Mol Biotechnol (2008) 38:109–119
DOI 10.1007/s12033-007-9008-5

RESEARCH

Naked Plasmid DNA-Based a-Galactosidase A Gene
Transfer Partially Reduces Systemic Accumulationof Globotriaosylceramide in Fabry Mice
Gen Nakamura Æ Hiroki Maruyama Æ Satoshi Ishii Æ
Masaaki Shimotori Æ Shigemi Kameda Æ Toru Kono Æ
Jun-ichi Miyazaki Æ Ashok B. Kulkarni Æ Fumitake Gejyo

Published online: 13 October 2007
 Humana Press Inc. 2007

Abstract Fabry disease is an X-linked recessive inborn
metabolic disorder in which a deficiency in lysosomal
enzyme a-galactosidase A (Gal A) causes the systemic
accumulation of globotriaosylceramide (Gb3). Althoughmany investigators have attempted to treat a-Gal A knockout mice (Fabry mice) with gene therapy, no report has
demonstrated therapeutic effects by the retrograde renalvein injection of naked DNA. We recently developed anaked plasmid vector-mediated kidney-targeted genetransfer technique. A solution containing naked plasmid
G. Nakamura  M. Shimotori  S. Kameda  F. Gejyo
Division of Clinical Nephrology and Rheumatology, Niigata
University Graduate School of Medical and Dental Sciences,
1-757 Asahimachi-dori, Niigata 951-8510, Japan
H. Maruyama (&)
Department of Clinical Nephroscience, Niigata University
Graduate School of Medical and Dental Sciences, 1-757
Asahimachi-dori, Niigata 951-8510, Japane-mail: hirokim@med.niigata-u.ac.jp
S. Ishii
Department of Agricultural and Life Sciences, Obihiro
University of Agriculture and Veterinary Medicine, Obihiro
080-8555, JapanT. KonoDivision of Gastroenterology, Department of Surgery II,
Asahikawa Medical College, 2-1 Midorigaoka-higashi,
Asahikawa 078-8510, Japan
J.-i. Miyazaki
Division of Stem Cell Regulation Research, G6, Osaka
University Medical School, 2-2 Yamadaoka, Suita 565-0871,
Japan
A. B. Kulkarni
Functional Genomics Section, CDBRB NIDCR, NIH, 30
Convent Drive, MSC # 4395, Bethesda, MD 20892, USA

DNA encoding human a-Gal A (pKSCX-a-Gal A) was
rapidly injected into the left kidney of Fabry mice
(pKSCX-a-Gal A mice). pKSCX was used for mocktransfections (pKSCX mice). We confirmed that vectorderived human a-Gal A mRNA was present in the left
kidney but not in other tissues, by reverse transcriptase
polymerase chain reaction. Compared with the pKSCX
mice, the pKSCX-a-Gal A mice showed partial therapeutic
effects: increased a-Gal A activity in the injected kidney
and in the liver, heart, and plasma, and decreased Gb3 in
the injected kidney, contralateral kidney, liver, heart, andspleen. Our results demonstrated that, although further
studies are needed to improve the outcome, this method has
promise as a potential treatment option for Fabry disease.
Keywords Naked plasmid DNA  Fabry disease Catheter-based gene transfer  Renal vein injection 
CAG promoter  Hydrodynamics-based transfection

Introduction
Fabry disease is a pan-ethnic, X-linked, lysosomal storage
disorder (LSD) with an estimated frequency of 1 in 40,000
male births [1]. It is caused by a deficiency in the lysosomal
enzyme a-galactosidase A (Gal A) [2]. Undigested glycosphingolipids with terminal a-galactosyl moieties, mainly
globotriaosylceramide (Gb3), progressively accumulate inmost organs. Patients with classical Fabry disease have little
or no detectable Gal A enzyme activity and suffer fromangiokeratoma, hypohidrosis, and episodic pain crises inthe extremities during childhood or adolescence. Withadvancing age there is increased morbidity and mortality
due to renal failure, cardiac disease, and early stroke. Heterozygous female Fabry patients manifest a wide spectrum

110of disease severity, ranging from a virtually symptom-freecourse [3] to a course comparable to that in males [4].
The rationale behind the gene therapy for Fabry diseaseis as follows. Overexpressed hydrolytic enzymes implicated in LSDs, including a-Gal A, are secreted from thetransduced cells, and the circulating enzymes are thentransferred to other cells by mannose-6-phosphate receptormediated endocytosis, a phenomenon called metaboliccooperativity. In gene therapy, expressed a-Gal A could betransferred into uncorrected cells by the same mechanism.
Relatively low levels of enzyme correction may suffice toreduce the Gb3 storage in a number of clinically relevantorgans [5, 6]. A tight regulation of enzyme productionlevels is unlikely to be necessary, in part because the acidic
pH optimum for enzymatic activity probably means the
enzyme is inactive in the circulation at neutral pH [7].
In gene therapy studies using a-Gal A knock-out mice
(Fabry mice), several organs have been used as depots forthe production of a-Gal A by a variety of vectors [5, 8–16].
The liver was targeted by injecting adenoviral vector via
the tail vein [8] or by injecting adeno-associated vectors via
the portal vein [9], or the tail vein [10, 14]; the lungs weretargeted by the pulmonary instillation of an adenoviral
vector [11] or by injecting lipoplex via the tail vein [12];the muscle was targeted by injecting adeno-associatedvector [5] and naked plasmid vector [16]; and systemicorgans were targeted by injecting a lentiviral [13] oradenoviral [15] vector.
The kidney is the one of the main organs affected by
Fabry disease [17, 18]. We recently developed a novel
kidney-targeted gene transfer method using hydrodynamics-based transfection in rats [19] and mice [20]. We
demonstrated that this method can transfer naked plasmid
DNA into renal interstitial fibroblasts near the peritubular
capillaries (PTC) and that the kidney can serve as a depot
organ for the production of therapeutic proteins such as rat
erythropoietin [19] and mouse interleukin 10 with the Fc
protein of mouse IgG2a, IL10/Fc [20]. Here, to decrease
the Gb3 level in the kidneys, with an expected concomitant
reduction of Gb3 in other organs, we introduced the humana-Gal A gene directly into the left kidney of Fabry mice.

Materials and Methods
Plasmid DNA
Plasmid pKSCX-a-Gal A was constructed by inserting
human a-Gal A cDNA into the unique EcoRI site between
the CAG promoter (cytomegalovirus immediate-earlyenhancer/chicken b-actin hybrid) and the 30 -flanking
sequence of the rabbit b-globin gene of the pKSCX
expression vector [21], which is based on pCAGGS but withMol Biotechnol (2008) 38:109–119

a kanamycin-resistance gene as the selective marker. Plasmids were grown in Escherichia coli JM109 (Toyobo,
Osaka, Japan) and prepared as described previously [22].
The empty pKSCX plasmid was used for mock transfections.

Transient a-Gal A Expression in COS7 Cells
COS7 cells (RIKEN BRC Cell Bank No. 0539, Ibaraki,
Japan) were cultured as described previously [21]. The
COS7 cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according
to the manufacturer’s directions. About 24 hlater, the cells
were harvested by trypsin treatment, rinsed with phosphatebuffered saline, and homogenized in 0.2 ml water with a
Physcotron (NS-310E, Niti-on, Chiba, Japan). The supernatant was obtained by centrifugation at 10,000 g and usedas the enzyme source.

Western Blotting
Supernatants of cell homogenates were used for Western
blotting with an anti-a-Gal A antibody raised in rabbits[23] and a horseradish peroxidase-conjugated secondary
antibody (Amersham, Piscataway, NJ, USA). The protein
concentration was determined using the DC Protein Assaykit (Bio-Rad Hercules, CA, USA) with bovine serum
albumin as the standard. Samples were separated by electrophoresis on 10% SDS-polyacrylamide gels (Ready GelJ; Bio-Rad). Following electrophoresis, the proteins were
electrotransferred to nitrocellulose (Hybond-P; Amersham), and the signals were visualized using enhanced
chemiluminescence (Amersham).

Mice
Fabry mice [24] were bred at Niigata University. This
experiment was reviewed by the Committee on the Ethicsof Animal Experimentation at Niigata University. Hemizygous affected males were bred to homozygous affected
females, yielding only affected offspring. Genotypes were
determined by PCR analyses [25]. In this study we used
only female mice because of possible sex-related differences in the levels of testosterone-induced Gb3 (see Fig. 6,
below). About 9-week-old C57BL/6J mice were purchasedfrom Charles River Japan (Tokyo, Japan).
DNA Injection
The plasmid DNA was diluted in Ringer’s solution (Ohtsuka, Tokushima, Japan). The mice were anesthetized, andMol Biotechnol (2008) 38:109–119

an incision was made in the median section of the abdomen. Immediately before injection of the DNA solution,
the left renal vein and artery were clamped with angledtype Diethrich bulldog clamps (Muromachi Kikai, Tokyo,
Japan). It is technically difficult to perform renal vein
injection in mice, because their renal vein is thin and short.
To overcome this problem, a 1-ml-capacity Myjector syringe with a 29-gauge needle was used (Terumo, Tokyo,
Japan). The naked plasmid DNA solution was injected into
the vein within 2 sec, and blood flow was re-established
immediately afterwards. To avoid narrowing the internal
diameter of the renal vein, care was taken not to pull on thekidney as the needle was inserted. Hemostasis was seen at
the infected site after applying pressure for 5 sec.

Total Tissue RNA Extraction and RT-PCR Analysis
At 1, 2, 4, and 8 weeks after gene transfer, the mice were
killed and their injected kidneys were harvested. Total
RNA of the tissue samples was obtained with Isogen(Nippon Gene, Tokyo, Japan). The sequences of theprimers were: human a-Gal A gene, backward primer(a-Gal A backward primer), 50 -TCCATTGTCCAGTGCT
CTAG-30 ; and forward primer (a-Gal A forward primer),
50 -TCTGACTGACCGCGTTACTC-30 .
Glyceraldehyde
3-phosphate dehydrogenase (G3PDH) mRNA was also
detected by RT-PCR [20]. The a-Gal A primer set
encompassed the intron between the CAG promoter andthe human a-Gal A cDNA, allowing us to distinguish PCR
products from contaminating plasmid DNA. The length ofthe expected product was 201 bp for a-Gal A mRNA and452 bp for G3PDH mRNA. The RT-PCR conditions were
60C for 30 min, 94C for 2 min, then 40 cycles of 94C
for 1 min, 60C for 1.5 min, and 60C, for 7 min.

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mRNA. These PCR products were directly inserted into thepGEM-T Easy Vector (Promega), to create pGEM-T-a-Gal
A or pGEM-T-G6PDH, respectively. These plasmids weregrown in E. coli DH5a cells and prepared with a QIAprep
Spin Miniprep kit (Qiagen) for use as the external standard.
To determine the levels of transgene-derived a-Gal A
mRNA in the injected kidney, we performed quantitative
real-time PCR analysis using the LightCycler Quick system 330 (Roche Diagnostics, Mannheim, Germany). PCR
was performed with LightCycler-FastStart DNA Master
SYBR Green I (Roche Diagnostics), according to the
manufacturer’s protocol. The PCR reaction consisted of95C for 10 min, then a total of 40 cycles of 95C briefly,
62C for 10 sec, and 72C for 13 sec with a transition rateof 20C/sec between temperature plateaus. The data were
quantified by the LightCycler analysis software (version 3,
Roche Diagnostic). The results were expressed initially as
the number of target molecules/2 ll cDNA. To standardize
the results for variability in RNA and cDNA quantity andquality, we quantified the total number of G6PDH transcripts in each sample as an internal control. The
normalized levels of the a-Gal A transcripts were calculated as the ratio of the number of a-Gal A transcripts to
G6PDH transcripts. We confirmed the RT-PCR products
by 4% agarose gel electrophoresis.

Enzyme Assay
Tissue samples (approximately 0.1 gwet weight) were
homogenized in 0.3 ml water using a Physcotron and spunat 10,000 gfor 5 min. The protein concentration wasdetermined as above. The a-Gal A activity was assayedwith an artificial substrate, 4-methylumbelliferyl a-Dgalactoside (4MU-a-Gal), as described by Fan et al. [26].

Quantitative Real-Time PCR Analysis for a-Gal A
mRNA in the Injected Kidneys

Detection of Neutral Glycosphingolipids by Thin-Layer
Chromatography (TLC) Analysis

The total RNA was used for the synthesis of first-strand
cDNA with Moloney Murine Leukemia Virus reverse
transcriptase (RT; Gibco BRL, Rockville, MD, USA) andrandom hexamers (Promega, Madison, WI, USA). The RT
product was amplified by PCR with Taq DNA polymerase(Promega) and specific primers for the human a-Gal A
gene, which were identical to the a-Gal A backward andforward primers, and for the mouse housekeeping gene,
G6PDH: backward primer, 50 -TTCTTGGTCATCATCTT
GGTGTAT-30 ; forward primer, 50 -TATCTCAGAGGTGG
AAACTGACAA-30 . The primer set for the detection of
G6PDH mRNA was also designed to span introns. The
length of the expected product was 342 bp for the G6PDH

Tissues from Fabry or wild-type mice were minced withscissors and then homogenized in water. After the protein
content was measured, the crude lipids were extracted
from homogenates containing 5–10 mg protein with 20
volumes of chloroform–methanol (2:1, v/v). The lipids
were dried under a stream of nitrogen and subjected tomild alkaline treatment with 1 ml of 0.2 N NaOH inmethanol at 40̊C for 2 h. The solution was neutralizedwith glacial acetic acid, and the glycosphingolipids were
subjected to Folch’s partition (chloroform–methanol–H2O,
8:4:3 in v/v/v), recovered in the lower phase and applied
to Thin-Layer Chromatography (TLC) plates. TLC analyses were performed quantitatively with high-performance

112

TLC (HPTLC)-Silica gel 60 plates (Merck & Co., Inc.,Whitehouse Station, NJ, USA) and a solvent system ofchloroform–methanol–water (60:35:8, v/v/v). Glycosphingolipids were visualized by spraying the plates withorcinol–sulfuric acid reagent, and their band intensities
were determined using Scion Image software. Standard
Gb3 from porcine erythrocytes was purchased from
Nacalai Tesque, Inc. (Kyoto, Japan).

Histology
For light microscopy, 1 week after the injection, the mice
were sacrificed, and the harvested kidneys were immersion
fixed for 4 days in 10% phosphate-buffered formalin,embedded in paraffin, and 10-lm tissue sections were cut
and stained with Methylene Blue. For electron microscopy,
the mice were placed under general anesthesia, and perfused through the aorta with 10 ml of PBS followed by
10 ml periodate-lysine-paraformaldehyde using a gravityfeed system. The tissue blocks were fixed in 4% formaldehyde/1% glutaraldehyde in phosphate buffer at 4̊C for
24 h. The samples were washed three times in 0.1 M
cacodylate buffer with 0.2 M sucrose and postfixed in 1%
osmium tetroxide buffered with symcollidine (pH 7.2) at
4̊C for 1 h. After dehydration in serial alcohol and propylene oxide solutions, the samples were infiltrated withand embedded in Epok 812 (Oken, Tokyo, Japan), which isequivalent to Epon. Ultrathin (100 nm) sections were
stained with uranyl acetate and lead citrate, and examinedwith an H-600A electron microscope (Hitachi, Ibaragi,
Japan).

Mol Biotechnol (2008) 38:109–119

twice that of an age-matched pKSCX mouse at the same
dilution.

Statistical Analysis
The data are presented as mean values ± standard deviation. All data were analyzed using the StatView statisticalprogram for Macintosh (SAS, Cary, NC, USA). Statisticalsignificance was evaluated using Student’s t-test. P valuesof \ 0.05 were considered to be statistically significant.

Results
In Vitro Expression of a-Gal A
We first tested the expression of human a-Gal A protein in
COS7 cells by Western blot analysis. A robust signal of
46 kD, the expected molecular mass, was obtained (Fig. 1).
The biological activity of pKSCX-a-Gal A was also
examined by measuring the a-Gal A activity in COS7 cells
and in their medium (secreted a-Gal A) before and after
transfection. The intracellular and secreted a-Gal A activityof COS7 cells significantly increased after the introductionof pKSCX-a-Gal A (Fig. 2). These in vitro results confirmed that this construct delivered functional transgene tothe cells and that the overexpressed enzyme was secreted.

Enzyme-Linked Immunosorbent Assay (ELISA) for the
Detection of Anti-Human-a-Gal A IgG Antibodies
Enzyme-Linked Immunosorbent Assay (ELISA) plates(96-well; Nunc, Maxisorb, Roskilde, Denmark) were
coated with 1 lg/ll agalsidase beta (Fabrazyme, Genzyme
Corp., Cambridge, USA). Serial dilutions of plasma wereadded to the coated wells. Bound anti-human-a-Gal A IgG
antibodies were detected using horseradish peroxidaselabeled goat antihuman IgG anti-body (Cappel, Eschwege,
Germany), then incubating with a substrate solution containing 3,30 ,5,50 -tetramethylbenzidine (TMB) and H2O2(TMB microwell peroxidase substrate system, KPL, Gaithersburg, MD, USA) for 10 min for color development.
Optical density was measured at 450 nm on an ELISA
plate reader (Ultrospec Visible Plate Reader; Amersham
Pharmacia Biotech, Wals/Satzburg, Austria). The titer wasdetermined by the maximum dilution at which the plasmaof the pKSCX-a-Gal A mouse had an absorbance at least

Fig. 1 Western blot of a-Gal A expressed in COS7 cells. Lane 1,
empty pKSCX transfection. Lane 2, pKSCX-a-Gal A transfection

Mol Biotechnol (2008) 38:109–119

113

Fig. 3 Brain, heart, lungs, liver, spleen, kidneys, small intestine,
muscle, and skin harvested 1 day, and 2, 4, and 8 weeks after
transfection RT-PCR analysis for a-Gal A mRNA and control
G3PDH mRNA 1 day after pKSCX-a-Gal A injection in majororgans (a) and 2, 4, and 8 weeks after injection in bilateral kidneys(b). B, brain; H, heart; Lu, lungs; Li, liver; Sp, spleen; LK, left
kidney; RK, right kidney; Si, small intestine; Mu, muscle; Sk, skin(Fig. 4). This finding confirmed that an approximately
constant level of a-Gal A expression continued for 8 weeks
in the injected kidneys of the treated mice.

a-Gal A Levels

Fig. 2 a-Gal A activity in COS7 cells transfected with pKSCX-a-Gal
A (a) and in their culture medium (secreted a-Gal A) (b). n = 5 ineach group. *, P \ 10–5 compared with empty pKSCX

We evaluated the time course of a-Gal A activity in thekidneys, heart, liver, spleen, small intestine, and plasma inthe treated mice. The pKSCX mice exhibited only low

RT-PCR Analysis for a-Gal A mRNA in Major Organs
We detected the transgene-derived a-Gal A mRNA by RTPCR only in the left kidney on day 1 after the injection(Fig. 3a). We did not detect the transgene-derived a-Gal A
mRNA in the kidneys of the pKSCX mice (data notshown), although the control G3PDH mRNA was detected
in all the injected kidneys. The expression of transgenic aGal A mRNA was sustained in the injected kidney for atleast 8 weeks (Fig. 3b).

Quantitative Real-Time PCR Analysis for a-Gal A
mRNA in the Injected Kidneys
Quantitative real-time PCR analysis was performed at
different time points following the injection of pKSCX-aGal A to determine the quantity of transgene-derived
human a-Gal A mRNA in the injected kidneys. The
quantity of the expressed gene was highest at 2 weeks andthen declined over time, but there were no statistical differences between the a-Gal A levels at each time point

Fig. 4 The levels of a-Gal A mRNA and G6PDH mRNA in theinjected kidneys of pKSCX-a-Gal A mice were determined by realtime PCR analysis at 1, 2, 4, and 8 weeks after transfection.
Normalized levels of the a-Gal A transcripts were calculated as theratio of the number of a-Gal A transcripts to G6PDH transcripts.
n = 3 in each group114

levels of a-Gal A activity in the tissues assayed (Fig. 5).
This residual enzyme activity was probably attributable toa-galactosidase B [27]. The level of this residual activity inthe spleen and small intestine of the pKSCX mice varied byapproximately twofold at the three time points shown, andthis difference was significant for the small intestine for thevalues obtained at 2 weeks and at 4 weeks. The reason forthese differences, and whether the significant Gb3 reduction in the spleen at the 1- and 2-week time points (seeFig. 7, below) has any bearing on this observation, areunclear at present.
In the pKSCX-a-Gal A mice at 1 week post-injection,
the injected kidney showed the highest levels of a-Gal A
expression (Fig. 5). Much lower a-Gal A levels weredetected in the heart, liver, and plasma, but they weresignificantly greater than in the pKSCX mice. In the otherorgans, no significant difference in enzyme activity was
found between the pKSCX-a-Gal A and pKSCX mice.
About 2 weeks after the injection, there was no significant
difference between the pKSCX-a-Gal A and pKSCX mice,

Fig. 5 a-Gal A activity intissues from pKSCX, pKSCX-aGal A, and age-matched
C57BL/6J mice. n = 5 in eachgroup. *, P \ 0.05, and **,P \ 10–2 compared with
pKSCX mice

Mol Biotechnol (2008) 38:109–119

even in the injected kidney. Thus, low and transient levelsof a-Gal A were expressed in the injected kidney, thesecreted enzyme entered the bloodstream, and detectableamounts of enzyme were taken up by the heart and liver.

Gb3 Analysis
We observed an intense signal corresponding to ceramide
disaccharide in the kidneys of male C57BL/6J and Fabry
mice (Fig. 6a, b). In male C57BL/6J kidneys, a relatively
intense signal corresponding to Gb3, compared with thefemale kidneys, was also observed (Fig. 6a). The male
C57BL/6J kidney contained about three times as much Gb3
as the female kidney (Fig. 6c). This abundant Gb3 in themale kidney was probably induced by testosterone [28].
The ceramide disaccharides in the male C57BL/6J kidney
were mainly galabiglycosylceramide, which is also induced
by testosterone [28], and a small amount of lactosylceramide. The Fabry mouse [24] is a chimera resulting fromMol Biotechnol (2008) 38:109–119

115

order of magnitude to that seen in the injected kidney at thesame time point. Although the half-life of a-Gal A in thekidney is 0.73 days that of a-Gal A in the spleen is
5.62 days [29]. We therefore assume that the Gb3 level inthe spleen was reduced because of the prolonged enzyme
half-life in this organ, even though the amount of enzyme
was undetectable in the present study. In the injected kidney, and in the liver and spleen, the reductions in Gb3 were
sustained for 2 weeks; by 4 weeks, Gb3 had reaccumulated
to some extent. In the heart, there was some reduction in
Gb3 levels at 4 weeks. The basis for this delayed effect isunclear at present. The levels of Gb3 in the pKSCX tissues
remained unchanged or increased slightly over the 4-weekinterval, except for an initial drop in and recovery of Gb3
content in the spleen 2 weeks after the transfection. The
reductions in Gb3 levels in the different pKSCX-a-Gal A
tissues were closely correlated with the levels of a-Gal A inthese organs.

Histology

Fig. 6 TLC of neutral glycosphingolipids of C57BL/6J (a) and Fabry(b) kidneys. Ga2, galabiglycosylceramide. LacCer, lactosylceramide.
Gb3 content of C57BL/6J and Fabry (c) kidneys. n = 3 in each group.
*, P \ 10–2 for males compared with females

modified J1 cells injected into a C57BL/6 background.
Therefore, to avoid the possibility of skewing our data withtestosterone-induced glycosphingolipids, we used only
female mice in all of our gene therapy experiments. We
observed no testosterone-induced glycosphingolipids inother organs.
Figure 7 shows the effect of the retrograde injection ofpKSCX-a-Gal A into the left renal vein on the Gb3 levels
in various organs. A statistically significant reduction in
Gb3 levels was observed at 1 week in the injected kidney.
A significant reduction in Gb3 was also seen in the spleen,with small but significant reductions in the liver and uninjected kidney, even though no a-Gal A activity was
detected in the spleen or uninjected kidney at any time
point. The observed reduction in Gb3 in organs that had nodetectable enzyme activity may have been owing to atransient, undetectable enzyme activity that reduced theGb3 level. Subsequently, the reaccumulation of Gb3 inthese organs was observed. The Gb3 reduction was especially striking in the spleen at 1 week, being of a similar

To investigate which cell compartments were involved in
reducing the Gb3 content and to evaluate histologically thetherapeutic effects in the kidney, we analyzed renal specimens of pKSCX-a-Gal A and pKSCX mice by light andelectron microscopy. As described elsewhere [24], lipid
inclusions with electron-dense concentric lamellar structures in the lysosomes were observed only in the renal
tubular cells of Fabry mice. These inclusions appeared not
to be affected by our treatment (data not shown), so we
could not assess its subcellular effects.

Anti-Human-a-Gal A Antibody
Plasma samples were collected at various time points andanalyzed for the presence of anti-human-a-Gal A antibodies by ELISA. pKSCX-a-Gal A mice (n = 5) 1-weekpost-transfection showed no antibody titer. However, boththe number of mice that developed antibodies and theantibody titer increased over time (Fig. 8).

Body Weight and Growth
Compared with C57BL/6J females (mean 21.5 g, range
20.2–22.2 g), the female Fabry mice had a low bodyweight (mean 20.6 g, range 19.4–21.0 g) (p \ 0.05), butotherwise appeared normal. Although the treated anduntreated Fabry mice grew and gained weight during the
8-week duration of the experiment, the pKSCX-a-Gal A

116

Mol Biotechnol (2008) 38:109–119

Fig. 7 Gb3 levels in tissues of
pKSCX, pKSCX-a-Gal A, andage-matched C57BL/6J mice.
n = 5 in each group. *,P \ 0.05, and **, P \ 10–2, and***, P \ 10–3 compared with
pKSCX mice. #, not detected.
Columns are not shown forsome data points because of the
low Gb3 levels in the C57BL/6J
mice

mice did not gain as much weight as the same-age wildtype mice.
Discussion

Fig. 8 Anti-human-a-Gal A IgG antibodies were detected by ELISA.
Five animals were used for each time point. About 1 week after
transfection, no antibodies were detected. At 2 weeks, one of fivemice developed a low antibody titer. At 4 weeks,two of five mice
showed a low antibody titer. At 8 weeks, four of five mice had
developed antibodies, and the titers were relatively high. No
antibodies were detected in the pKSCX mice (data not shown)

The present results demonstrate that a pKSCX-a-Gal A
gene that was transferred into the kidney by hydrodynamics-based transfection expressed a-Gal A exclusively
in the injected kidney of Fabry mice. Although only a small
amount of a-Gal A was expressed, the pKSCX-a-Gal A
mice showed partial therapeutic effects in the injectedkidney, and therapeutic effects to varying extents in otherorgans through metabolic cooperativity.
Since kidney involvement is an important cause ofmorbidity and mortality in patients with Fabry disease [17,18], the kidneys should be one of the main organs targeted
for treatment. However, the Gb3 in the Fabry mouse kidney is relatively resistant to enzyme replacement therapy

Mol Biotechnol (2008) 38:109–119(ERT) by systemic intravenous injection [30, 31]. In ERT,
most (65 * 70%) of the injected enzyme is recovered in
the liver via the hepatic asialoglycoprotein and mannose
receptors, and less than roughly 1% of the injected dose isdetected in the Fabry mouse kidney [32].
The effect of gene therapy in the Fabry mouse kidney
seems to depend on the transgene method and/or transduced cells: some studies [11–13] reported no benefit,
some [8–10] showed partial therapeutic effects (60 * 74%of the Gb3 levels in the untreated Fabry mouse kidney),and some [5, 14, 15] demonstrated a significant reductionin Gb3 levels (3 * 25% of the untreated Fabry mouse
kidney). Although Takahashi et al. [5] found that the
intramuscular injection of a recombinant adeno-associatedvector construct led to sustained circulating levels of a-Gal
A and reduced Gb3 in the kidney, Park et al. [14], using asimilar but not identical method, obtained no systemic a-Gal
A activity. Clearly, additional experiments are required toclarify why completely different results were obtained inthese two studies.
Although a significant therapeutic effect in the kidney
can be obtained using viral vectors, potential problems
include the humoral immunological reaction against viral
vectors [33] and the possible presence of pre-existing antiviral antibodies, which could influence the transduction
efficiency [34]. Plasmid vectors have several advantages
over viral vectors. A large quantity of highly purified
plasmid DNA can be obtained easily and inexpensively,
and gene transfer can be repeated without any apparent
immune response to the DNA vector. The central problem
with plasmid DNA transfection is efficiency, as most of thetransferred DNA does not get into the target cells or isquickly degraded. Also, because the plasmid DNA does not
integrate with genomic DNA, expression of the transgene
is transient. However, since low enzyme activity is sufficient to clear the Gb3 deposits in various organs [5, 6],
direct gene transfer to the kidney by naked plasmid vector,
to provide a direct therapeutic effect in the transfectedkidney and in other visceral organs through metaboliccooperativity, appears to be a rational strategy for treating
Fabry disease.
Replagal1 (agalsidase alfa, Transkaryotic Therapies,
Cambridge, MA, USA) and Fabrazyme1 (agalsidase beta,
Genzyme, Cambridge, MA, USA) are genetically engineered forms of a-Gal A. These two glycoproteins haveidentical amino acid sequences but are produced in different cell lines (Replagal1 in a genetically engineered
human cell line, and Fabrazyme1 in a Chinese hamster
ovary cell line), resulting in different glycosylations at the
N-linked carbohydrate attachment sites. Fabrazyme1
contains a higher fraction of sialylated and phosphorylated
carbohydrate; Replagal1 contains a greater amount ofcomplex carbohydrate [32]. Interestingly, following a117

single intravenous bolus injection of 3 mg/kg of Replagal1
or Fabrazyme1 into the Fabry mouse, approximately twice
as much Fabrazyme1 is detected in the kidney as Replagal1 [32]. In Fabrazyme1, the carbohydrate at N215,
mostly biphosphorylated oligomannose, extends away fromthe protein, in an ideal position to bind the mannose-6phosphate receptor, but in Replagal1 the carbohydrate at
this site is mostly singly phosphorylated oligomannose[35]. Since these two polypeptides are derived from thesame human a-Gal A gene, these different post-translational modifications, especially the carbohydratecomposition, are probably responsible for their differencesin tissue distribution and dose response. We have shownthat a minimal expression of a-Gal A reduced the Gb3
levels in the injected kidney, and that secreted a-Gal A
reduced Gb3 in the contralateral kidney (where no detectable a-Gal A activity was observed). Our present results aredifferent from those of previous studies, in which no
therapeutic effects in the Fabry mouse kidney wereobserved using other transgene methods [11–13]. Different
post-translational modifications in the transgene-expressing
cells could explain these discrepancies.
A decrease in glycolipid inclusions is observed in therenal vascular endothelium, but not in other renal cellcompartments, in response to ERT [36]. An in vitro study
using immortalized human renal tubular epithelial cells
demonstrated that these cells have a relatively low affinity
for a-Gal A [37]. Consistent with these findings, we did notobserve a reduced number of inclusions in the renal tubularcells of the a-Gal A-treated Fabry mice. However, Thurberg
and colleagues observed a significant reduction in Gb3
levels in mesangial cells and a modest one in podocytes
within 5 months of initiating ERT [38]. Theoretically, inhumans, circulating a-Gal A cannot pass through glomerular capillary walls because this barrier normally traps
molecules greater than 50 Å [39], and a-Gal A is 75 Å
· 75 Å · 50 Å [34]. It is possible that the injured glomerular endothelium allowed some protein to pass through thebarrier in Thurberg’s study [38]. Nevertheless, the findingthat circulating a-Gal A cannot be efficiently taken up by
renal cells supports the strategy of transducing the a-Gal A
gene and expressing a-Gal A in the Fabry kidney.
Although we confirmed that a relatively constant levelof transgene expression was maintained for at least
8 weeks, a-Gal A activity could be detected for only
1 week after the transfection, and Gb3 had reaccumulated
in the kidneys 4 weeks after the transfection. We detected
some anti-human-a-Gal A antibodies in the pKSCX-a-Gal
A mice, probably because Fabry mice do not express
endogenous a-Gal A. We speculate that the deactivation of
a-Gal A was mainly due to the anti-human-a-Gal A antibodies, as is the case with anti-human factor VIII
antibodies in Hemophilia A model mice [40].

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In this study, we could not reduce the Gb3 to the levels
in C57BL/6J mice, even in the injected kidney, and new
methods are needed to improve the outcomes. However,
although the safety of this approach needs to be further
investigated in large animals, we demonstrated that thepKSCX-a-Gal A gene transferred by hydrodynamics-basedtransfection has the potential to be an alternative treatment
option for Fabry disease, especially targeting the kidney,alone or in combination with ERT.
Acknowledgments This work was partly supported by a Grant-inaid for Scientific Research (C) from the Ministry of Education, Science, Sports, and Culture and a Yujin Memorial Grant to Hiroki
Maruyama.

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