In vitro
gene therapy of mucopolysaccharidosis type I by lentiviral
vectors
Paola Di Natale
1
, Carmela Di Domenico
1
, Guglielmo R. D. Villani
1
, Angelo Lombardo
2
, Antonia Follenzi
2
and Luigi Naldini
2
1
Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II; Italy
2
Laboratory for Gene Transfer
and Therapy, Institute for Cancer Research and Treatment, University of Turin, Italy
Mucopolysaccharidosis type I (MPS I) results from a defi-
ciency in the enzyme a-
L
-iduronidase (IDUA), and is char-
acterized by skeletal abnormalities, hepatosplenomegaly and
neurological dysfunction. In this study, we used a late gen-
eration lentiviral vector to evaluate the utility of this vector
system for the transfer and expression of the human IDUA
cDNA in MPS I fibroblasts. We observed that the level of
enzyme expression in transduced cells was 1.5-fold the level
found in normal cells; the expression persisted for at least

difference in severity is due primarily to the effect of
various mutations in the human IDUA gene, located on
the short arm of chromosome 4 [2–4]. Homozygosity as
well as compound heterozygosity for some mutations (e.g.
W402X and Q70X) results in the most severe phenotype,
Hurler syndrome, while some alterations permit residual
enzyme activity that results in the mild phenotype (for
reviews see [1,5]). Recently, a large mutational analysis was
described including in vitro expression of missense muta-
tions [6]; our group contributed to the characterization of
defects in Italian population [7].
Therapies for MPS I include allogeneic bone marrow
transplantation [8–10] and enzyme replacement therapy
(ERT) [11]; in vitro transduction of IDUA cDNA in
cultured cells gave promising results [12–17]. Correction of a
metabolic defect is based on early biochemical findings:
lysosomal enzymes are post-translationally processed to
contain mannose 6-phosphate residues that bind to man-
nose 6-phosphate receptors, which target enzymes to
lysosomes. Receptors are also present on the cell membrane
and are able to bind circulating extracellular enzymes and
deliver them to the lysosomes [18]. Results obtained with
ERT were encouraging although inconvenient, because the
therapy has to be performed weekly. Thus, the search for
alternative therapies is motivated, first to be tested in vitro or
on animal models. A naturally occurring canine model has
been useful in testing direct enzyme replacement [19] and the
murine knock-out model [20] represents a promising tool
for the development of new therapies.
The availability of the MPS I murine model and the

from the Gaslini Institute, Genoa, Italy. The cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM,
Life Technologies) containing 10% fetal bovine serum
(Sigma), 100 UÆmL
)1
penicillin, 100 mgÆmL
)1
streptomycin
and 2 m
ML
-glutamine. Cells were cultured at 37 °Cina5%
CO
2
humidified incubator.
Lentiviral vector containing IDUA cDNA
The human IDUA cDNA was excised from expression
plasmid pBSIIKS-hIdu obtained from E. F. Neufeld,
UCLA School of Medicine, Los Angeles, CA, USA.
The 2.2-Kb human IDUA cDNA was excised from this
plasmid by digestion with XbaI followed by filling-in with
klenow polymerase and a second digestion with MluI. The
purified band was then subcloned into plasmid
pRRLsinPPT.CMV.WPRE after double digestion of this
vector with MscIandMluI to obtain the self inactivating
(sin) gene transfer construct PRRLsinPPT.CMV.
IDUA.WPRE. The packaging of the vector was obtained
as described previously [23,24] by cotransfection of 293T
cells with four different constructs: the pMDLg/pRRE, a
multiple attenuated packaging construct containing
the RRE sequence coding for HIV-1 gag and pol genes,

0.1
M
glycine in NaCl/P
i
, twice with NaCl/P
i
and once with
H
2
O. The fluorescence was visualized under an Axioplan
fluorescence microscope (Zeiss).
Detection of the integrated lentiviral-IDUA construct
by Alu-PCR
Cell extracts from transduced and untransduced MPS I
fibroblasts were prepared as described previously [25]. For
Alu-PCR reactions, two different primers pairs were used:
one at the 5¢ end and the other at the 3¢ end of the vector
genome. Primers for the 5¢ end were Alu3¢ sense [25] and
5NC2 antisense: 5¢-GAGTCCTGCGTCGAGAGAG-3¢
[26]; the other pair at the 3¢ end was Alu278 antisense [25]
and Wpre sense: 5¢-CTTTCCATGGCTGCTCCC-3¢ [26].
The PCR procedure required a 10 min initial denaturation
step (95 °C) followed by 30 cycles at 94 °Cfor1min,57°C
(for the 5¢Alu-PCR) or 49 °C(forthe3¢Alu-PCR) for
1min,and72°C for 1 min. After this first amplification, a
nested PCR was performed using 10 lL of the first PCR
product with two different internal primers in the vector
genome. For the 5¢ nested PCR the primers were:
LTR9 (sense: 5¢-GCCTCAATAAAGCTTGCCTTG-3¢)
and U5PBS (antisense: 5¢-GGCGCCACTGCTAGAGAT

formate pH 3.2, with 2 m
M
4-methyl-
umbelliferyl-a-
L
-iduronide (Calbiochem), at 37 °Cfor1h.
The reaction was stopped by adding 0.5
M
Na
2
CO
3
/
NaHCO
3
buffer, pH 10.7. The liberated 4
M
U was detected
fluorimetrically with 365-nm excitation and 448-nm emis-
sion filters.
Correction of metabolic defect in transduced MPS I
fibroblasts
MPS I fibroblasts were plated in replicate 6-cm plates and
transduced with 50 ng p24, as described above. Seven and
14 days after viral infection, transduced and untransduced
cells were treated with SO
4
-free medium (ICN Biomedicals)
containing 10% fetal bovine serum dialyzed for 24 h. Cells
were then incubated in the same medium in the presence of

35
S-Express Protein Labeling Mix (NEN Life
Science Products). After a 2-h labeling period (pulse), the
cells were chased for 24 h in DMEM medium supplemented
with 0.3 gÆL
)1
nonradioactive methionine and cysteine. The
medium was then collected and concentrated to 0.5 mL
using a Millipore Centricon Centrifugal Filter Device, with
YM-30 membrane, by spinning 2 h at 2200 g in a Beckman
centrifuge CS-6R. Cells were washed with NaCl/P
i
and lysed on ice for 30 min in 1 mL lysis buffer (10 m
M
Tris/HCl buffer pH 7.4, 150 m
M
NaCl, 1 m
M
EDTA
pH 8.0, 0.1% Triton X-100) containing 0.2 m
M
phenyl-
methanesulfonyl fluoride, 1 l
M
pepstatin A and 1 l
M
leupeptin. Labeled a-
L
-iduronidase was immunopreci-
pitated from cells and medium using 1 lL of specific

conditional packaging system [23]. The proviral form of the
vector is shown in Fig. 1. The packaging system contains
the self-inactivating transducing constructs (sin) obtained
after a 400-bp deletion including the enhancer and promoter
from U3. The system conserves only three of the nine HIV-1
genes and relies on four separate transcriptional units for the
production of transducing particles. This system offers
significant advantages for its biosafety and allows the
production of high-titer HIV-derived vector stocks. In
addition, this late-generation gene transfer construct con-
tains the polypurine tract (PPT), the structural element from
pol of HIV-1 virus encompassing the central PPT and
termination sequences. These were previously reported to
enhance gene transfer into primary cells including peripheral
blood lymphocytes, macrophages, fibroblasts and endo-
thelial cells [24].
Evaluation of transgene expression and transduction
efficiency
To analyse lentiviral-vector-mediated IDUA transduction
and expression, a set of experiments were performed using
MPS I fibroblasts. Cells were transduced with three differ-
ent doses (1, 10, and 50 ng) of p24 viral protein, in the
absence or in the presence of 5 lgÆmL
)1
of polybrene
(Table 1), as described in Materials and methods. Trans-
duced fibroblasts, in triplicate plates, showed an increased
enzymatic activity, both in the absence of polybrene (means
of 6, 22 and 117 nmolÆh
)1

added to MPS I fibroblasts as indicated. Cells, in triplicate plates, were
incubated for 2.5 days before harvesting for IDUA activity. Data
show mean ± SD. Untreated MPS I fibroblasts have an enzyme
activity of 0.25 ± 0.03 nmolÆh
)1
Æmg
)1
;normalfibroblastshavean
enzyme activity of 98 ± 13 nmolÆh
)1
Æmg
)1
.
IDUA Vector
(ng p24)
a-
L
-Iduronidase activity (nmolÆh
)1
Æmg
)1
)
– Polybrene + Polybrene
1 6 ± 1 20 ± 4.5
10 22 ± 5.5 32 ± 4.1
50 117 ± 19.31 155 ± 18.1
2766 P. Di Natale et al. (Eur. J. Biochem. 269) Ó FEBS 2002
fluorescent cells over the total number of cells and was
estimated to be 70% (data not shown).
Long-term IDUA transgene expression in transduced

PCR to amplify sequences from the vector DNA. As
expected, both for 3¢ and 5¢-Alu-PCR no bands were visible
after the first amplification (Figs 2B, lanes 1–3); visible bands
were obtained only after nested PCR (Figs 2B, lanes 4–7).
Reactions corresponding to the transduced MPS I fibro-
blasts amplified a single band of 166 bp from the 3¢ end and
a 121-bp fragment from the 5¢ end (Figs 2B, lane 5), while no
fragments were visible for the untreated cells (Figs 2B, lane
6) or for the control using as template cell lysate not subjected
to the first amplification (Figs 2B, lane 7).
Correction of metabolic defect in transduced MPS I
fibroblasts
To verify if the GAG accumulation could be corrected by
treatment with lentiviral vector, cells were infected and then
cultured in the presence of H
35
2
SO
4
. The GAG level
measured in transduced cells 1 or 2 weeks after infection
resulted decreased, approaching the level found in normal
cells (Table 2). The results showed the correction of the
defective glycosaminoglycan catabolism after treatment
with vector.
Metabolic labeling of recombinant IDUA enzyme
in transduced MPS I fibroblasts
The maturation of the recombinant IDUA enzyme was
studied through metabolic labeling experiments in trans-
duced fibroblasts as described in Materials and methods.

3¢ end and the other at the 5¢ end of the vector genome. After the first
amplification (lanes 1–3) no band was visible. The nested reaction
(lanes 4–6) amplified a fragment of 166 and 121 bp at-3¢ and 5¢ end of
the vector DNA, respectively. M: 100 bp marker; lanes 1 and 4: blank
reaction; lanes 2 and 5: transduced fibroblasts; lanes 3 and 6: untreated
cells; lane 7: control reaction amplified only with nested PCR.
Ó FEBS 2002 Lentiviral vector-mediated IDUA gene transfer (Eur. J. Biochem. 269) 2767
of 116 nmolÆh
)1
Æmg
)1
, vs. an activity of 0.3 nmolÆh
)1
Æmg
)1
found in untransduced cells. Taking into account the total
enzyme units (70) measured in the extracellular medium and
the total enzyme units (10) recovered in the recipient cells
after co-culture, a value of 14% endocytosis was calculated.
In the presence of 5 m
M
mannose 6-phosphate, the enzyme
activity in the recipient cells reached the basal levels of
untreated MPS I fibroblasts, showing a strong inhibition of
the uptake by mannose 6-phosphate (Fig. 4).
In another set of experiments, co-culture was performed
in the presence of a labeling protein mixture to see whether
the recaptured enzyme was correctly processed. The results
are shown in Fig. 5. The enzyme released in the culture
medium, the precursor form of 76 kDa, was correctly

-glycosaminoglycan accumulation and protein content (mean ± SD, n ¼ 3)asdescribedinMaterials
and methods. U, untreated; T, transduced; N, normal fibroblasts.
35
S-Glycosaminoglycans (c.p.m. per mg)
UT N
Experiment 1 127 000 ± 17 473 10 000 ± 4509 8340 ± 3120
Experiment 2 40 000 ± 6658 7322 ± 1761 6820 ± 2318
Fig. 3. Synthesis of recombinant a-
L
-iduronidase in transduced MPS I
fibroblasts. Untreated and transduced fibroblasts were grown to sub-
confluence and metabolically labeled with 300 lCi of
35
S-Express
Protein Labeling Mix for 2 h. The labeling medium was then removed
and the cells were chased in growth medium for 24 h. After this time
labeled cells and corresponding medium were harvested, immunopre-
cipitated and analysed via SDS/PAGE and autoradiography. The
molecular masses of the protein standards are indicated on the left. U,
untreated fibroblasts; T, transduced fibroblasts; M, medium; C, cell
lysate. The arrows on the right indicate the 76 kDa precurson form
and the 66 kDa mature form of the enzyme.
Fig. 4. Correction of MPS I fibroblasts by enzyme released from
transduced deficient cells. Transduced fibroblasts, which can secrete the
IDUA enzyme, were cultured in the presence of recipient deficient cells
in separate chambers of a trans-well system as described in Materials
and methods. After 72 h of coculture, recipient cells were harvested to
measure enzyme activity.
2768 P. Di Natale et al. (Eur. J. Biochem. 269) Ó FEBS 2002
IDUA activity in patients will moderate the severe clinical

imply that the introduction of a therapeutic IDUA gene
by lentiviral vector into a small portion of target cells
may result in the release of the expressed enzyme from
these transduced cells with a subsequent uptake by
unmodified cells and tissues and correction of the
lysosomal metabolism.
In vitro correction of mucopolysaccharidosis type I cells
has been obtained in the last few years using retroviral
vectors [12–16] or an adeno-associated vector [17] trans-
ducing the IDUA cDNA. Anson et al. [12] first reported
an expression of high levels of human iduronidase after
retroviral transduction of MPS I fibroblasts. The trans-
duction of hematopoietic stem cells was studied by
Fairbain et al. [13], who demonstrated retrovirus-mediated
IDUA gene transfer into MPS I CD34
+
cells, with high
levels of IDUA activity detectable in a significant
percentage of these cells. Huang et al.[14]characterized
a series of retroviral IDUA vectors that also exhibited
efficient gene transfer into MPS I bone marrow cells;
Stewart et al. [15] showed that primary neuronal and
astrocyte cultures were capable of taking up the enzyme
from the supernatant of fibroblasts transduced with an
IDUA retroviral vector; Pan et al. [16] compared the
efficacy of different IDUA retroviral constructs in expres-
sing IDUA cDNA in MPS I CD34
+
cells and in MPS I
fibroblasts; Hartung et al.[17]usedanIDUAadeno-

ACKNOWLEDGEMENTS
We thank Prof Elizabeth F. Neufeld, UCLA School of Medicine, for
providing us with the pBSIIKS-hIdu plasmid and anti-IDUA anti-
bodies.
We thank the ÔLaboratorio di Diagnosi Pre-Postnatale Malattie
Metaboliche ÔIstituto G. Gaslini for providing us with specimens from
the collection ÔCell lines and DNA bank from patients affected by
Genetic disease, supported by Telethon grants (project C.52) and Mrs
R. Baldoni for typewriting.
This work was supported by a grant from MURST to Prof P. Di
Natale and Prof. Franco Zacchello (University of Padua, Italy).
Fig. 5. Uptake of secreted recombinant IDUA into MPS I fibroblasts.
Transduced MPS I fibroblasts were cocultured in the presence of
untreated deficient cells in a trans- well system and labeled for 72 h,
with labeling protein mixture in the absence or in the presence of 5 m
M
mannose 6-phosphate, as described in Materials and methods. The
conditioned medium from the upper chambers was then collected and
concentrated; the recipient cells in the lower chambers were harvested.
IDUA molecules were immunoprecipitated and subjected to SDS/
PAGE and autoradiography. The molecular masses of protein
standards are indicated on the left. M, medium; C, cell lysate. The
arrows on the right indicate the 76 kDa precursor form and the
66 kDa mature form of the enzyme.
Ó FEBS 2002 Lentiviral vector-mediated IDUA gene transfer (Eur. J. Biochem. 269) 2769
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