enough GAA protein, to allow for the systemic cross-correction of
multiple muscle groups.
Bone-marrow transplantation has not been successful. However, recent data suggested that bone-marrow cells can participate in the repair
process of muscle39,40, and donor cells have been found to be incorporated into mdx (muscular dystrophy, X-linked) mouse muscles following transplantation of bone-marrow-derived mesenchymal stem cells
from normal mice41. It is possible that bone marrow transplantation in
the modern era might be more effective for GSD-II.
References
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maltase) deficiency. In The Metabolic and Molecular Basis of Inherited Disease
(Scriver, C.R. et al., eds), pp. 2443–2464, McGraw–Hill
02 Reuser, A.J.J. et al. (1995) Glycogenosis type II (acid maltase deficiency). Muscle
Nerve (Suppl.) 3, S61–S69
03 Raben, N. et al. (1995) Genetic defects in patients with glycogenosis type II (acid
maltase deficiency). Muscle Nerve 3, 570–574
250
•
•
•
hepatic gene therapy? Is the mannose 6-phosphate
receptor primarily involved, or do other carbohydrate
receptors and/or other mechanisms play a significant role?
Can existing muscular damage be cured, or will current
approaches only prevent further damage? How much
muscle regeneration can be expected in the various forms
of GSD-II?
Will the correction of the muscular symptoms of GSD-II
reveal underlying neurological involvement in treated
patients?
Will immune responses interfere with the effectiveness of
the therapies?
Is the human liver as amenable to viral-mediated gene
transfer as some animal species’ livers appear to be?
04 Martiniuk, F. et al. (1998) Carrier frequency for glycogen storage disease type II in
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05 Ausems, M.G. et al. (1999) Frequency of glycogen storage disease type II in the
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08 de Barsy, T. et al. (1973) Enzyme replacement in Pompe disease: an attempt with
purified human acid α-glucosidase. Birth Defects Orig. Art. Ser. 9, 184–190
09 Sabatini, D. and Adesnik, M.B. (1995) The biogenesis of membranes and organelles.
In The Metabolic and Molecular Basis of Inherited Disease (Scriver, C.R. et al.,
eds), pp. 459–553, McGraw–Hill
10 Kornfeld, S. (1992) Structure and function of the mannose 6-phosphate/insulin-like
growth factor II receptors. Annu. Rev. Biochem. 61, 307–330
11 Tsuji, A. et al. (1988) Intracellular transport of acid alpha-glucosidase in human
fibroblasts: Evidence for involvement of phosphomannosyl receptor-independent
system. J. Biochem. 104, 276–278
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Past Review articles covering aspects of gene therapy in
Molecular Medicine Today
Genes influencing HDL metabolism: new perspectives and implications for atherosclerosis prevention
by Daniel J. Rader and Cyrille Maugeais
Gene therapy strategies for colon cancer
by Guy A. Chung-Faye, David J. Kerr, Lawrence S. Young and Peter F. Searle
RNA viruses: emerging vectors for vaccination and gene therapy
by Roger Hewson
The HDL receptor SR-BI: a new therapeutic target for atherosclerosis
by Susan L. Acton, Karen F. Kozarsky and Attilio Rigott
Gene therapy strategies to facilitate organ transplantation
by Shaoping Deng and Kenneth L. Brayman
Gene therapy using HVJ-liposomes: the best of both worlds?
Yasufumi Kaneda, Yoshinaga Saeki and Ryuichi Morishita
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