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

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 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 IDUA gene to cells in vivo for 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 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 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 IDUA cDNA and have tested it in vitro on fibroblasts from affected patients. Here, we describe the capacity of this vector to mediate high levels of IDUA expression in transduced cells. In addition, we demonstrate that the IDUA enzyme 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) Eur. J. Biochem. 269, 2764–2771 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02951.x MATERIALS AND METHODS 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 )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, 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 IDUA vector (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 37 °C, 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/P i , washed once with 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 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 m M and 0.5 l M , respectively) and in the annealing temperature, which was 55 °Cforthe5¢ nested PCR and 58 °Cforthe3¢ 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/P i , 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 0.1 M 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 40 · 10 6 counts per min (c.p.m.) of H 35 2 SO 4 (Amersham Pharmacia Biotech) per plate. After 48 h at 37 °C, cells were harvested and washed three times in NaCl/P i ; the pellet was resuspended in 10 m M sodium phosphate pH 5.8 contain- ing 0.5% NP-40, and lysed by three cycles of freezing and Ó FEBS 2002 Lentiviral vector-mediated IDUA gene transfer (Eur. J. Biochem. 269) 2765 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 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 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 IDUA vector. 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 m M . In a similar set of experiments, transduced fibroblasts in the upper chambers were starved 2 h with methionine-cysteine-free medium and labeled with 35 S-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. RESULTS Lentiviral vector expressing human IDUA cDNA A third-generation VSV-pseudotyped lentiviral vector con- taining IDUA cDNA was 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 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 Æmg )1 ) or in the presence of polybrene (means of 20, 32, and 155 nmolÆh )1 Æmg )1 ). The IDUA activity obtained after infection was 1.5 the level observed in normal control cells (98 nmolÆh )1 Æmg )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 IDUA cDNA with 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 IDUA vector were 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 MPS I fibroblasts To investigate the IDUA expression 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 IDUA activity showed a linear increase, with a highest point found 3 weeks after transduction (520 nmolÆh )1 Æmg )1 ), and persisted at high levels (about 400 nmolÆh )1 Æmg )1 ) for almost 8 weeks, when the experiment was concluded due to cell aging (Fig. 2A). The increase of IDUA activity 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 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. 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 IDUA was 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 IDUA enzyme 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. 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 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 IDUA enzyme that can enter enzyme- deficient cells by the well characterized mannose 6-phos- phate mechanism [18]. DISCUSSION MPS I has always been considered a candidate for gene therapy because high levels of IDUA expression 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)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 symptoms related to Hurler syndrome [1]. A third generation lentiviral vector was constructed to transduce human a- L -iduronidase cDNA into 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 IDUA activity (1.5-fold the normal control cells) and reduced levels of 35 S-labeled glycos- aminoglycans, approaching those observed in normal control cells. IDUA activity 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 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- associated vector to transduce 293 cells and MPS I fibroblasts, obtaining high levels of IDUA activity and intercellular metabolic cross-correction. Here, we have extended these transfer studies to verify the lentivirus as an effective system for IDUA gene 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 A cDNA into the brain of metachromatic leukodystrophy mice [21] and b-glucuronidase cDNA into 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 IDUA expression 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. 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 REFERENCES 1. Neufeld, E.F. & Muenzer, J. (2001) The mucopolysaccharidoses. In Scriver, C.R., Beaudet, A.L., Sly, W.S. & Valle, D., eds. The Metabolic and Molecular Bases of Inherited DiseaseÕ, 8th edn. pp. 3421–3452. McGraw-Hill, New York: 2. Scott,H.S.,Ashton,L.J.,Eyre,H.J.,Baker,E.,Brooks,D.A., Callen, D.F., Sutherland, G.R., Morris, C.P. & Hopwood, J.J. (1990) Chromosomal localization of the human alpha- L -iduroni- dase gene (IDUA) to 4p16.3. Am.J.Hum.Genet.47, 802–807. 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: cDNA isolation and expression. Proc. Natl Acad. Sci. USA 88, 9695–9699. 4. Scott, H.S., Guo, X.H., Hopwood, J.J. & Morris, C.P. (1992) Structure and sequence of the human a- L -iduronidase gene. Genomics 13, 1311–1313. 5. Scott, H.S., Bunge, S., Gal, A., Clarke, L.A., Morris, C.P. & Hopwood, J.J. (1995) Molecular genetics of mucopolysacchari- dosis type I: diagnostic, clinical, and biological implication. Hum. Mutat. 6, 288–302. 6. Beesley, C.E., Meaney, C.A., Greenland, G., Adams, V., Vellodi, A., Young, E.P. & Winchester, B.G. (2001) Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations. Hum. Genet. 109, 503–511. 7. Gatti, R., Di Natale, P., Villani, G.R.D., Filocamo, M., Muller, V., Guo, X.H., Nelson, P.V., Scott, H.S. & Hopwood, J.J. (1997) Mutations among Italian mucopolysaccharidosis type I patients. J. Inherit. Metab. Dis. 20, 803–806. 8. Withley, C.B., Belani, K.G., Chang, P.N., Summers, C.G., Blazar, B.R., Tsai, M.Y., Latchaw, R.E., Ramsay, N.K. & Kersey, J.H. (1993) Long-term outcome of Hurler syndrome following bone marrow transplantation. Am.J.Med.Genet.46, 209–218. 9. Vellodi, A., Young, E.P., Cooper, A., Wraith, J.E., Winchester, B., Meaney, C., Ramaswami, U. & Will, A. (1997) Bone marrow transplantation for mucopolysaccharidosis type I: experience of two British centers. Arch.Dis.Child.76, 92–99. 10. Peters, C., Shapiro, E.G., Anderson, J., Henslee-Downey, P.J., Klemperer, M.R., Cowan, M.J., Saunders, E.F., DeAlarcon, P.A., Twist, C., Nachman, J.B., Hale, G.A., Harris, R.E., Rozans, M.K., Kurtzberg, J., Grayson, G.H., Williamson, T.E., Lenarsky, C., Wagner, J.E. & Krivit, W. (1998) Hurler syndrome. Outcome of HLA-genotypically identical sibling and HLA-haploidentical related donor bone marrow transplantation in fifty-four children. The storage Disease Collaborative Study Group. Blood 91, 2601– 2608. 11. Kakkis, E.D., Muenzer, J., Tiller, G.E., Waber, L., Belmont, J., Passage,M.,Izykowski,B.,Phillips,J.,Doroshow,R.,Walot,I., Hoft, R. & Neufeld, E.F. (2001) Enzyme-replacement therapy in mucopolysaccharidosis I. N. Engl. J. Med. 344, 182–188. 12. Anson, D.S., Bielicki, J. & Hopwood, J.J. (1992) Correction of mucopolysaccharidosis type I fibroblasts by retroviral-mediated transfer of the human alpha- L -iduronidase gene. Hum. Gene Ther. 3, 371–379. 13. Fairbairn, L.J., Lashford, L.S., Spooncer, E., McDermott, R.H., Lebens, G., Arrand, J.E., Arrand, J.R., Bellantuono, I., Holt, R., Hatton, C.E., Cooper, A., Besley, G.T., Wraith, J.E., Anson, D.S., Hopwood, J.J. & Dexter, T.M. (1996) Long-term in vitro correc- tion of a- L -iduronidase deficiency (Hurler syndrome) in human bone marrow. Proc. Natl Acad. Sci. USA 93, 2025–2030. 14. Huang, M.M., Wong, A., Yu, X. & Kohn, D.B. (1997) Retrovi- rus-mediated transfer of the human a- L -iduronidase cDNA into human hematopoietic progenitor cells leads to correction in trans of Hurler fibroblasts. Gene Ther. 4, 1150–1159. 15. Stewart, K., Brown, O.A., Morelli, A.E., Fairbairn, L.J., Lash- ford, L.S., Cooper, A., Hatton, C.E., Dexter, T.M., Castro, M.G. & Lowenstein, P.R. (1997) Uptake of a- L -iduronidase produced by retrovirally transduced fibroblasts into neuronal and glial cells in vitro. Gene Ther. 4, 63–75. 16. Pan, D., Aronovich, E.L., McIvor, R.S. & Whitley, C.B. (2000) Retroviral vector design studies toward hematopoietic stem cell gene therapy for mucopolysaccharidosis type I. Gene Ther. 7, 1875–1883. 17. Hartung, S.D., Reddy, R.G., Whitley, C.B. & McIvor, R.S. (1999) Enzymatic correction and cross-correction of mucopolysaccari- dosis type I fibroblasts by adeno-associated virus mediated transduction of the a- L -iduronidase gene. Hum. Gene. Ther. 10, 2163–2172. 18. Kornfeld, S. & Mellman, I. (1989) The biogenesis of lysosomes. Ann. Rev. Cell. Biol. 5, 483–525. 19. Kakkis, E.D., McEntee, M.F., Schmidtchen, A., Neufeld, E.F., Ward, D.A., Gompf, R.E., Kania, S., Bedolla, C., Chien, S.L. & Shull, R.M. (1996) Long-term and high-dose trials of enzyme replacement therapy in the canine model of mucopolysacchari- dosis I. Biochem. Mol. Med. 58, 156–167. 20. Clarke, L.A., Russel, C.S., Pownall, S., Warrington, C.L., Borowski,A.,Dimmick,J.E.,Toone,J.&Jirik,F.(1997)Murine mucopolysaccharidosis type I: targeted disruption of the murine a- L -iduronidase gene. Hum. Mol. Genet. 64, 503–511. 21. Consiglio, A., Quattrini, A., Martino, A., Bensadoun, J.C., Dolcetta, D., Troiani, A., Benaglia, G., Marchesini, S., Cestari, V., Oliverio, A., Bordignon, C. & Naldini, L. (2001) In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice. Nat. Med. 7, 310–316. 22. Bosch, A., Perret, E., Desmaris, N., Trono, D. & Heard, J.M. (2000) Reversal of pathology in the entire brain of mucopolysac- charidosis type VII mice after lentivirus-mediated gene transfer. Hum. Gene Ther. 11, 1139–1150. 23. Dull, T., Zufferey, R., Kelly, M., Mandel, R.J., Nguyen, M., Trono, D. & Naldini, L. (1998) A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463– 8471. 24. Follenzi, A., Ailles, L.E., Bakovic, S., Geuna, M. & Naldini, L. (2000) Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat. Genet. 25, 217–222. 25. Sonza, S., Maerz, A., Deacon, N., Meanger, J., Mills, J. & Crowe, S. (1996) Human immunodeficiency virus type 1 replication is blocked prior to reverse transcription and integration on freshly isolated peripheral blood monocytes. J. Virol. 70, 3863–3869. 26. Follenzi, A., Sabatino, G., Lombardo, A., Boccaccio, C. & Naldini, L. (2001) Efficient gene delivery and targeted expression to hepatocytes in vivo by improved lentiviral vectors. Hum. Gene Ther. in press. 27. Hopwood, J.J., Muller, V., Smithson, A. & Baggett, N. (1979) A fluorimetric assay using 4-methylumbelliferyl alpha- L -iduronide for the estimation of alpha- L -iduronidase activity and the detection of Hurler and Scheie syndromes. Clin. Chim. Acta 92, 257–265. 28. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. & Randall, K.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 256–276. 29. Kakkis, E.D., Matynia, A., Jonas, A.J. & Neufeld, E.F. (1994) Overexpression of human lysosomal enzyme alpha- L -iduronidase in Chinese hamster ovary cells. Protein Expr. Purif. 5, 225–232. 30. Myerowitz, R. & Neufeld, E.F. (1981) Maturation of a- L -iduronidase in cultured human fibroblasts. J. Biol. Chem. 256, 3044–3048. 31. Naldini, L. & Verma, I.M. (2000) Lentiviral vectors. Adv. Virus Res. 55, 599–609. 32. Negre, D., Mangeot, P.E., Duisit, G., Blanchard, S., Vidalain, P.O., Leissner, P., Winter, A.J., Rabourdin-Combe, C., Mehtali, 2770 P. Di Natale et al. (Eur. J. Biochem. 269) Ó FEBS 2002 M., Moullier, P., Darlix, J.L. & Cosset, F.L. (2000) Characteri- zation of novel safe lentiviral vectors derived from simian immu- nodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells. Gene Ther. 7, 1613–1623. 33. Johnson, L.G., Olsen, J.C., Naldini, L. & Boucher, R.C. (2000) Pseudotyped human lentiviral vector-mediated gene transfer to airway epithelia in vivo. Gene Ther. 7, 568–574. 34. Richter, J. & Karlsson, S. (2001) Clinical gene therapy in hema- tology: past and future. Int. J. Hematol. 73, 162–169. 35. Pfeifer, A., Kessler, T., Yang, M., Baranov, E., Kootstra, N., Cheresh, D.A., Hoffman, R.M. & Verma, I.M. (2001) Transduc- tion of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging. Mol. Ther. 3, 319–322. Ó FEBS 2002 Lentiviral vector-mediated IDUA gene transfer (Eur. J. Biochem. 269) 2771 . b-glucuronidase cDNA into the brain of mucopolysaccharidosis type VII mice [22], showing that the in vivo transfer of genes by lentiviral vectors reverts the disease. In vitro gene therapy of mucopolysaccharidosis type I by lentiviral vectors Paola Di Natale 1 , Carmela Di Domenico 1 , Guglielmo R. D. Villani 1 ,