Gene Therapy (1997) 4, 442–448
 1997 Stockton Press All rights reserved 0969-7128/97 $12.00
In vitro correction of iduronate-2-sulfatase deficiency
by adenovirus-mediated gene transfer
C Di Francesco1, C Cracco1, R Tomanin1, L Picci1, L Ventura1, F Zacchello1, P Di Natale 2,
DS Anson3, JJ Hopwood3, FL Graham4 and M Scarpa1
1
Department of Pediatrics and Center for Biotechnology CRIBI, University of Padova, Italy; 2Department of Biochemistry and
Medical Biotechnology, University of Napoli ‘Federico II’, Italy; 3 Lysosomal Disease Research Unit, Department of Biochemistry,
Adelaide Children’s Hospital, Adelaide, Australia; and 4Departments of Pathology and Biology, McMaster University, Hamilton,
Ontario, Canada
Hunter syndrome is a lethal lysosomal storage disorder
caused by the deficiency of iduronate-2-sulfatase and
characterized by severe skeletal and neurological symptoms. Only symptomatic treatments are available and,
although bone marrow transplantation has been suggested, no encouraging results have been obtained so far.
Therefore, gene therapy might be a route to be pursued
for treatment of the disease. In this respect, one major goal
to achieve is the generation of an overexpressing vector
able to correct, in particular, central nervous system (CNS)
cells. Adenoviruses have been shown to infect CNS cells
efficiently with minor or even absent immunological
response. We describe the generation of a replicationdefective adenoviral vector, AdRSVIDS, which is able to
express in vitro high levels of iduronate-2-sulfatase. After
infection, accumulation of mucopolysaccharides in treated
Hunter cells was normalized. Furthermore, endocytosis of
the transduced IDS did occur via the mannose-6-phosphate (M6P) receptor. Since no animal model for the disease is available, we developed a system based on the
generation of derma-equivalents which enabled us to verify
the expression of high levels of sulfatase up to 30 days
after infection.
Keywords: gene therapy; mucopolysaccharidosis type II; adenovirus; derma-equivalents
Introduction
Mucopolysaccharidoses (MPS) are a group of 10 disorders caused by the deficiency of lysosomal enzymes
(four glycosidases, five sulfatases and one non-hydrolytic
transferase) needed for the catabolism of glycosaminoglycans (GAG): dermatan-, heparan-, keratan- and chondroitin-sulfate (mucopolysaccharides). Their accumulation in
lysosomes results in cell, tissue or organ dysfunction
determining various chronic and progressive patterns of
clinical severity, even within each enzyme deficiency.1
All MPS, except Hunter syndrome, are inherited as
autosomal recessive disorders. Hunter syndrome, MPS
type II, is a rare X-linked inborn error of metabolism
characterized by the deficiency of iduronate-2-sulfatase
(IDS) (E.C. 3.1.6.13), which removes the sulfate group in
dermatan- and heparan-sulfate. The defect is due to point
mutations or deletions in the 24 kb gene, mapping on
Xq28.2. 2 The cDNA was cloned as a 2.3 kb sequence,3,4
and successfully used to produce a recombinant active
enzyme in CHO cells.5
Hunter syndrome occurs in a severe and a mild form.
The severe form is characterized by progressive somatic
and neurological involvements. The onset of the disease
usually occurs between the second and fourth year of
Correspondence: M Scarpa, Department of Pediatrics and CRIBI, Via
Trieste 75, 35121 Padova, Italy
Received 5 November 1996; accepted 8 January 1997
age. Facial features, hepatosplenomegaly, short stature,
skeletal deformities, joint stiffness, severe retinal
degeneration and hearing impairment are coupled with
an incremental deterioration of the neurological system.
Death generally occurs between the ages of 10 and 14
years.
No mental impairment is characteristic of the mild
form, however, skeletal deformities can be present to the
same degree as in the severe form. Retinal and hearing
problems are milder than in the severe form and the
patient can survive until the fifth or sixth decade. Usually
cardiac failure or airway obstruction are the cause of
death. Only symptomatic treatments are available for
MPS in general. Transient improvement of patient conditions have been obtained with leukocyte and plasma
infusions,6,7 while fibroblast and amnion transplantations
have not been successful in restoring, even partially, any
enzyme activity or improving the objective signs.8–10
Bone marrow transplantation (BMT) has been suggested as a potential method for enzyme supplement for
MPS patients,11,12 since enzyme replacement therapy is
not available yet. In the case of MPSII, however, the value
of BMT still has to be investigated.
Correction by gene therapy might represent another
route to be pursued. High levels of recombinant human
lysosomal enzymes were obtained in vitro 13,14 and in
vivo15 by retrovirus-mediated gene transfer in different
MPS as well as a successful metabolic correction of
Hunter lymphoblastoid cell lines. 16 A phase I clinical trial
aimed at increasing the enzyme level in Hunter patients
Gene transfer in mucopolysaccharidosis type II
C Di Francesco et al
affected by the mild form of the disease was also
approved.17
In order to improve considerably the life condition of
patients affected by the severe form of Hunter syndrome,
local production of the enzyme lacking in the brain might
be required. In fact, the capability of IDS enzyme to cross
the blood–brain barrier still needs to be ascertained.
Since adenoviral vectors have been shown to be
adequate and safe delivery systems to transfer normal
sequences also to nonproliferative cells,18–21 we generated
a replication-defective adenovirus vector, derived from
human adenovirus type 5, expressing the human IDS
(AdRSVIDS).
Infection experiments performed on primary Hunter
cells showed that AdRSVIDS was able to normalize the
intralysosomal GAG accumulation; furthermore, the
recombinant enzyme secreted in the extracellular
compartment was endocytosed by deficient cells via the
mannose-6-phosphate receptor (M6P).22
Because of the lack of animal models to perform longterm expression experiments, we included infected primary fibroblasts from Hunter patients into collagen matrices (derma-equivalents).23,24 This technique enabled us to
show expression of the virus-transduced IDS up to 30
days after infection, confirming that AdRSVIDS might be
a valuable vector for gene therapy of Hunter syndrome.
Results
Construction of pXCRSVIDSpA
The 578 bp Rous sarcoma virus long terminal repeat
(RSV-LTR) sequence was isolated from pRSVLuc25 by
NdeI–XbaI restriction, treated with Klenow polymerase
and cloned as blunt end fragment into the filled in XbaI
site of pXCJL1.26 The new plasmid was called pXCRSV.
The SV40 polyA sequence containing the small intron
was isolated as an 876 bp HindIII–BamHI fragment from
the pSV23p construct,27 filled in and cloned into pXCRSV
after treatment with Klenow of the unique ClaI site. This
plasmid was called pXCRSVpA.
The 1814 bp IDS cDNA was isolated from the construct
pLX-IDS (DS Anson and JJ Hopwood, unpublished) by
ClaI–SalI restriction and filled in. The cDNA was cloned
into the filled in SalI site of pXCRSVpA. The final
plasmid, named pXCRSVIDSpA (Figure 1), was used to
generate the viral vector AdRSVIDS.
Molecular analysis of infected Hunter cells
The efficiency of adenovirus infection on Hunter cells
was first assessed by using AdHCMVsp1lacZ vector at
100 p.f.u. per cell. Nearly 100% of cells were found to
express b-galactosidase 24 h after infection (data not
shown).
To assess the transduced IDS activity in the short and
prolonged period, primary Hunter fibroblasts were
subsequently infected with the vector AdRSVIDS and
analysed 48 h and 30 days after infection, respectively.
The amplification of IDS cDNA was performed with IDSspecific oligonucleotides 5 and 340. A 334 bp band was
detected only in infected cells (data not shown).
The amplification of reverse transcribed total RNA
extracted from Hunter transduced cells, performed with
IDS-specific oligonucleotides 423a and 424a showed a
585 bp band only in infected cells. No amplification
443
Figure 1 The vector pXCRSVIDS pA used to cotransfect 293 cells with
the plasmid pJM17 to generate AdRSVIDS. AmpR: ampicillin resistance;
Ad5: 5′ sequence 1–452 bp; RSV: Rous sarcoma virus LTR; preproins.
leader seq.: 5′ rat preproinsulin leader sequence: 48 bp; IDS: IDS cDNA;
SV40 pA: SV40 polyA containing the small intron; Ad5: Ad5 3′ sequence
3328–5788 bp.
was obtained on non-reverse transcribed total RNA
(Figure 2).
IDS enzyme activity in normal and transduced
fibroblasts
To show that AdRSVIDS was able to express a functional
recombinant enzyme, IDS activity was tested on noninfected and infected Hunter cells compared with normal
Figure 2 RT-PCR on two different Hunter fibroblasts infected with
AdRSVIDS. Twenty micrograms of total RNA were retrotranscribed with
oligo dT and amplified with IDS-specific oligonucleotides 423a and 424a.
A 585 bp product was detected only from Hunter infected cells. Lane C−:
amplification of noninfected Hunter cells. Lanes 1 and 3: amplification of
total RNA nontreated with reverse transcriptase. Lanes 2 and 4: PCR
product from retrotranscribed RNA of infected Hunter cells. C+: positive
control, pXCRSVIDS pA. M: molecular weight marker VI (Boehringer
Mannheim). B: blank.
Gene transfer in mucopolysaccharidosis type II
C Di Francesco et al
444
Table 1 IDS activity detection in infected Hunter cells
Hunter fibroblasts
Control fibroblasts
Hunter cells +
AdRSVIDS
IDS expression
(U/mg) in cultured
fibroblastsa
IDS expression
(U/mg) in
derma-equivalentsb
,5
81 (±10)
,5
111 (±10)
1758 (±120)
2580 (±85)
a
IDS activity was determined 48 h after infection.
Fibroblasts were included in collagen matrices and IDS activity
determined 30 days after infection.
Values represent a mean ± s.d. of three independent experiments on two different normal primary fibroblasts and four different Hunter primary cells.
b
AdRSVIDS. Figure 3 shows that, as expected, a three-fold
increased level (286 ± 56 c.p.m.) (bar 1) of accumulation
in noninfected Hunter cells with respect to normal ones
(103 ± 8 c.p.m.) (bar 3) was measured. GAG levels in
Hunter cells were back to normal (95 ± 13 c.p.m.) after
infection (bar 2).
Secretion and endocytosis of recombinant IDS in Hunter
cells
To evaluate IDS secretion from Hunter-infected cells, IDS
activity was determined in IDS-conditioned medium
detecting 33 ± 8 U/ml. Such levels were 10-fold higher
than those measured in the medium collected from normal fibroblasts (,5 U/ml). Table 3 shows that IDS
activity was readily achieved in Hunter cells cultured
with IDS-conditioned medium, while no enzyme activity
was detected when Hunter cells were maintained in
medium obtained from normal fibroblasts. Furthermore,
ones (Table 1). No basal IDS activity (,5 U/mg) was
observed in Hunter cells (H130, H423, H435 and H452),
while normal cells showed standard IDS activity (81 ± 10
U/mg). In comparison, up to 20-fold higher expression
was detected in infected Hunter cells (1758 ± 120 U/mg).
In order to assay prolonged IDS expression, fibroblasts
were included into collagen matrices and maintained for
30 days. We observed that 30 days after infection Hunter
cells were still able to express up to 20-fold higher levels
of enzyme (2580 ± 85 U/mg) with respect to normal
fibroblasts (110 ± 10 U/mg) (Table 1). Owing to progressive decrease of cell viability, analysis beyond 30 days
could not be performed (data not shown).
Expression of lysosomal enzymes in infected Hunter
fibroblasts
To evaluate whether overexpression of transduced IDS
might alter the level of expression of other lysosomal
enzymes, activity of a-N-acetyl-glucosaminidase, bgalactosidase, a-l-iduronidase, N-acetylglucosamino-6sulfatase, galacto-6-sulfatase, acetyl-CoA:a-glucosaminide-N-acetyltransferase, were measured in Hunter fibroblasts before and after infection. Table 2 shows that the
overexpression of IDS induced by AdRSVIDS does not
seem to interfere with the expression and activity of
endogenous lysomal enzymes.
35
SO4 -GAG accumulation in Hunter fibroblasts before
and after infection with AdRSVIDS
To evaluate the capability of the recombinant enzyme to
correct the metabolic defect, 35SO4 -GAG levels were
evaluated in Hunter cells before and after infection with
Figure 3 Mucopolysaccharides accumulation analysis by 35SO4-GAG
labeling assay in Hunter cells before and after infection. Before infection
Hunter cells (bar 1) are accumulating three-fold more GAG than normal
cells (bar 3). After infection the level of GAG is back to normal (bar 2).
Each lane represents a mean ± s.d. of three independent experiments each
one performed on two different normal primary fibroblasts and four different Hunter primary cells. See Materials and methods for details.
Table 2 Lysosomal enzymes assayed in infected Hunter cells
a-N-acetylglucosaminidase
(nmol/mg/h)
b-Galactosidase
(nmol/mg/h)
a-l-iduronidase
(nmol/mg/h)
N-acetyl-glucose
amino-6-sulfatase
(nmol/mg/17 h)
Galacto-6-sulfatase
(nmol/mg/17 h)
Acetyl-CoA:
a-glucosaminideN-acetyl-transferase
Hunter +
AdRSVIDS
7.3 ± 1.5
784 ± 85
137 ± 20.7
34.1 ± 5.3
14.4 ± 5.0
49.9 ± 5.2
Hunter
9.3 ± 2.0
602 ± 100
128 ± 25.5
31.0 ± 9.2
15.4 ± 3.0
45.6 ± 4.6
Cells
Values represent a mean ± s.d. of four different Hunter primary cells.
Gene transfer in mucopolysaccharidosis type II
C Di Francesco et al
Table 3 Secretion and endocytosis
expressed by AdRSVIDS
of
recombinant
IDS
IDS activity
Conditioned medium for normal
fibroblasts
Conditioned medium from Hunter
cells infected with AdRSVIDS
Hunter cells cultured with conditioned
medium from normal fibroblasts
Hunter cells cultured with conditioned
medium from Hunter infected
fibroblasts
Without mannose-6-phosphate
With mannose-6-phosphate
,5 U/ml
33 ± 8 U/ml
,5 U/mg
235 ± 12 U/mg
,5 U/mg
Values represent a mean ± s.d. of three independent experiments on two different normal primary fibroblasts and four different Hunter primary cells.
IDS endocytosis is blocked by competition with M6P; no
increased IDS activity was, in fact, detected in Hunter
cells cultured with IDS-conditioned medium in the
presence of M6P.
Discussion
Somatic gene transfer is a promising strategy for the
treatment of many metabolic disorders. Essential to any
gene therapy protocol is the expression of the transferred
gene which has to be targeted to the major cellular sites
of pathology in sufficient amounts to correct the metabolic defect.
For the severe form of Hunter syndrome the target
organ is represented by CNS. Although retroviral vectors
have been shown to be the safest and more reliable vectors used in the ongoing clinical trials, they cannot be
used for gene transfer of differentiated neuronal cells. In
fact, efficient retrovirus-mediated gene transfer relies
strictly on cell replication. On the other hand, adenovirus
vectors have been shown to be reliable tools to infect CNS
cells in vitro and in vivo18,19,28 and were able to maintain
in vivo expression up to 6 months after infection.18,19
In this article, we describe the in vitro correction of
Hunter cells and the overexpression of the human IDS
cDNA mediated by the replication defective adenovirus
vector AdRSVIDS. The vector was able to express a
recombinant IDS protein that could substantially
decrease GAG substrate accumulation. Furthermore, it
appeared that recombinant IDS, as the normal enzyme,
was endocytosed into cells by the M6P receptor,
as shown by the lack of IDS activity in Hunter cells cultured with IDS-conditioned medium in the presence
of M6P.
AdRSVIDS has been shown to express high levels of
recombinant enzyme, up to 20-fold over normal levels,
which was maintained for at least 30 days. The overexpression of the recombinant IDS might be due to the
insertion of a 45 bp fragment of the rat preproinsulin
leader sequence cloned as substitution of the 5′-noncoding region of IDS. This insertion was necessary since
clones generated by stable transfection of unmodified IDS
cDNA were producing low levels of enzyme.5 IDS over-
expression from infected cells seemed to be crucial to
allow cross-correction of noninfected Hunter cells. In fact,
the use of IDS-conditioned medium obtained from normal fibroblasts was not successful in transferring IDS into
Hunter cells. The reason for this might be that IDS, a
housekeeping enzyme, is produced by normal cells
mostly for intracellular metabolic need. This might be a
further explanation to be taken into consideration,
together with antibody generation against implanted
cells, low number of implanted cells and shortness of the
enzyme half-life, to explain the failure of early clinical
trials that were unable to ameliorate Hunter patient conditions significantly by supplying fibroblasts, amnion or
plasma.6–10 However, besides overexpression, the set-up
of a gene therapy protocol for Hunter syndrome also
requires a vector able to produce high levels of enzyme
over a prolonged period. Because of the nonintegration
of adenoviruses into infected cell genome, prolonged
expression analysis is not feasible if replicating cells are
used. Experiments performed infecting Hunter fibroblasts with AdHCMVsp1lacZ showed that infected cells
were highly positive to X-gal staining for a few days after
infection (3 days), but totally negative within a few cell
passages (10 days). Since Hunter primary fibroblasts
were the only cells available for this study, a reproducible
system, based on derma-equivalents, able to allow prolonged expression analysis was developed. It was previously shown that keratinocytes or fibroblasts mixed
with collagen type I contract into a tissue.23 Within the
tissue, cells can be maintained for prolonged periods of
time and can reach a high degree of differentiation exhibiting a bipolar morphology.24 Cell division is blocked at
phases G1 and G2 and the synthesis of macromolecules
(collagen, noncollagen proteins, glycosaminoglycans) can
be identified.29 The generation of derma-equivalents is a
well established technique, which has also been exploited
for medical applications30,31 and for the construction of
secreting neo-organs for gene therapy purposes.32,33 In
this study, the use of derma-equivalents allowed us to
detect IDS activity up to 30 days after infection. The
expression was stable and comparable to the levels measured 48 h after infection. To our knowledge, this is the
first time that a prolonged term expression analysis has
been performed in vitro by using adenovirus vectors.
Therefore, derma-equivalents can be proposed as a
reliable, easy, cheap system for the analysis of adenovirus-transduced genes in the absence of a suitable
animal model.
The vector AdRSVIDS was able to correct the metabolic
alteration typical of MPSII and therefore it might be considered a valuable tool for gene therapy of patients affected by Hunter syndrome.
Although our final goal will be the correction of the
enzyme defect in CNS of Hunter patients, in the absence
of an animal model, clinical studies aimed to correct the
IDS deficiency in CNS should be preceded by experiments performed in other areas severely affected by the
disease (ie joints). Such experiments should allow evaluation of the vector safety and hopefully its immunogenicity. In this respect, the next step will be preclinical
experiments aimed at evaluating the ability of the vector
to overexpress IDS, its safety and its immunogenicity in
areas other than CNS in healthy animals.
445
Gene transfer in mucopolysaccharidosis type II
C Di Francesco et al
446
Materials and methods
DNA cloning
Cloning was performed following standard procedures.34
All enzymes for DNA manipulation were purchased
from Boehringer Mannheim (Mannheim, Germany).
Cell cultures
Hunter (H130, H423, H435 and H452) and normal primary fibroblasts were obtained from skin biopsies.
Hunter cells were obtained from patients affected by the
severe form of MPSII; primary cells obtained from three
healthy subjects were used as a control. Cells were cultured in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 2 mm l-glutamine, Hepes 5 g/l, nonessential amino acids, penicillin
(50 U/ml) streptomycin sulfate (50 mg/ml) and Fungizone (ICN, Costa Mesa, CA, USA) (125 mg/ml)
(complete medium). Cultures were incubated in a
humidified atmosphere at 5% CO2 at 37°C. All reagents
were obtained from GIBCO BRL (Gaithersburg, MD,
USA) except for FCS, obtained from Boehringer
Mannheim.
Generation and purification of AdRSVIDS
To generate AdRSVIDS, pXCRSVIDSpA and pJM1735
were cotransfected in 293 cells,36 by the Ca-P coprecipitation technique.37 Purification and titration were performed by standard procedures.26,38 The virus genome was
analyzed by enzymatic restriction. Correct plaques were
purified and analyzed once more. Medium from the
second purification was collected and used to build up a
high titer stock in 293 cells.
Adenovirus infection of Hunter cells
Hunter primary fibroblasts were infected with 100 p.f.u.
per cell of either AdRSVIDS or AdHCMVSp1lacZ,39 the
latter expressing the E. coli lacZ gene.
For short-term experiments using AdRSVIDS, cells
were infected and analyzed 48 h later. For prolonged
experiments, infected cells were included in derma-equivalents for 30 days, and analyzed after collagenase digestion as described below.
The infection and IDS transcription was determined by
PCR reaction using the following virus-specific oligonucleotides: No. 340 forward: 5′ TAC GAT CGT GCC TTA
TTA GG 3′, priming within the RSV promoter; and IDS
cDNA oligonucleotides No. 5 antisense: 5′ ACG TTC
AGA GCA TCT GTG GTC GAG TTG GCC 3′, priming
at position 210–239 of the IDS cDNA;3 No. 423a forward
CAT CAG CAA GCA GGT CAT T, priming at position
20–38 of the rat preproinsulin leader sequence;5 No. 424a
reverse: CCA CAG GGC AAA GCA GGT T, priming at
position 560–578 of the IDS cDNA.3 Taq polymerase was
purchased from Roche Molecular Systems (Branchburg,
NJ, USA). DNA oligonucleotides were synthesized with
a Beckman SM oligosynthesizer (Beckman Instruments,
Palo Alto, CA, USA); H235SO4 was purchased from Amersham (Buckinghamshire, UK).
Total DNA was isolated from cells as previously
described.34 IDS cDNA was obtained by reverse transcription of 20 mg of total RNA obtained by using RNAZol B treatment (BIOTECX, Houston, TX, USA) according
to the manufacturer’s protocol. Amplification of trans-
duced IDS was performed with oligonucleotides 340 and
5 by 35 cycles at 94°C for 1 min, 62°C for 45 s, 72°C for
1 min with a 5′ extended elongation time at the last cycle.
Amplification of IDS transcript was performed with oligonucleotides 423a and 424a by 35 cycles at 94°C for 1
min, 59°C for 45 s, 72°C for 1 min with a 5′ extended
elongation time at the last cycle. Hunter cells infected
with AdHCMVsp1lacZ were analyzed 24 h after infection
for b-gal expression by using X-gal staining procedure.40
Collagen preparation
Collagen type I was obtained from rat tails as described.23
Briefly, rat tails were stored in 70% EtOH until use, tendons were isolated and exposed overnight to UV light.
The day after, tendons were put in 1% glacial acetic acid
at 4°C for 48 h. Collagen was centrifuged at 15 000 g for
90 min at 4°C and dialyzed against 0.1% acetic acid solution for 6 days. Collagen concentration was determined
by measuring hydroxyproline content as described.41
Reconstitution of derma-equivalents
Normal, Hunter and virus-infected cells were mixed with
collagen type I to reconstitute derma-equivalents. As previously described,23 1.2 × 106 cells were plated in complete medium to which collagen and 0.1 n sodium
hydroxide were added and incubated in a humidified
atmosphere at 5% CO2 at 37°C. Derma-equivalents were
digested by adding 2 mg/ml Collagenase Type 1A
(Sigma, St Louis, MO, USA) from Clostridium histolyticum
and incubated for 40 min at 30°C. Cells were counted,
pellets resuspended in 150 mm NaCl and stored at −80°C
until used. Cell viability was assayed by trypan blue
dye exclusion.
Determination of IDS enzyme activity
Normal, Hunter and AdRSVIDS-infected cells were
tested for IDS activity as previously described.42 Fortyeight hours after infection, cells were trypsinized, centrifuged and lysed by six cycles of freezing–thawing. Lysates were dialyzed in distilled water for 16 h at 4°C before
assaying. Protein concentration was determined according to Bradford.43 IDS activity was assayed using the
radiolabeled disaccharide substrate L-O-(a-iduronic acid
2-sulphate)-(1-.4)-D-O-2,5-anhydrol 3 H mannitol 6-sulphate. Briefly, 15 ml of substrate solution (radioactive disulfated disaccharide, 0.22 mm, 2.6 × 106 c.p.m./min 0.27
m sodium acetate buffer pH 4.0/13 mm NaN 3) were
mixed with suitable enzyme aliquots and assayed in 80
ml final volume. Reaction was stopped after 24 h incubation at 37°C, with 1 ml 1 mm Na2 HPO4 , samples were
loaded on an ECTEOLA-23 column (FLUKA, Buchs,
Switzerland) and the product was eluted by adding 5 ml
70 mm sodium formiate. Scintillation liquid Pico Fluor 40
(INSTA-GEL Packard, Meriden CT, USA) was added and
samples were counted by a scintillation counter. Enzymatic activity was expressed as U/mg of proteins. One
unit of IDS activity is the amount of enzyme required to
catalyse the hydrolysis of 1% 3 H substrate per hour.
Determination of lysosomal enzyme activities
Hunter and AdRSVIDS-infected Hunter cells were
assayed for the following lysosomal enzymes: a-N-acetylglucosaminidase, b-galactosidase, a-l-iduronidase, Nacetyl-glucosamino-6-sulfatase, galacto-6-sulfatase, acetyl-CoA:a-glucosaminide-N-acetyltransferase according
Gene transfer in mucopolysaccharidosis type II
C Di Francesco et al
to standard procedures.44,45 Activities were expressed as
nmol/mg/h for a-N-acetyl-glucosaminidase, b-galactosidase, a-l-iduronidase, as nmol/mg/17 h for N-acetylglucosamino-6-sulfatase, galacto-6-sulfatase, and as
U/mg protein for acetyl-CoA:a-glucosaminide-N-acetyltransferase.
35
SO4-GAG accumulation assay
Studies assessing GAG metabolism were performed as
previously described.46 Infected fibroblasts were grown
for 48 h in MgSO4 defective medium (Basal Medium
Eagle Diploid Modified, (BMEDM); ICN Biomedicals,
Costamesa, CA, USA) supplemented with 2 mm l-glutamine and 10% FCS, the latter dialyzed against sterile
water and PBS for 1 week and 2 days, respectively. Cellular GAG were metabolically labeled by addition of
H2 35SO4 (Amersham) (4 mCi/ml; 1 Ci = 37 GBq) to culture
medium for 48 h. Fibroblasts were then washed with PBS,
harvested by trypsinization, collected, centrifuged at
1500 g, washed with PBS and centrifuged once more.
Two milliliters of 80% ethanol were added. Samples were
boiled for 5 min and centrifuged at 1500 g for 3 min. Ethanol extraction was done twice. 0.5 ml 10% NaOH were
added and samples were heated at 100°C. 35SO4 incorporation was assessed by counting 0.25 ml from each sample
in PicoFluor 40 with the scintillation counter.
Secretion and endocytosis of recombinant IDS in Hunter
cells
To assess whether transduced IDS was secreted, fibroblasts were infected with AdRSVIDS for 24 h, washed to
eliminate free virus and re-fed with complete medium
for 24 h more. This medium, called IDS-conditioned
medium, was collected, filtered and used to feed Hunter
cells. IDS activity was measured as described above.
Medium collected from normal fibroblasts was used as
control. To assess whether recombinant IDS was endocytosed via the M6P receptor, 19 fibroblasts were plated
and allowed to reach confluence, incubated for 24 h in
10 ml IDS-conditioned medium with or without 5 mm
M6P. Cell lysates were dialyzed against distilled water
and then analyzed for total protein content and IDS
activity as described above.
Acknowledgements
We wish to thank J Rudy for the excellent technical assistance. The work was supported in part by the Italian Mucopolysaccharidoses Association (IMA), the ‘Salus Pueri’
Foundation and by Regione Veneto ‘Ricerca Finalizzata’,
Venice, Italy, 579/01/95. C Cracco is recipient of an IMA
fellowship. P Di Natale is supported by the TelethonItalia research grant No. 085. FL Graham’s research was
supported by a grant from the Medical Research Council
of Canada. FL Graham is a Terry Fox Research Scientist
of the National Cancer Institute of Canada. DS Anson
and JJ Hopwood are supported by a National Health and
Medical Research Council of Australia program grant.
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