In Vitro Correction of Iduronate-2-Sulfatase Deficiency By
Total Page：16
File Type：pdf, Size：1020Kb
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 Natale2, DS Anson3, JJ Hopwood3, FL Graham4 and M Scarpa1 1Department of Pediatrics and Center for Biotechnology CRIBI, University of Padova, Italy; 2Department of Biochemistry and Medical Biotechnology, University of Napoli ‘Federico II’, Italy; 3Lysosomal 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 response. We describe the generation of a replication- caused by the deficiency of iduronate-2-sulfatase and defective adenoviral vector, AdRSVIDS, which is able to characterized by severe skeletal and neurological symp- express in vitro high levels of iduronate-2-sulfatase. After toms. Only symptomatic treatments are available and, infection, accumulation of mucopolysaccharides in treated although bone marrow transplantation has been sug- Hunter cells was normalized. Furthermore, endocytosis of gested, no encouraging results have been obtained so far. the transduced IDS did occur via the mannose-6-phos- Therefore, gene therapy might be a route to be pursued phate (M6P) receptor. Since no animal model for the dis- for treatment of the disease. In this respect, one major goal ease is available, we developed a system based on the to achieve is the generation of an overexpressing vector generation of derma-equivalents which enabled us to verify able to correct, in particular, central nervous system (CNS) the expression of high levels of sulfatase up to 30 days cells. Adenoviruses have been shown to infect CNS cells after infection. efficiently with minor or even absent immunological Keywords: gene therapy; mucopolysaccharidosis type II; adenovirus; derma-equivalents Introduction age. Facial features, hepatosplenomegaly, short stature, skeletal deformities, joint stiffness, severe retinal Mucopolysaccharidoses (MPS) are a group of 10 dis- degeneration and hearing impairment are coupled with orders caused by the deficiency of lysosomal enzymes an incremental deterioration of the neurological system. (four glycosidases, five sulfatases and one non-hydrolytic Death generally occurs between the ages of 10 and 14 transferase) needed for the catabolism of glycosaminogly- years. cans (GAG): dermatan-, heparan-, keratan- and chondro- No mental impairment is characteristic of the mild itin-sulfate (mucopolysaccharides). Their accumulation in form, however, skeletal deformities can be present to the lysosomes results in cell, tissue or organ dysfunction same degree as in the severe form. Retinal and hearing determining various chronic and progressive patterns of problems are milder than in the severe form and the 1 clinical severity, even within each enzyme deficiency. patient can survive until the fifth or sixth decade. Usually All MPS, except Hunter syndrome, are inherited as cardiac failure or airway obstruction are the cause of autosomal recessive disorders. Hunter syndrome, MPS death. Only symptomatic treatments are available for type II, is a rare X-linked inborn error of metabolism MPS in general. Transient improvement of patient con- characterized by the deficiency of iduronate-2-sulfatase ditions have been obtained with leukocyte and plasma (IDS) (E.C. 3.1.6.13), which removes the sulfate group in infusions,6,7 while fibroblast and amnion transplantations dermatan- and heparan-sulfate. The defect is due to point have not been successful in restoring, even partially, any mutations or deletions in the 24 kb gene, mapping on enzyme activity or improving the objective signs.8–10 2 3,4 Xq28.2. The cDNA was cloned as a 2.3 kb sequence, Bone marrow transplantation (BMT) has been sug- and successfully used to produce a recombinant active gested as a potential method for enzyme supplement for 5 enzyme in CHO cells. MPS patients,11,12 since enzyme replacement therapy is Hunter syndrome occurs in a severe and a mild form. not available yet. In the case of MPSII, however, the value The severe form is characterized by progressive somatic of BMT still has to be investigated. and neurological involvements. The onset of the disease Correction by gene therapy might represent another usually occurs between the second and fourth year of route to be pursued. High levels of recombinant human lysosomal enzymes were obtained in vitro13,14 and in vivo15 by retrovirus-mediated gene transfer in different Correspondence: M Scarpa, Department of Pediatrics and CRIBI, Via MPS as well as a successful metabolic correction of Trieste 75, 35121 Padova, Italy Hunter lymphoblastoid cell lines.16 A phase I clinical trial Received 5 November 1996; accepted 8 January 1997 aimed at increasing the enzyme level in Hunter patients Gene transfer in mucopolysaccharidosis type II C Di Francesco et al 443 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 long- term expression experiments, we included infected pri- Figure 1 The vector pXCRSVIDS pA used to cotransfect 293 cells with the plasmid pJM17 to generate AdRSVIDS. AmpR: ampicillin resistance; mary fibroblasts from Hunter patients into collagen matr- Ad5: 5′ sequence 1–452 bp; RSV: Rous sarcoma virus LTR; preproins. 23,24 ices (derma-equivalents). This technique enabled us to leader seq.: 5′ rat preproinsulin leader sequence: 48 bp; IDS: IDS cDNA; show expression of the virus-transduced IDS up to 30 SV40 pA: SV40 polyA containing the small intron; Ad5: Ad5 3′ sequence days after infection, confirming that AdRSVIDS might be 3328–5788 bp. a valuable vector for gene therapy of Hunter syndrome. Results was obtained on non-reverse transcribed total RNA (Figure 2). Construction of pXCRSVIDSpA The 578 bp Rous sarcoma virus long terminal repeat (RSV-LTR) sequence was isolated from pRSVLuc25 by IDS enzyme activity in normal and transduced NdeI–XbaI restriction, treated with Klenow polymerase fibroblasts and cloned as blunt end fragment into the filled in XbaI To show that AdRSVIDS was able to express a functional site of pXCJL1.26 The new plasmid was called pXCRSV. recombinant enzyme, IDS activity was tested on nonin- The SV40 polyA sequence containing the small intron fected and infected Hunter cells compared with normal 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. Figure 2 RT-PCR on two different Hunter fibroblasts infected with The amplification of IDS cDNA was performed with IDS- AdRSVIDS. Twenty micrograms of total RNA were retrotranscribed with specific oligonucleotides 5 and 340. A 334 bp band was oligo dT and amplified with IDS-specific oligonucleotides 423a and 424a. − detected only in infected cells (data not shown). A 585 bp product was detected only from Hunter infected cells. Lane C : The amplification of reverse transcribed total RNA amplification of noninfected Hunter cells. Lanes 1 and 3: amplification of total RNA nontreated with reverse transcriptase. Lanes 2 and 4: PCR extracted from Hunter transduced cells, performed with product from retrotranscribed RNA of infected Hunter cells. C+: positive IDS-specific oligonucleotides 423a and 424a showed a control, pXCRSVIDS pA. M: molecular weight marker VI (Boehringer 585 bp band only in infected cells. No amplification Mannheim). B: blank. Gene transfer in mucopolysaccharidosis type II C Di Francesco et al 444 AdRSVIDS. Figure 3 shows that, as expected, a three-fold Table 1 IDS activity detection in infected Hunter cells increased level (286 ± 56 c.p.m.) (bar 1) of accumulation in noninfected Hunter cells with respect to normal ones IDS expression IDS expression ± (U/mg) in cultured (U/mg) in (103 8 c.p.m.) (bar 3) was measured. GAG levels in fibroblastsa derma-equivalentsb Hunter cells were back to normal (95 ± 13 c.p.m.) after infection (bar 2). Hunter fibroblasts ,5 ,5 Control fibroblasts 81 (±10) 111 (±10) Secretion and endocytosis of recombinant IDS in Hunter Hunter cells + cells ± ± AdRSVIDS 1758 ( 120) 2580 ( 85) To evaluate IDS secretion from Hunter-infected cells, IDS activity was determined in IDS-conditioned medium aIDS activity was determined 48 h after infection. ± b detecting 33 8 U/ml.