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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.

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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.

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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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