Powell Gene Therapy Center, and Departments of Molecular Genetics and Microbiology and Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32610, USA
* Author for correspondence (e-mail: bbyrne{at}ufl.edu)
Accepted 15 March 2004
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SUMMARY |
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Key words: In utero, rAAV, Gene therapy, GSDII, Acid alpha-glucosidase, Diaphragm
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Introduction |
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Glycogen storage disease type II (GSDII) or Pompe disease is a rare
autosomal recessive disorder caused by a defect in the gene encoding acid
-glucosidase (GAA) (Hirschhorn and
Reuser, 2000
; Raben et al.,
2002
). In the lysosome, the 110 kDa precursor form of GAA is
successively cleaved at both termini to produce 95, 76 and 67 kDa peptides of
which the 76 and 67 kDa isoforms are catalytically active
(Hoefsloot et al., 1990
;
Wisselaar et al., 1993
). GAA
reduces lysosomal glycogen and maltose to glucose by hydrolyzing
-1,4
and
-1,6 linkages at acid pH. In the absence of GAA, glycogen
accumulates within the lysosomes and cytoplasm of all cells. Pathophysiology
of the disease is limited to striated muscle, where accumulated glycogen
causes disruption of the contractile apparatus, eventually leading to muscle
weakness (Hers, 1963
;
Pompe, 1932
). The age of
onset, as well as the severity and progression of muscle weakness, is usually
dependent on the level of residual GAA activity. Mutations leading to no
detectable active GAA protein cause infantile-onset GSDII. This form of the
disease manifests as severe cardiac and skeletal myopathies, resulting in
death before 1 or 2 years of age due to cardiorespiratory failure. Individuals
diagnosed with late-onset GSDII typically have residual levels of GAA
activity. These patients show less cardiac involvement, and instead suffer
from respiratory and proximal muscle weakness with death eventually resulting
from respiratory insufficiency (Hirschhorn
and Reuser, 2000
).
The knockout mouse model used in these studies was produced by interrupting
exon 6 of the mouse GAA cDNA with a neomycin cassette
(Raben et al., 1998). In this
mouse model, glycogen is found highly concentrated in heart, skeletal muscle,
diaphragm and liver as early as 3 weeks of age. By 18 months, the mice exhibit
extreme muscle wasting, resulting in kyphosis, anterior and posterior
weakness, and difficulty breathing (Raben
et al., 1998
; Raben et al.,
2000
).
Currently, there is no widely accessible treatment for GSDII. Enzyme
replacement therapy (ERT) has progressed significantly in the treatment of
lysosomal storage disorders and is currently being assessed for the treatment
of GSDII in a Phase I/II clinical trial
(http://clinicaltrials.gov/ct/show/NCT00053573)
(Amalfitano et al., 2001;
Eng et al., 2001
;
Kakkis et al., 2001
;
Van den Hout et al., 2001
).
The premise of ERT is based on the uptake of circulating enzyme through
mannose 6-phosphate receptor-mediated endocytosis.
We, and others, have shown the potential of viral vectors in treating GSDII
(Chen and Amalfitano, 2000).
Direct intramuscular, intracardiac or portal vein injections of
replication-defective, E1-deleted adenoviral vectors containing the human
GAA cDNA (rAd-GAA) into animal models of the disease
resulted in production of therapeutic levels of GAA and corresponding
reduction of intracellular glycogen stores
(Amalfitano et al., 1999
;
Ding et al., 2001
;
Ding et al., 2002
;
Martin-Touaux et al., 2002
;
Pauly et al., 1998
;
Pauly et al., 2001
;
Tsujino et al., 1998
).
Cross-correction resulting from uptake of secreted enzyme by distant organs,
such as heart and skeletal muscle, was achieved through high-level
rAd-GAA transduction of the liver in GAA-deficient animals.
Regrettably, anti-GAA antibody titers increased, resulting in a 100-fold
reduction in the initial level of GAA enzyme
(Ding et al., 2002
). Clinical
applications of gene replacement strategies using recombinant adenoviral
vectors are currently limited because of toxicity and transient expression due
to host immune responses.
A newer class of gene therapy vectors, based on the adeno-associated virus
(AAV), has become a popular vehicle for delivering genes to cells for the
treatment of several diseases. Two recent studies demonstrated the use of
recombinant AAV vectors encoding the human GAA cDNA for gene
replacement in deficient human cells and in two animal models
(Fraites et al., 2002;
Lin et al., 2002
). Lin et al.
injected rAAV directly into the pectoral muscle of the Japanese quail model
and demonstrated expression of GAA, correlating with a decrease in
intracellular glycogen content and an increase in muscle performance as
measured by wing motion (Lin et al.,
2002
). Our group showed the effectiveness of intramyocardial and
intramuscular delivery of a similar vector to the exon 6-knockout mouse model
of GSDII (Fraites et al.,
2002
). Both delivery methods established that near normal levels
of GAA activity could be achieved, and, in the case of the direct
intramuscular injections, the increase in GAA activity correlated with an
increase in muscle function.
Our objective was to deliver rAAV vectors encoding human GAA to
the developing mouse model of GSDII in an effort to prevent the disease state
in target organs. However, there is very limited information on murine in
utero gene delivery. The first murine in utero vector transduction experiments
were conducted using recombinant adenoviral vectors. Pioneer studies involved
using adenovirus to deliver the lacZ, CFTR or Factor IX gene into the
amniotic fluid of fetal Cftr-/- mice at 15 days
post-coitum (pc), three-quarters of the way through the gestation period.
Expression of the transgene was detected in the liver, epidermis, lung and
gastrointestinal tract (Cohen et al.,
1998; Douar et al.,
1997
; Holzinger et al.,
1995
; Larson et al.,
1997
; Schneider et al.,
1999
). Several in utero CFTR-treated knockout mice were
rescued from lethal intestinal obstruction
(Cohen et al., 1998
). Other
studies using adenovirus were directed toward liver transduction via
intrahepatic, intraperitoneal, yolk sac vessel or retro-orbital injections to
midgestation mice. These studies involved the delivery of the luciferase or
lacZ reporter gene under transcriptional control of the
cytomegalovirus (CMV) promoter (Lipshutz
et al., 1999a
; Lipshutz et
al., 1999b
; Lipshutz et al.,
1999c
; Schachtner et al.,
1999
). When the animals reached a month of age, only minimal
transgene expression was detected after a gradual decline in activity
(Lipshutz et al., 1999b
).
Other gene therapy studies in adult mice indicated that CMV transcriptional
activity was inactivated a few weeks after initial expression
(Loser et al., 1998
). It is
not known whether expression in the liver after in utero vector delivery
decreases because of promoter inactivation, because of an immunologic response
to adenoviral transduction, or because of a dilution of vector genomes as the
cells of the liver divide during fetal development.
Even less is known about in utero delivery of AAV to the developing murine
fetus. To date, three published manuscripts and a handful of abstracts address
this issue. Similar CMV-lacZ or CMV-luciferase expression cassettes
were used in the context of rAAV. In cases where the vector was delivered
intrahepatically or intraperitoneally, expression was detected mainly in the
liver or the luminal wall of the peritoneal cavity, respectively. This
activity dropped significantly over time
(Lipshutz et al., 2000;
Schneider et al., 2002
).
However, studies involving intramuscular injections of vector containing
CMV-driven lacZ or human factor IX expression cassettes indicated
that the level of transgene expression was maintained throughout the course of
the experiment (Mitchell et al.,
2000
; Schneider et al.,
2002
).
We treated Gaa-/- mice in utero with a genetic therapy
that would prevent glycogen accumulation and maintain normal muscle function.
We used in utero delivery of recombinant AAV to introduce the human
GAA cDNA into Gaa-/- diaphragm muscle at an early
stage in development. Active GAA protein was produced in the diaphragm and
prevented glycogen from accumulating and causing long-term irreversible
damage. This method of in utero vector delivery could be extended to other
knockout models such as Mtpa-/-,
Serca1-/- and Cypher-/-, where
expression of the normal gene product specifically in the diaphragm would
permit the mice to survive the neonatal stage and allow investigators to study
the disease pathology in other muscle types
(Ibdah et al., 2001;
Pan et al., 2003
;
Zhou et al., 2001
).
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Materials and methods |
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In utero viral delivery
Animal procedures were performed in accordance with the guidelines of the
University of Florida Institutional Animal Care and Use Committee. On day 15
of gestation, pregnant females were anesthetized using 0.03 mL/gm total body
weight of 20 mg/mL Avertin (tribromoethanol in tert-amyl alcohol diluted in
PBS) administered intraperitoneally. A midline laparotomy was performed on
each pregnant female, with the abdominal wall being retracted to expose the
peritoneal cavity. Each horn of the uterus was exposed individually onto a
pre-warmed saline-moistened sponge and up to 10 µL of saline, beads or
virus was injected into each fetus. A preloaded Hamilton syringe, bearing a
33-gauge needle with a beveled end and side pore (Hamilton Company, Reno, NV),
was inserted through the uterine wall into the fetal liver or peritoneal
cavity. After the injections, the abdominal muscle layer was sewn using 5-0
prolene and the skin layer was closed using 5-0 vicryl. Ampicillin (2.4
µL/gm body weight of 0.1 g/mL stock) and Buprenorphine (0.1 mg/kg) were
administered after the surgery to control infection and pain. Mothers were
monitored until they regained consciousness, after which they were returned to
the colony and permitted to proceed with the pregnancy. Newborn pups were kept
with their mothers for 1 month before weaning.
Perfusion, necropsy, histology and electron microscopy
After animals were perfused with PBS to remove excess blood, organs were
successively removed from the animal using sterile surgical utensils, first
beginning with skeletal muscle removed from lower extremities, then gonad,
spleen, kidney, liver, diaphragm, lung, heart, tongue and brain. The tissues
were snap frozen in liquid nitrogen and stored at -80°C until analyzed by
activity assays, western analysis and rAAV genome copy number.
Tissues isolated for electron microscopy and histology were taken after first perfusing the mice with PBS for 5 minutes, followed by 5 minutes of fixative (2% paraformaldehyde/1% glutaraldehyde in PBS, pH 7.4). Skeletal muscle, liver, diaphragm and heart were removed, dissected into very small cubes and stored overnight in 2% glutaraldehyde. They were rinsed in 0.1 M sodium cacodylate buffer and incubated at 4°C in 2% osmium tetroxide in cacodylate buffer for 1 hour. They were then rinsed twice in cacodylate buffer, dehydrated in a series of graded alcohol solutions, rinsed in 100% propylene oxide and embedded in TAAB resin (Marivac, Halifax, Canada). All other reagents were purchased from Electron Microsopy Sciences (Fort Washington, PA). Thick sections (1 µm) were stained with Schiff's reagent, followed by Toluidine Blue, and photographed using light microscopy. Thin sections (0.1 µm) were stained with lead citrate and uranyl acetate, and photographed with a Zeiss EM10 transmission electron microscope at 80 kV.
Biochemical assays
Luciferase expression assay
The Luciferase Assay System (Promega, Madison, WI) was used to quantify the
expression of luciferase. The samples were prepared by homogenization in 300
µL of water. Then 20 µL of the supernatant, and 100 µL of luciferase
assay substrate diluted in assay buffer, was added to a glass test tube and
incubated at room temperature for 20 minutes. The intensity of light emitted
from the reaction was detected using the Monolight® 2010 luminometer (BD
Biosciences, Mississauga, ON). Luciferase expression was reported as relative
light units per µg protein, as determined by DC Protein Assay
(Bio-Rad, Hercules, CA).
Acid -glucosidase activity assay
GAA naturally cleaves the 1,4 bond of glycogen, and in this
fluorimetric assay converts synthetic substrate
4-methylumbelliferyl-
-D-glucopyranoside (4-MUG; Calbiochem-Novabiochem,
San Diego, CA) to 4-methylumbelliferone (4-MU) and glucose. Snap frozen
tissues were homogenized in water, and cell pellets were resuspended in water
and lysed by three freeze/thaw cycles. Lysates were centrifuged and 20 µl
of clarified supernatant was added to each well in triplicate of a black
96-well plate. The reaction was initiated by the addition of 40 µl of
substrate solution [2.2 mM 4-MUG in 0.2 M sodium acetate (pH 3.6)] and was
incubated for 1 hour at 37°C before the reaction was stopped with 200
µl of 0.5 M sodium carbonate (pH 10.7). Fluorescence (360 nm/460 nm) was
then measured using an FLx800 Microplate Fluorescence Reader (Biotek
Instruments, Winooski, VT). GAA specific activity was quantified in nmoles of
substrate hydrolyzed (nmoles 4-MUG/hr/mg protein), based on a standard curve
of 4-MU concentrations and standardized by protein concentration determination
by DC Protein Assay (Bio-Rad, Hercules, CA).
Acid -glucosidase staining of tissues
GAA was detected in fixed tissues by cytochemical staining using the
synthetic substrate 5-bromo-4-chloro-3-indolyl--D-glucopyranoside
(X-Gluc; Calbiochem-Novabiochem, San Diego, CA), which when cleaved releases a
blue product. After washing with PBS, X-Gluc stain (0.25 mM potassium
ferricyanide, 0.25 mM potassium ferrocyanide, 1 mM magnesium chloride, 1 mg/mL
X-Gluc in PBS reduced to pH 3.6) was added and the samples incubated at room
temperature overnight. The tissues were photographed using a digital camera
attached to a dissecting microscope.
Western blotting
Rabbit polyclonal antiserum was raised against placentally derived human
GAA as previously described (Pauly et al.,
1998). The antiserum was used for western blotting to detect hGAA
protein. A total of 5 µg of protein from tissue homogenates was applied to
Novex® 8% Tris-Glycine gels (Invitrogen Life Technologies, Carlsbad, CA)
and separated at 125 V for approximately 2 hours. After transfer to
nitrocellulose filters, blots were probed with a 1:1000 dilution of primary
antibody followed by a 1:5000 dilution of peroxidase-labeled anti-rabbit IgG,
and detected using the ECL+Plus chemiluminescence kit (Amersham Biosciences,
Piscataway, NJ). Human placental GAA was included on each blot as a positive
control.
Quantification of genome copies by quantitative-competitive PCR
Competitor plasmid construct, p43.2-hGAA2.8-5'del, was
created in which approximately 350 nucleotides from the 5' end of the
GAA gene were removed. The 5' primer
(5'-GCTAGCCTCGAGAATTC-3') was located in the multiple cloning site
after the CMV promoter of p43.2, and the 3' primer
(5'-CGGTTCTCAGTCTCCATCAT-3') was positioned beginning at
nucleotide 514 of the hGAA coding sequence. These primers were
designed to amplify 595 nucleotides of rAAV-CMV-hGAA genomic DNA and
239 bp of the p43.2-hGAA2.8-5'del competitor template.
Total DNA was isolated from snap-frozen specimens using the DNeasy® tissue kit (Qiagen, Valencia, CA). An RNase digestion step was included to remove any mRNA species that may contaminate the QC-PCR. Reactions were arranged by adding 200 ng of total DNA, competitor plasmid DNA (ranging from 0 to 108 copies), 20 pmol of each primer, and water to Ready-To-GoTM PCR beads (Amersham Biosciences, Piscataway, NJ). The reaction contained 1.5 mM MgCl in a total volume of 25 µL according to the manufacturer's suggestions. Samples were subjected to 30 cycles of denaturation at 95°C for 30s seconds, annealing at 60°C for 30 seconds, and elongation at 72°C for 30 seconds, using a RoboCycler® Gradient 96 thermocycler (Stratagene, La Jolla, CA).
QC-PCR samples were separated on a 2% agarose gel and photographed using the Eagle EyeTM II imaging system (Stratagene, La Jolla, CA). The amplified products were quantified using ImagequantTM software (Amersham Biosciences, Piscataway, NJ). Intensities of products from amplified genomic rAAV-CMV-hGAA and competitor plasmid DNA were plotted on the same graph using SigmaPlot 2001 software (SPSS, Chicago, IL). The point where both lines crossed was considered the point of equal amplification. Given that the amount of competitor and sample template is equal at this point, we approximated the number of vector genome copies present in the sample. Data were reported as vector genome copies/diploid cell after converting from vector genome copies/200 ng DNA using a conversion factor of 5 pg DNA/diploid nucleus.
In vitro assessment of diaphragm contractile function
Mice were anesthetized via intraperitoneal (IP) injection of sodium
pentobarbital (65 mg/kg). After reaching a surgical plane of anesthesia, the
diaphragm was surgically excised, with the ribs and central tendon attached,
and placed in a cooled dissecting chamber containing Krebs-Henseleit solution
equilibrated with a 95% O2/5% CO2 gas mixture. A single
muscle strip (3-4 mm in width) was cut from the ventral costal diaphragm
parallel to the connective tissue fibers. Segments of the rib and central
tendon were used to attach the strip to two lightweight Plexiglas clamps. The
muscle strip was vertically suspended between the two lightweight Plexiglas
clamps, connected in series to a force transducer (Model FT03, Grass
Instruments, West Warwick, RI) in a water-jacketed tissue bath (Radnoti,
Monrovia, CA) containing Krebs-Henseleit solution equilibrated with a 95%
O2/5% CO2 gas mixture (bath, 37±0.5°C;
pH,
7.4±0.05; osmolality,
290 mOsmol). Transducer outputs
were amplified and differentiated by operational amplifiers, and underwent A/D
conversion for analysis using a computer-based data acquisition system
(Polyview, Grass Instruments).
In vitro contractile measurements began with empirical determination of the
optimal length (Lo) of the muscle for isometric tetanic tension
development. The muscle was field-stimulated (Model S48, Grass Instruments)
along its entire length with platinum electrodes. Using a micrometer, muscle
length was progressively increased until maximal isometric twitch tension was
obtained. Once the highest twitch force was achieved, all contractile
properties were measured isometrically at Lo. The force-frequency
relationship was examined using previously described methods
(Brooks and Faulkner, 1988;
Fraites et al., 2002
;
Staib et al., 2002
).
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Results |
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Localization of fluorescent beads to the liver after in utero hepatic injection
We focused on in utero hepatic delivery of rAAV with the aim of achieving
high-level gene expression of GAA in the liver. GAA produced in the liver
could be secreted and dispersed via the circulation to target tissues such as
the heart, diaphragm, and skeletal muscle. In the target tissue, the protein
would be escorted to the lysosome by mannose 6-phosphate receptor-mediated
endocytosis. Localization of the injected medium after in utero hepatic
injections was investigated using 10 µL of 0.1% (w/v) 30 nm fluorescent
beads. The beads were introduced by injecting through the uterine wall and
into the red-pigmented liver of a 15 pc CD-1 fetus. Fluorescent beads were
found localized in the liver at the site of injection (data not shown). This
was important as the diameter of the fluorescent beads (30 nm) and rAAV
(approximately 25 nm) are similar. From these results, we were confident we
could successfully deliver rAAV to the liver of the developing murine fetus,
and that the fetus could be carried to term.
Survival study of Gaa-/- in utero injections
Our group has injected a total of 294 Gaa-/- fetuses at
15 days gestation from 50 timed-pregnant females, of which 167 fetuses were
brought to term leading to a surgery survival rate of 60.5%, compared with
100% normal birth rate. Of the 148 injected mice allowed to reach a weaning
age of 3 weeks, 108 remained. This indicated a post-birth survival rate of
73.0%, a rate similar to animals of this strain not treated in utero. Most of
these deaths were due to maternal neglect or cannibalization, which is
normally seen in this and other knockout strains.
High-level transduction of diaphragm muscle through in utero delivery of rAAV serotype 2 to the liver and peritoneal cavity
We delivered rAAV containing the luciferase reporter gene driven by the
chicken ß-actin promoter plus the CMV enhancer (CBA) to fetal liver.
Several reports demonstrate the use of the CBA promoter in hepatic gene
transfer studies for high-level transduction
(Daly et al., 2001;
Song et al., 2001
;
Xiao et al., 1998
;
Xu et al., 2001
). The level of
luciferase expression was determined in several tissue types 1 month after in
utero hepatic delivery of 3x107 infectious particles of
rAAV2-CBA-Luc to Gaa-/- fetuses on day 15 of gestation.
Expression levels were highest in the diaphragm and liver
(Fig. 2A,B), whereas no
significant expression was detected in kidney, spleen, skeletal muscle, gonad,
lung, heart, brain or tongue of 1-month-old Gaa-/-
vector-treated mice (data not shown). In
Fig. 2, luciferase expression
levels of individual samples are shown by black circles, whereas averaged
activity values of saline and rAAV2-CBA-Luc-treated tissues are indicated by
white and gray bars, respectively. More than 100-fold higher luciferase
expression was detected in rAAV in utero-treated diaphragms compared with in
saline-treated mice. As the liver was the site of delivery, we were surprised
to find drastically lower luciferase expression in rAAV2-CBA-Luc-treated
livers compared with treated diaphragms. This could be attributed to a
dilution effect when nonintegrated or episomal forms of the vector are
dispersed to daughter cells as the liver divides during development. It is
likely that high-level diaphragmatic transduction occurred through
intraperitoneal exposure to the rAAV2 vector.
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Higher level expression achieved using rAAV serotype 1
Based on previous experimentation, we discovered that rAAV serotype 1 is
highly effective at transducing muscle tissue when delivering the human
GAA cDNA to the Gaa-/- mouse
(Fraites et al., 2002). In
addition, we purified serotype 1 vector that was higher in titer than the
serotype 2 vector used in this study (8.14x1012 particles/ml
of rAAV1-CMV-hGAA compared with 9.4x1010
particles/ml of rAAV2-CMV-hGAA). We sought to determine whether in
utero delivery of 8.14x1010 particles of
rAAV1-CMV-hGAA to Gaa-/- fetuses at 15 days
gestation could result in a higher level of transduction than
1x109 infectious particles of rAAV2-CMV-hGAA. After
allowing the vector-treated pups to reach 1 month of age, they were sacrificed
to isolate liver, kidney, spleen, skeletal muscle, gonad, diaphragm, lung,
heart, brain and tongue for GAA activity assays. Once again, GAA activity was
detected only in diaphragm. No other tissues tested expressed a significant
level of GAA activity (data not shown). In several cases, diaphragmatic
transduction with rAAV serotype 1 resulted in almost 10-fold higher GAA
activity, surpassing both normal controls as well as rAAV serotype 2 in
utero-treated Gaa-/- animals
(Fig. 4B).
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The level of GAA activity determined from the other half of the diaphragm indicated that the amount of staining was relative to the level of activity (Fig. 4B). The diaphragms with the highest intensity of staining reached over 100 nmol 4-MUG/hour/mg protein (rAAV1-CMV-hGAA-2, rAAV1-CMV-hGAA-6 and rAAV1-CMV-hGAA-8), with one yielding 824 nmol 4-MUG/hour/mg protein (rAAV1-CMV-hGAA-2); those with intermediate staining attained normal levels of approximately 20 nmol 4-MUG/hour/mg protein (1 and 5); and those that lacked staining had no detectable GAA activity (rAAV1-CMV-hGAA-3, rAAV1-CMV-hGAA-4 and rAAV1-CMV-hGAA-7).
By western analysis, we discovered that the 76 kDa mature form of GAA was responsible for the observed activity (Fig. 4C). The intensity of the 76 kDa band observed in each of the in utero-treated diaphragms was consistent with what was determined by activity assay and X-Gluc staining, with the exception of rAAV1-CMV-hGAA-5. Although an intermediate level of X-Gluc staining was observed in rAAV1-CMV-hGAA-5 (Fig. 4A), GAA activity analysis revealed that only 18 nmol 4-MUG/hour/mg protein of active protein was present (Fig. 4B). Correlating with the activity assay, western analysis indicated a very low level of mature enzyme was present. However, there was a predominant band of a molecular weight higher than the 95 kDa intermediate form visible in the placental control lane (Fig. 4C, lane 5). This was likely to be the 110 kDa precursor form of the protein. This partially explains why the X-gluc staining of this diaphragm did not correlate with the activity assay. The higher molecular weight species detected by western analysis may be able to enzymatically cleave the X-Gluc substrate more efficiently than 4-MUG, which was used in this activity assay. The 110 kDa precursor form of GAA exhibits low level activity on particular substrates, but this activity increases as the protein is further processed.
It is not known why the predominant species in rAAV1-CMV-hGAA-5 was the precursor form when in every other treated tissue the mature form prevailed. The presence of a similar sized precursor was also detected in other rAAV1-CMV-hGAA treated diaphragms, but the ratio of the precursor to the mature form was reversed. We conclude that this animal experienced an error in processing that prevented the precursor from being efficiently cleaved into its mature form. Further studies are necessary to determine what kind of processing error was responsible.
Prevention of lysosomal glycogen accumulation in GAA-/- mice treated in utero with rAAV2-hGAA
As rAAV-hGAA in utero-treated Gaa-/- animals
were exposed to vector-produced hGAA enzyme at an early stage in development,
we wanted to determine whether lysosomal glycogen accumulation associated with
GSDII and observed in this animal model was prevented in the treated animals.
For this purpose, periodic acid-Schiffs (PAS) reagent was used to stain
intracellular glycogen deposits of normal C57B6/129-SvJ, untreated
Gaa-/- and rAAV1-CMV-hGAA-treated
Gaa-/- diaphragm sections from 1-month-old mice
(Fig. 5A-C). At this early
stage in development, glycogen inclusions were evident in the diaphragm of
untreated Gaa-/- mice. Numerous pink-stained
glycogen-filled lysosomes scattered the field of the untreated
Gaa-/- diaphragm (Fig.
5B). Lysosomes swollen with undegraded glycogen were found both at
the cell periphery and among the fibers of the microtubes. Conversely, all of
the myfibers of the normal and vector-treated Gaa-/-
diaphragms were free of stain, making it impossible to differentiate between
the two (Fig. 5A,C). These
findings were confirmed by electron microscopy analysis. Extremely large
lysosomes full of glycogen were present among the muscle fibers of untreated
Gaa-/- diaphragm (Fig.
5E), but were not seen in normal tissue
(Fig. 5D) or in treated samples
expressing normal levels of GAA (Fig.
5F). However, we were not successful in transducing the diaphragm
to act as a factory for producing secreted GAA to treat other tissues. For
instance, heart tissue from the same animal whose diaphragm is pictured in
Fig. 5C had significant
PAS-positive material (Fig.
5I). Control heart tissue from C57B6/129-SvJ and untreated
Gaa-/- mice was also included
(Fig. 5G,H).
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The 595 bp rAAV1-CMV-hGAA amplified product was detected in each treated diaphragm sample, rAAV1-CMV-hGAA-1 through rAAV1-CMV-hGAA-8, but to varying levels (data not shown). All samples indicated the presence of vector genomes whether or not GAA protein was detected by staining, enzyme assay or western analysis (Fig. 4A-C). The 595 bp rAAV1-CMV-hGAA amplified product was not detected in untreated Gaa-/- animals (Fig. 6). Densitometry was performed to more accurately determine vector genome copy number present within 200 ng of diaphragm DNA, and this value was converted into vector copies/diploid genome based on a conversion factor of 5 pg total DNA/cell. Representative quantitative-competitive PCR experiments for diaphragm samples rAAV1-CMV-hGAA-1, rAAV1-CMV-hGAA-2 and rAAV1-CMV-hGAA-7 are summarized in Fig. 6. Listed beside each sample number, is the vector copy number reported as estimated vector copies per diploid genome, the GAA activity value as previously determined, and the raw data from each QC-PCR experiment. Control reactions were completed in which ß-actin was amplified from 200 ng DNA from each sample. This showed that the amount of DNA added to each QC-PCR reaction was relatively the same (data not shown).
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We also performed QC-PCR on the four livers from the treated mice in which the diaphragms exhibited high GAA activity (rAAV1-CMV-hGAA-2, rAAV1-CMV-hGAA-5, rAAV1-CMV-hGAA-6 and rAAV1-CMV-hGAA-8). Three of the livers tested had positive amplification signals, and were found to contain, on average, 0.1 estimated vector copies per diploid genome (data not shown). Even though it was uncertain whether the livers sampled in this experiment were actually the lobes directly injected, this indicated that there were vector genomes present in most of the livers tested. For the most accurate representation of vector genome copies in the liver, QC-PCR should be performed on DNA representative of the entire liver.
Diaphragmatic transduction following in utero delivery of rAAV1-CMV-hGAA results from intraperitoneal exposure to the vector
To determine whether intraperitoneal exposure of rAAV-hGAA after
hepatic in utero injections was the source of diaphragmatic transduction, we
performed several intraperitoneal in utero injections. We delivered
8.14x1010 particles rAAV1-CMV-hGAA to 15 day pc
Gaa-/- fetuses via the intraperitoneal cavity and
harvested the diaphragms from three animals (1-3) at 1 month of age. The
tissues were assayed by X-Gluc staining, GAA activity, western analysis and
QC-PCR. Each diaphragm was positive to a varying extent for X-Gluc staining
(data not shown), GAA activity and vector genomes
(Table 1).
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In utero delivery of rAAV2-CBA-hGAA preserves diaphragm muscle contractile force in Gaa-/- mice
Many patients suffering from GSDII succumb to respiratory insufficiencies
associated with diaphragm muscle weakness. We determined that near-normal
diaphragm muscle function can be preserved in 6-month-old
Gaa-/- mice when treated in utero with
rAAV2-CMV-hGAA. Using isometric force-frequency relationships as an
index of contractile function, we tested the contractile properties of
diaphragm muscle strips from age-matched knockout and normal mice
(Fig. 7). At 6 months
postpartum, impairment of contractile function was observed in
Gaa-/- diaphragms, as evidenced by their decreased
developed tensions over a range of stimulation frequencies (circles). By
contrast, 6-month-old Gaa-/- mice treated in utero with
2x108 infectious particles of rAAV2-CBA-hGAA did not
exhibit the same functional pathology and maintained near-normal contractile
properties (triangles) compared with normal controls (squares). The same
diaphragm strips used to study contractile function were also assayed for GAA
expression by X-Gluc staining and revealed several X-Gluc positive myofibers
(data not shown). GAA activity in the diaphragms of these
rAAV2-CBA-hGAA-treated 6-month-old Gaa-/- animals
was 84.0, 74.0 and 7.6 nmol 4-MUG/hour/mg protein. Western analysis revealed
the presence of the mature form of GAA (data not shown). These studies showed
that long-term expression of GAA can be achieved after in utero delivery of
rAAV-hGAA.
|
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Discussion |
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Limited work has been reported on the in utero delivery of rAAV. Two groups
reported high-level gene expression in several tissues on the first day of
life after liver-targeted delivery of rAAV2 to the mouse at day 15 of
gestation (Lipshutz et al.,
2001; Schneider et al.,
2002
). The authors also reported a decrease in expression as
development of the animal continues, with low-level expression being
maintained after 1 month of age. The current hypothesis for loss of expression
is that as cells divide throughout development, the unintegrated vector
genomes are dispersed to daughter cells and the effective dose of copies per
cell dramatically decreases over time. This hypothesis also explains why a
steady level of expression is maintained after the initial phases of
development, and after target cells differentiate. A dilution effect similar
to that observed in the liver is not seen in the diaphragm because, even
though transduced embryonic myoblasts of the diaphragm may be diluted after
cell division, these myoblasts fuse to create multi-nucleated myotubes,
thereby reversing the dilution effect. However, other possibilities may
explain why liver is difficult to transduce in utero. For example, rAAV2 may
not transduce embryonic liver with high efficiency because a majority of the
cells in the fetal liver are hematopoietic progenitors. Cells of hematopoietic
lineage are not easily transduced with rAAV2
(Srivastava, 2002
). This may
be because these cells do not express high enough levels of the appropriate
rAAV receptors, although there are many other possible reasons. Our study
focused on expression analysis at one month of age, after early development
and after the initial dilution of vector genomes has already occurred.
We found that expression was concentrated to the diaphragm of in utero hepatic-injected mice and that very little to no expression was found in the liver, the site of injection. We believe that during these hepatic injections, vector was released into the peritoneal cavity, allowing for direct transduction of the diaphragm. Delivery of the human GAA gene under transcriptional control of the CMV promoter by rAAV serotype 2 resulted in levels of GAA expression reaching and exceeding normal levels in diaphragm. Diaphragms transduced with the same expression cassette, but by rAAV serotype 1 and using almost a hundred times more total viral particles, expressed GAA at even higher levels, reaching over 10-fold above normal levels. Chemical staining of diaphragms treated with rAAV1-CMV-hGAA showed that transduction of the entire muscle can be achieved by this delivery method. In both cases, the predominant form of GAA detected by western analysis was the 76 kDa mature form. In diaphragms in which GAA expression reached normal or above normal levels, most of the fibers were clear of lysosomal glycogen deposits and normal muscle structure was preserved.
The diaphragm of animals treated with a lower dose of rAAV2-CBA-hGAA were assayed for contractile function at 6 months of age. Significant improvement was observed when these force-frequency relationships were compared with those of untreated age-matched controls. Diaphragms from adult animals treated with rAAV2-CMV-hGAA and rAAV1-CMV-hGAA are similarly expected to show significant functional improvement, and possibly preservation of normal contractile function, as these tissues reached normal or above normal levels of GAA expression after in utero transduction. The studies in adult animals are ongoing in the laboratory.
QC-PCR experiments, used to quantify vector genomes present in a particular tissue, indicated that up to an estimated 50 vector copies per diploid genome could be achieved in diaphragm when serotype 1 vectors were used. This correlated with GAA activity levels reaching over 10-fold higher than those observed in normal diaphragm. Livers from hepatic in utero-treated mice were assayed for the presence of vector DNA by the same method. On average, 0.1 estimated vector copies per diploid genome were detected. This correlated with the undetectable enzymatic activity. This result supports the hypothesis that upon direct delivery of vector to the liver, unintegrated vector genomes are diluted as they are dispersed to daughter cells by repeated cell divisions during liver development.
Results from adult mouse diaphragmatic transduction experiments showed very
limited transduction of muscle fibers by either direct intramuscular
injection, or by tail vein administration followed by clamping of the vena
cava below the diaphragm (Decrouy et al.,
1997; Petrof et al.,
1995
; Petrof,
1998
). The transduction levels attained in diaphragm muscle
presented in this study surpassed other published results several fold. We
showed that transduction of the entire diaphragm muscle was possible through
either in utero hepatic delivery or direct intraperitoneal delivery of rAAV.
We found that intraperitoneal delivery of rAAV1-CMV-hGAA resulted in
diaphragm transduction in all of the treated mice tested. GAA activity in all
cases reached normal, or above normal, levels. We believe in utero
intraperitoneal injection of rAAV-GAA to be an efficient delivery
method to achieve high-level diaphragm transduction, and it should be explored
further as a possible treatment method of GSDII. In addition, this technique
will be helpful for those studying other skeletal myopathies in which the
knockout mouse models are perinatal lethal because of respiratory distress
caused by lack of specific proteins being produced in the diaphragm
(Ibdah et al., 2001
;
Pan et al., 2003
;
Zhou et al., 2001
).
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amalfitano, A., McVie-Wylie, A. J., Hu, H., Dawson, T. L.,
Raben, N., Plotz, P. and Chen, Y. T. (1999). Systemic
correction of the muscle disorder glycogen storage disease type II after
hepatic targeting of a modified adenovirus vector encoding human
acid-alpha-glucosidase. Proc. Natl. Acad. Sci. USA
96,8861
-8866.
Amalfitano, A., Bengur, A. R., Morse, R. P., Majure, J. M., Case, L. E., Veerling, D. L., Mackey, J., Kishnani, P., Smith, W., McVie-Wylie, A. et al. (2001). Recombinant human acid alpha-glucosidase enzyme therapy for infantile glycogen storage disease type II: results of a phase I/II clinical trial. Genet. Med. 3,132 -138.[Medline]
Brooks, S. V. and Faulkner, J. A. (1988). Contractile properties of skeletal muscles from young, adult and aged mice. J. Physiol. 404,71 -82.[Abstract]
Chen, Y. T. and Amalfitano, A. (2000). Towards a molecular therapy for glycogen storage disease type II (Pompe disease). Mol. Med. Today 6,245 -251.[CrossRef][Medline]
Cohen, J. C., Morrow, S. L., Cork, R. J., Delcarpio, J. B. and Larson, J. E. (1998). Molecular pathophysiology of cystic fibrosis based on the rescued knockout mouse model. Mol. Genet. Metab. 64,108 -118.[CrossRef][Medline]
Daly, T. M., Ohlemiller, K. K., Roberts, M. S., Vogler, C. A. and Sands, M. S. (2001). Prevention of systemic clinical disease in MPS VII mice following AAV-mediated neonatal gene transfer. Gene Ther. 8,1291 -1298.[CrossRef][Medline]
Decrouy, A., Renaud, J. M., Davis, H. L., Lunde, J. A., Dickson, G. and Jasmin, B. J. (1997). Mini-dystrophin gene transfer in mdx4cv diaphragm muscle fibers increases sarcolemmal stability. Gene Ther. 4,401 -408.[CrossRef][Medline]
Ding, E. Y., Hodges, B. L., Hu, H., McVie-Wylie, A. J., Serra, D., Migone, F. K., Pressley, D., Chen, Y. T. and Amalfitano, A. (2001). Long-term efficacy after [E1-, polymerase-] adenovirus-mediated transfer of human acid-alpha-glucosidase gene into glycogen storage disease type II knockout mice. Hum. Gene Ther. 12,955 -965.[CrossRef][Medline]
Ding, E., Hu, H., Hodges, B. L., Migone, F., Serra, D., Xu, F., Chen, Y. T. and Amalfitano, A. (2002). Efficacy of gene therapy for a prototypical lysosomal storage disease (GSD-II) is critically dependent on vector dose, transgene promoter, and the tissues targeted for vector transduction. Mol. Ther. 5, 436-446.[CrossRef][Medline]
Douar, A. M., Adebakin, S., Themis, M., Pavirani, A., Cook, T. and Coutelle, C. (1997). Fetal gene delivery in mice by intra-amniotic administration of retroviral producer cells and adenovirus. Gene Ther. 4,883 -890.[CrossRef][Medline]
Eng, C. M., Guffon, N., Wilcox, W. R., Germain, D. P., Lee, P.,
Waldek, S., Caplan, L., Linthorst, G. E. and Desnick, R. J.
(2001). Safety and efficacy of recombinant human
alpha-galactosidase A - replacement therapy in Fabry's disease. N.
Engl. J. Med. 345,9
-16.
Fraites, T. J., Schleissing, M. R., Shanely, R. A., Walter, G. A., Cloutier, D. A., Zolotukhin, I., Pauly, D. F., Raben, N., Plotz, P. H., Powers, S. K. et al. (2002). Correction of the enzymatic and functional deficits in a model of Pompe disease using adeno-associated virus vectors. Mol. Ther. 5,571 -578.[CrossRef][Medline]
Hers, H. G. (1963). Alpha-glucosidase deficiency in generalized glycogen-storage disease (Pompe's disease). Biochem J. 86,11 .
Hirschhorn, R. and Reuser, A. J. (2000). Glycogen Storage Disease Type II: Acid alpha-Glucosidase (Acid Maltase) Deficiency. In The Metabolic and Molecular Basis of Inherited Disease (ed. C. R. Scriver, A. L. Beaudet, W. S. Sly and D. Valle), pp. 3389-3420. New York: McGraw-Hill.
Hoefsloot, L. H., Willemsen, R., Kroos, M. A., Hoogeveen-Westerveld, M., Hermans, M. M., Van der Ploeg, A. T., Oostra, B. A. and Reuser, A. J. (1990). Expression and routeing of human lysosomal alpha-glucosidase in transiently transfected mammalian cells. Biochem. J. 272,485 -492.[Medline]
Holzinger, A., Trapnell, B. C., Weaver, T. E., Whitsett, J. A. and Iwamoto, H. S. (1995). Intraamniotic administration of an adenoviral vector for gene transfer to fetal sheep and mouse tissues. Pediatr. Res. 38,844 -850.[Abstract]
Ibdah, J. A., Paul, H., Zhao, Y., Binford, S., Salleng, K.,
Cline, M., Matern, D., Bennett, M. J., Rinaldo, P. and Strauss, A. W.
(2001). Lack of mitochondrial trifunctional protein in mice
causes neonatal hypoglycemia and sudden death. J. Clin.
Invest. 107,1403
-1409.
Kakkis, E. D., Muenzer, J., Tiller, G. E., Waber, L., Belmont,
J., Passage, M., Izykowski, B., Phillips, J., Doroshow, R., Walot, I. et
al. (2001). Enzyme-replacement therapy in
mucopolysaccharidosis I. N. Engl. J. Med.
344,182
-188.
Larson, J. E., Morrow, S. L., Happel, L., Sharp, J. F. and Cohen, J. C. (1997). Reversal of cystic fibrosis phenotype in mice by gene therapy in utero. Lancet 349,619 -620.[Medline]
Lin, C. Y., Ho, C. H., Hsieh, Y. H. and Kikuchi, T. (2002). Adeno-associated virus-mediated transfer of human acid maltase gene results in a transient reduction of glycogen accumulation in muscle of Japanese quail with acid maltase deficiency. Gene Ther. 9,554 -563.[CrossRef][Medline]
Lipshutz, G. S., Flebbe-Rehwaldt, L. and Gaensler, K. M. (1999a). Adenovirus-mediated gene transfer in the midgestation fetal mouse. J. Surg. Res. 84,150 -156.[CrossRef][Medline]
Lipshutz, G. S., Flebbe-Rehwaldt, L. and Gaensler, K. M. (1999b). Adenovirus-mediated gene transfer to the peritoneum and hepatic parenchyma of fetal mice in utero. Surgery 126,171 -177.[CrossRef][Medline]
Lipshutz, G. S., Sarkar, R., Flebbe-Rehwaldt, L., Kazazian, H.
and Gaensler, K. M. (1999c). Short-term correction of factor
VIII deficiency in a murine model of hemophilia A after delivery of adenovirus
murine factor VIII in utero. Proc. Natl. Acad. Sci.
USA 96,13324
-13329.
Lipshutz, G. S., Flebbe-Rehwaldt, L. and Gaensler, K. M. (2000). Reexpression following readministration of an adenoviral vector in adult mice after initial in utero adenoviral administration. Mol. Ther. 2,374 -380.[CrossRef][Medline]
Lipshutz, G. S., Gruber, C. A., Cao, Y., Hardy, J., Contag, C. H. and Gaensler, K. M. (2001). In utero delivery of adeno-associated viral vectors: intraperitoneal gene transfer produces long-term expression. Mol. Ther. 3, 284-292.[CrossRef][Medline]
Loser, P., Jennings, G. S., Strauss, M. and Sandig, V.
(1998). Reactivation of the previously silenced cytomegalovirus
major immediate-early promoter in the mouse liver: involvement of NFkappaB.
J. Virol. 72,180
-190.
Martin-Touaux, E., Puech, J. P., Chateau, D., Emiliani, C.,
Kremer, E. J., Raben, N., Tancini, B., Orlacchio, A., Kahn, A. and Poenaru, L.
(2002). Muscle as a putative producer of acid
alpha-glucosidase for glycogenosis type II gene therapy. Hum. Mol.
Genet. 11,1637
-1645.
Mitchell, M., Jerebtsova, M., Batshaw, M. L., Newman, K. and Ye, X. (2000). Long-term gene transfer to mouse fetuses with recombinant adenovirus and adeno-associated virus (AAV) vectors. Gene Ther. 7,1986 -1992.[CrossRef][Medline]
Pan, Y., Zvaritch, E., Tupling, A. R., Rice, W. J., de Leon, S.,
Rudnicki, M., McKerlie, C., Banwell, B. L. and MacLennan, D. H.
(2003). Targeted disruption of the ATP2A1 gene encoding the
sarco(endo)plasmic reticulum Ca2+ ATPase isoform 1 (SERCA1) impairs diaphragm
function and is lethal in neonatal mice. J. Biol.
Chem. 278,13367
-13375.
Pauly, D. F., Johns, D. C., Matelis, L. A., Lawrence, J. H., Byrne, B. J. and Kessler, P. D. (1998). Complete correction of acid alpha-glucosidase deficiency in Pompe disease fibroblasts in vitro, and lysosomally targeted expression in neonatal rat cardiac and skeletal muscle. Gene Ther. 5,473 -480.[CrossRef][Medline]
Pauly, D. F., Fraites, T. J., Toma, C., Bayes, H. S., Huie, M. L., Hirschhorn, R., Plotz, P. H., Raben, N., Kessler, P. D. and Byrne, B. J. (2001). Intercellular transfer of the virally derived precursor form of acid alpha-glucosidase corrects the enzyme deficiency in inherited cardioskeletal myopathy Pompe disease. Hum. Gene Ther. 12,527 -538.[CrossRef][Medline]
Petrof, B. J., Acsadi, G., Jani, A., Massie, B., Bourdon, J., Matusiewicz, N., Yang, L., Lochmuller, H. and Karpati, G. (1995). Efficiency and functional consequences of adenovirus-mediated in vivo gene transfer to normal and dystrophic (mdx) mouse diaphragm. Am. J. Respir. Cell. Mol. Biol. 13,508 -517.[Abstract]
Petrof, B. J. (1998). Respiratory muscles as a
target for adenovirus-mediated gene therapy. Eur. Respir.
J. 11,492
-497.
Pompe, J. C. (1932). Over idiopatische hypertrophie van het hart. Ned. Tijdschr. 76, 304.
Raben, N., Nagaraju, K., Lee, E., Kessler, P., Byrne, B., Lee,
L., LaMarca, M., King, C., Ward, J., Sauer, B. and Plotz, P.
(1998). Targeted disruption of the acid alpha-glucosidase gene in
mice causes an illness with critical features of both infantile and adult
human glycogen storage disease type II. J. Biol. Chem.
273,19086
-19092.
Raben, N., Nagaraju, K., Lee, E. and Plotz, P. (2000). Modulation of disease severity in mice with targeted disruption of the acid alpha-glucosidase gene. Neuromuscul. Disord. 10,283 -291.[CrossRef][Medline]
Raben, N., Plotz, P. and Byrne, B. J. (2002). Acid alpha-glucosidase deficiency (glycogenosis type II, Pompe disease). Curr. Mol. Med. 2,145 -166.[Medline]
Schachtner, S., Buck, C., Bergelson, J. and Baldwin, H. (1999). Temporally regulated expression patterns following in utero adenovirus-mediated gene transfer. Gene Ther. 6,1249 -1257.[CrossRef][Medline]
Schneider, H., Adebakin, S., Themis, M., Cook, T., Douar, A. M., Pavirani, A. and Coutelle, C. (1999). Therapeutic plasma concentrations of human factor IX in mice after gene delivery into the amniotic cavity: a model for the prenatal treatment of haemophilia B.J. Gene Med. 1,424 -432.[CrossRef][Medline]
Schneider, H., Muhle, C., Douar, A. M., Waddington, S., Jiang, Q. J., von der, M. K., Coutelle, C. and Rascher, W. (2002). Sustained delivery of therapeutic concentrations of human clotting factor IX - a comparison of adenoviral and AAV vectors administered in utero. J. Gene Med. 4,46 -53.[CrossRef][Medline]
Song, S., Embury, J., Laipis, P. J., Berns, K. I., Crawford, J. M. and Flotte, T. R. (2001). Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors. Gene Ther. 8,1299 -1306.[CrossRef][Medline]
Srivastava, A. (2002). Obstacles to human hematopoietic stem cell transduction by recombinant adeno-associated virus 2 vectors. J. Cell Biochem. Suppl. 38, 39-45.[Medline]
Staib, J. L., Swoap, S. J. and Powers, S. K.
(2002). Diaphragm contractile dysfunction in MyoD
gene-inactivated mice. Am. J. Physiol Regul. Integr. Comp.
Physiol. 283,R583
-R590.
Tsujino, S., Kinoshita, N., Tashiro, T., Ikeda, K., Ichihara, N., Kikuchi, H., Hagiwara, Y., Mizutani, M., Kikuchi, T. and Sakuragawa, N. (1998). Adenovirus-mediated transfer of human acid maltase gene reduces glycogen accumulation in skeletal muscle of Japanese quail with acid maltase deficiency. Hum. Gene Ther. 9,1609 -1616.[Medline]
Van den Hout, J. M., Reuser, A. J., de Klerk, J. B., Arts, W. F., Smeitink, J. A. and Van der Ploeg, A. T. (2001). Enzyme therapy for pompe disease with recombinant human alpha-glucosidase from rabbit milk. J. Inherit. Metab. Dis. 24,266 -274.[CrossRef][Medline]
Wisselaar, H. A., Kroos, M. A., Hermans, M. M., van Beeumen, J.
and Reuser, A. J. (1993). Structural and functional changes
of lysosomal acid alpha-glucosidase during intracellular transport and
maturation. J. Biol. Chem.
268,2223
-2231.
Xiao, W., Berta, S. C., Lu, M. M., Moscioni, A. D., Tazelaar, J.
and Wilson, J. M. (1998). Adeno-associated virus as a vector
for liver-directed gene therapy. J. Virol.
72,10222
-10226.
Xu, L., Daly, T., Gao, C., Flotte, T. R., Song, S., Byrne, B. J., Sands, M. S. and Parker, P. K. (2001). CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice. Hum. Gene Ther. 12,563 -573.[CrossRef][Medline]
Zhou, Q., Chu, P. H., Huang, C., Cheng, C. F., Martone, M. E.,
Knoll, G., Shelton, G. D., Evans, S. and Chen, J. (2001).
Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of
congenital myopathy. J. Cell Biol.
155,605
-612.
Zolotukhin, S., Potter, M., Zolotukhin, I., Sakai, Y., Loiler, S., Fraites, T. J., Jr, Chiodo, V. A., Phillipsberg, T., Muzyczka, N., Hauswirth, W. W. et al. (2002). Production and purification of serotype 1, 2 and 5 recombinant adeno-associated viral vectors. Methods 28,158 -167.[CrossRef][Medline]