Báo cáo Y học: In vitro gene therapy of mucopolysaccharidosis type I by lentiviral vectors pdf

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 cDNAin MPS I fibroblasts.. Puls

In vitro gene therapy of mucopolysaccharidosis type I by lentiviral vectors

Paola Di Natale1, Carmela Di Domenico1, Guglielmo R D Villani1, Angelo Lombardo2, Antonia Follenzi2 and Luigi Naldini2

1

Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II; Italy2Laboratory 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

cDNAin 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

two months In addition, transduced MPS I fibroblasts were

capable of clearing intracellular radiolabeled

glycosami-noglycan (GAG) Pulse-chase experiments on transduced

fibroblasts showed that the recombinant enzyme was synthesized as a 76-kDa precursor form and processed to a 66-kDa mature form; it was released from transduced cells and was endocytosed into a second population of untreated MPS I fibroblasts via a mannose 6-phosphate receptor These results suggest that the lentiviral vector may be used for the delivery and expression of the IDUAgene to cells

in vivofor treatment of MPS I

Keywords: MPS I; Hurler syndrome; a-L-iduronidase; gene therapy

Mucopolysaccharidosis type I (MPS I) is a lysosomal

disease due to mutations in the gene encoding

a-L-iduronidase (IDUA, EC 3.2.1.76) The gene defect

causes a specific deficiency that results in the intracellular

accumulation and storage of the unprocessed

glycosami-noglycans (GAG), dermatan sulfate and heparan sulfate

Patients with MPS I show variable clinical phenotypes, the

most severe of which is Hurler’s syndrome, with

progres-sive neurological dysfunction and skeletal and soft tissue

anomalies that can lead to death within the first decade In

less severe forms of this disease, such as the intermediate

Hurler–Scheie syndrome or the mild Scheie syndrome, no

mental retardation and only mild symptoms occur [1] The

difference in severity is due primarily to the effect of

various mutations in the human IDUAgene, 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 IDUAcDNAin 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 Anaturally 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 recent results obtained with the use of lentiviral vectors on two lysosomal diseases [21,22] have allowed us to initiate a program aimed at developing gene therapy for MPS type I syndrome As a first step in this project, we have constructed

a lentiviral vector carrying the human IDUAcDNAand have tested it in vitro on fibroblasts from affected patients Here, we describe the capacity of this vector to mediate high levels of IDUAexpression in transduced cells In addition,

we demonstrate that the IDUAenzyme released from transduced cells is endocytosed and correctly processed in nontransduced cells, indicating the potential for metabolic cross-correction and for the therapeutic application of this system

Correspondence to P Di Natale, Department of Biochemistry and

Medical Biotechnologies, University of Naples Federico II, Via S.

Pansini, 5, 80131 Naples, Italy.

Fax: + 39 081 7463150, E-mail: dinatale@cds.unina.it

Abbreviations: IDUA, a- L -iduronidase; GAG, glycosaminoglycan;

MPS I, mucopolysaccharidosis type I; ERT, enzyme replacement

therapy; DMEM, Dulbecco’s modified Eagle’s medium; PPT,

polypurine tract.

(Received 2 January 2002, revised 19 March 2002,

accepted 22 April 2002)

M A T E R I A L S A N D M E T H O D S

Cells

Normal human diploid fibroblasts were obtained from skin

biopsies, while Hurler fibroblasts were cells 72/92 obtained

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)1penicillin, 100 mgÆmL)1streptomycin

and 2 mM L-glutamine Cells were cultured at 37C in a 5%

CO2humidified incubator

Lentiviral vector containing IDUA cDNA

The human IDUAcDNAwas excised from expression

plasmid pBSIIKS-hIdu obtained from E F Neufeld,

UCLA School of Medicine, Los Angeles, CA, USA

The 2.2-Kb human IDUAcDNAwas 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 MscI and MluI 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,

the pRSV-Rev construct expressing Rev protein, the

pMD2.G, which produces the VSV.G protein and

the plasmid pRRLsinPPT.CMV.IDUA.WPRE (or

pRRLsinPPT.CMV.GFP.WPRE as control construct)

The viral particles were obtained by concentration of

293T-conditioned media through ultracentrifugation at

50 000 g for 140 min Quantification of viral content was

performed by immunocapture of HIV-1 p24 antigen by

using HIV-1 p24 Core profile ELISA(NEN Life Science

Products)

Lentiviral infection of MPS I fibroblasts

MPS I fibroblasts were grown at
80% confluency, and

transduced with increasing doses of IDUAvector (1, 10

and 50 ng of p24 viral protein) in the absence or in the

presence of polybrene (5 lgÆmL)1) After a 24-h incubation

at 37C, the medium was changed, and the cells were

incubated for 2 additional days before harvesting For

expression persistence over the course of time, MPS I

fibroblasts were transduced with 50 ng p24, and replicate

plates were maintained and expanded until cells showed

aging (i.e 2 months) After harvesting, the IDUA enzyme

activity was measured in the cell lysates To evaluate the

transduction efficiency, control infections were performed

using 20 ng of green fluorescent protein (GFP) vector;

after incubation fibroblasts grown on coverslips were fixed

with 3.7% formaldehyde in NaCl/Pi, washed once with

0.1Mglycine in NaCl/Pi, twice with NaCl/Piand once with

H2O 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 (95C) followed by 30 cycles at 94 C for 1 min, 57 C (for the 5¢Alu-PCR) or 49 C (for the 3¢Alu-PCR) for

1 min, and 72C 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 TTT-3¢), amplifying a fragment of 121 bp [26] For the 3¢ nested PCR the primers used were: Dnef (sense: 5¢-CGAGCTCGGTACCTTTAAGACC-3¢) and LTR8 (antisense: 5¢-TCCCAGGCTCAGATCTGGTCTAAC-3¢) amplifying a fragment of 166 bp [26] Nested PCR condi-tions were similar to the first amplification, differing in dNTPs and primer concentrations (0.2 mM and 0.5 lM, respectively) and in the annealing temperature, which was

55C for the 5¢ nested PCR and 58 C for the 3¢ nested PCR As a control, a nested PCR was performed using 1 lL

of nonamplified lysate from transduced fibroblasts a-L-Iduronidase enzyme assay

a-L-Iduronidase activity in untreated and transduced fibro-blasts was determined as previously described [27] using a fluorogenic substrate Cells were harvested with trypsin, washed in NaCl/Pi, resuspended in 0.9% NaCl, and lysed

by six cycles of freezing-thawing The protein concentration was quantified using the Lowry assay [28] The reaction was performed in 0.1Mformate pH 3.2, with 2 mM 4-methyl-umbelliferyl-a-L-iduronide (Calbiochem), at 37C for 1 h The reaction was stopped by adding 0.5M Na2CO3/ NaHCO3buffer, pH 10.7 The liberated 4MU 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 SO4-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

40· 106counts per min (c.p.m.) of H352SO4 (Amersham Pharmacia Biotech) per plate After 48 h at 37C, cells were harvested and washed three times in NaCl/Pi; the pellet was resuspended in 10 mMsodium phosphate pH 5.8 contain-ing 0.5% NP-40, and lysed by three cycles of freezcontain-ing and

thawing Lysates were cleared by centrifugation at

6200 g for 5 min in an Eppendorf microfuge and the

supernatants were assessed for radioactivity and total cell

proteins

Metabolic labeling of recombinant IDUA in transduced

MPS I fibroblasts

Fibroblasts transduced with 50 ng p24 were metabolically

labeled 3 days after infection In detail, the cells were placed

in 6-cm plates and starved for 2 h in 1.5 mL

methionine-cysteine-free medium (DMEM, ICN Biomedicals)

supple-mented with 2% fetal bovine serum This medium was then

changed for one of the same composition containing

300 lCi of35S-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)1nonradioactive 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/Pi

and lysed on ice for 30 min in 1 mL lysis buffer (10 mM

Tris/HCl buffer pH 7.4, 150 mM NaCl, 1 mM EDTA

pH 8.0, 0.1% Triton X-100) containing 0.2 mM

phenyl-methanesulfonyl fluoride, 1 lM pepstatin Aand 1 lM

leupeptin Labeled a-L-iduronidase was

immunopreci-pitated from cells and medium using 1 lL of specific

antiserum The immunocomplexes were precipitated with

30 lL protein A-agarose (Santa Cruz Biotechnology),

washed four times with lysis buffer and analyzed by

SDS/PAGE followed by autoradiography

Correction of MPS I fibroblasts by enzyme released

from transduced cells

MPS I fibroblasts were grown in 6-cm plates and

trans-duced with 50 ng p24 of IDUAvector Three days after

viral infection, transduced cells were placed in the upper

chambers of a trans-well system (Costar, Cambridge, MA,

USA; 0.45 lm) and co-cultivated in the presence of deficient

cells grown in the lower chambers After a 72-h incubation,

cells were harvested and the enzyme activity was measured

in the cell lysates The experiments were performed in the

absence or in the presence of mannose 6-phosphate at a final

concentration of 5 mM In a similar set of experiments,

transduced fibroblasts in the upper chambers were starved

2 h with methionine-cysteine-free medium and labeled with

35S-Express Protein Labeling Mix for 72 h The recipient

untransduced cells in the lower chambers and the

condi-tioned medium from transduced fibroblasts were then

harvested, immunoprecipitated and processed as described

above

R E S U L T S

Lentiviral vector expressing human IDUA cDNA

Athird-generation VSV-pseudotyped lentiviral vector

con-taining IDUAcDNAwas constructed and prepared using a

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 IDUAtransduction 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)1Æmg)1) or in the presence of polybrene (means of 20, 32, and 155 nmolÆh)1Æmg)1) The IDUAactivity obtained after infection was 1.5 the level observed in normal control cells (98 nmolÆh)1Æmg)1) Transduction efficiency was evaluated by detection of fluorescence after infection of MPS I cells with 20 ng

of lentiviral-GFP; efficiency was calculated as number of

Fig 1 Diagram of lentiviral vector carrying the human IDUA cDNA Gene transfer vector includes the following elements from the 5¢ to the 3¢ end: viral cis-acting sequences (5¢ LTR region; splice donor site, 5¢ss; encapsidation signal, w; a portion of the HIV-1 gag gene, mut GAG; Rev response element, RRE; splice acceptor sites, 3¢ss; polypurinic and termination HIV-1 pol sequences enhancing nuclear translocation, PPT); the expression cassette for IDUAcDNAwith the promoter of the human cytomegalovirus CMV and the post-transcriptional regulatory element from woodchuck hepatitis virus WPRE; 3¢ LTR sequences.

Table 1 a- L -Iduronidase activity in MPS I fibroblasts after transduc-tion with lentiviral vector Increasing amounts of IDUAvector were added to MPS I fibroblasts as indicated Cells, in triplicate plates, were incubated for 2.5 days before harvesting for IDUAactivity Data show mean ± SD Untreated MPS I fibroblasts have an enzyme activity of 0.25 ± 0.03 nmolÆh)1Æmg)1; normal fibroblasts have an enzyme activity of 98 ± 13 nmolÆh)1Æmg)1.

IDUAVector (ng p24)

a- L -Iduronidase activity (nmolÆh)1Æmg)1) – Polybrene + Polybrene

50 117 ± 19.31 155 ± 18.1

fluorescent cells over the total number of cells and was

estimated to be 70% (data not shown)

Long-term IDUA transgene expression in transduced

MPS I fibroblasts

To investigate the IDUAexpression over the course of many

weeks, MPS I fibroblasts were transduced with 50 ng of p24

viral protein, in the presence of polybrene Transduced cells

were subcultured every week and maintained for 2 months,

during which time cell samples were collected every 5–7 days

In transduced cells, the specific IDUAactivity showed a

linear increase, with a highest point found 3 weeks after

transduction (520 nmolÆh)1Æmg)1), and persisted at high

levels (about 400 nmolÆh)1Æmg)1) for almost 8 weeks, when

the experiment was concluded due to cell aging (Fig 2A)

The increase of IDUAactivity to four to five times normal

levels from the initial levels after culture in the absence of

selection was probably due to some selective advantage

of metabolically corrected cells To assess vector integration

in transduced MPS I fibroblasts, Alu-PCR reactions were

performed, as described in Materials and methods, on

cellular extracts prepared from untreated and transduced

fibroblasts on day 27 after infection Reactions included a

first amplification using two different primer pairs specific

for Alu ubiquitous repeats and for 3¢ or 5¢ end of the

lentiviral vector genome, respectively, followed by a nested

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 H352SO4 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 IDUAenzyme was

studied through metabolic labeling experiments in

trans-duced fibroblasts as described in Materials and methods

Three days after infection, transduced cells were labeled for

a 2-h pulse period and harvested after a 24-h chase period

In addition, the medium surrounding cells was harvested

after the same 24 h chase period to see the precursor form of

the enzyme Radioactivity incorporated into IDUAwas

shown after immunoprecipitation and separation by SDS/

PAGE In the medium a precursor form of 76 kDa was

identified; in the cells a mature polypeptide of 66 kDa was detected, as shown after the 24 h chase (Fig 3) These results on the maturation of the recombinant protein are in agreement with those previously obtained on IDUA processing in cultured cells [29,30]

Correction of MPS I fibroblasts by enzyme released from transduced cells

To evaluate the capacity of transduced cells to release the IDUAenzyme to the extracellular environment, from which

it can be taken up and contribute to lysosomal metabolism

in nontransduced cells, coculture experiments were per-formed as described in Materials and methods As shown in Fig 4, the deficient cells cultured in the presence of transduced MPS I fibroblasts exhibited an enzyme activity

Fig 2 Persistence of vector-mediated transduction (A) Enzyme activity

in MPS I fibroblasts transduced with 50 ng p24 viral protein Replicate wells of MPS I cells were exposed to the indicated viral dose as des-cribed in Materials and methods and cells were collected at different time points from infection; lysates were assayed for protein content and enzyme activity (mean ± SD, n ¼ 3) (B) Alu-PCR analysis to test the lentiviral-vector integration Untreated and transduced cell extracts were assayed by PCR as described in Materials and methods Ampli-fication was performed with two different primer pairs, one at the 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.

of 116 nmolÆh)1Æmg)1, vs an activity of 0.3 nmolÆh)1Æmg)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 mMmannose 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 endocytosed by recipient cells to become the mature 66 kDa protein In the presence of mannose 6-phosphate, no mature form was immunoprecipitated from recipient cells, thus demonstrating that transduced MPS I fibroblasts can secrete a functional IDUAenzyme that can enter enzyme-deficient cells by the well characterized mannose 6-phos-phate mechanism [18]

D I S C U S S I O N

MPS I has always been considered a candidate for gene therapy because high levels of IDUAexpression may not be necessary to correct the lysosomal metabolism It has been reported, in fact, that the presence of less than 1% of normal

Table 2 Correction of 35 S-glycosaminoglycan accumulation in transduced MPS I fibroblasts MPS I fibroblasts in 6-cm dishes were transduced with

50 ng p24 viral protein; six (experiment 1) or 13 days (experiment 2) after transduction, treated and untreated cells were incubated in SO 4 -free medium overnight Fibroblasts were then added with fresh medium in the presence of 40 · 10 6 c.p.m of H 35

2 SO 4 per dish, and incubated for 48 h Cells were harvested and assayed for 35 SO 4 -glycosaminoglycan accumulation and protein content (mean ± SD, n ¼ 3) as described in Materials and methods U, untreated; T, transduced; N, normal fibroblasts.

35 S-Glycosaminoglycans (c.p.m per mg)

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 35S-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 releasedfrom transduced deficient cells Transduced fibroblasts, which can secrete the IDUAenzyme, 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.

IDUAactivity in patients will moderate the severe clinical

symptoms related to Hurler syndrome [1]

Athird generation lentiviral vector was constructed to

transduce human a-L-iduronidase cDNAinto MPS I

fibroblasts The vector contained only a fractional set of

HIV genes: gag, pol and rev, which are nonfunctional

outside the producer cells [23,24] High transduction

efficiency (70%) was found and the lentiviral vector

proved to be integrated in transduced cell, as seen by

Alu-PCR analysis Transduced MPS I fibroblasts

expres-sed high levels of IDUAactivity (1.5-fold the normal

control cells) and reduced levels of 35S-labeled

glycos-aminoglycans, approaching those observed in normal

control cells IDUAactivity persisted in transduced cells

for at least 2 months; after that time it was not

monitored due to cell aging In addition, transduced

fibroblasts showed correct processing of the IDUA

protein, from the precursor form of 76 kDa to the

mature form of 66 kDa Even more importantly, the

excess enzyme released into the surrounding medium of

transduced cells was endocytosed by the deficient cells

through the well-characterized mannose

6-phosphate-mediated receptor mechanism [17] and was trimmed

from the 76 kDa precursor form to the characteristic

intracellular mature form of 66 kDa These results are

particularly important in the gene transfer approaches to

treatment of mucopolysaccharidosis type I because they

imply that the introduction of a therapeutic IDUAgene

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 vitrocorrection 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 IDUAgene transfer into MPS I CD34+cells, with high levels of IDUAactivity detectable in a significant percentage of these cells Huang et al [14] characterized

a series of retroviral IDUAvectors 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 IDUAretroviral vector; Pan et al [16] compared the efficacy of different IDUAretroviral constructs in expres-sing IDUAcDNAin MPS I CD34+cells and in MPS I fibroblasts; Hartung et al [17] used an IDUA adeno-associated vector to transduce 293 cells and MPS I fibroblasts, obtaining high levels of IDUAactivity and intercellular metabolic cross-correction

Here, we have extended these transfer studies to verify the lentivirus as an effective system for IDUAgene delivery and expression at levels sufficient to provide cross-correction of co-incubated, untransduced cells Len-tiviral vectors have been shown to effectively transduce genes into a range of both dividing and nondividing cell types, including neurons, retinal cells, muscle cells and hematopoietic pluripotent cells [31–35] More recently, a late generation lentiviral vector was used to deliver arylsulfatase AcDNAinto the brain of metachromatic leukodystrophy mice [21] and b-glucuronidase cDNAinto the brain of mucopolysaccharidosis type VII mice [22], showing that the in vivo transfer of genes by lentiviral vectors reverts the disease phenotype in all areas investi-gated

In conclusion, the lentiviral vector reported here pro-vides high levels of IDUAexpression in recipient cells

in vitro and may also provide sufficient expression in deficient cells and tissues in vivo to similarly reduce or prevent storage accumulation in IDUA-deficient animals and then in MPS I patients The existing knock-out murine model [20] therefore provides an excellent test system Future studies will test the ability of this vector to mediate gene transfer and expression in appropriate targets

in vivo (muscle, brain, hematopoietic cells) and correct GAG storage in murine MPS I cells as a model for gene therapy of human MPS I

A C K N O W L E D G E M E N T S

We thank Prof Elizabeth F Neufeld, UCLASchool of Medicine, for providing us with the pBSIIKS-hIdu plasmid and anti-IDUAanti-bodies.

We thank the Laboratorio di Diagnosi Pre-Postnatale Malattie Metaboliche Istituto G Gaslini for providing us with specimens from the collection Cell lines and DNAbank 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 secretedrecombinant 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.

IDUAmolecules 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.

R E F E R E N C E S

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3421–3452 McGraw-Hill, New York:

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Callen, D.F., Sutherland, G.R., Morris, C.P & Hopwood, J.J.

(1990) Chromosomal localization of the human alpha- L

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3 Scott, H.S., Anson, D.S., Orsborn, A.M., Nelson, P.V., Clements,

P.R., Morris, C.P & Hopwood, J.J (1991) Human a- L

-iduroni-dase: cDNAisolation and expression Proc Natl Acad Sci USA

88, 9695–9699.

4 Scott, H.S., Guo, X.H., Hopwood, J.J & Morris, C.P (1992)

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