Proc. Natl. Acad. Sci. USA Vol. 93, pp. 65-70, January 1996 Genetics High-level production of recombinant human lysosomal acid a-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease JOHAN L. K. VAN HOVE*t, HELEN W. YANG*, JER-YUARN Wu*, RoscOE 0. BRADYt, AND YUAN-TSONG CHEN* *Department of Pediatrics, Duke University Medical Center, Durham, NC 27710; and *National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 Contributed by Roscoe 0. Brady, September 11, 1995 ABSTRACT Infantile Pompe disease is a fatal genetic transfected COS cells is taken up efficiently by cultured patient muscle disorder caused by a deficiency of acid v-glucosidase, cells through the mannose-6-phosphate receptor (3, 10-12). a glycogen-degrading lysosomal enzyme. We constructed a Because of the high abundance of these receptors in heart and plasmid containing a 5'-shortened human acid c-glucosidase muscle, bovine testis enzyme injected intravenously into mice is cDNA driven by the cytomegalovirus promoter, as well as the targeted to these tissues (13). Interspecies antigenicity requires aminoglycoside phosphotransferase and dihydrofolate reduc- the use of the human enzyme (7). The low abundance in human tase genes. Following transfection in dihydrofolate reductase- urine makes this source impractical (10). The isolation of a deficient Chinese hamster ovary cells, selection with Geneti- full-length acid a-glucosidase cDNA (1) makes production of cin, and amplification with methotrexate, a cell line producing recombinant human enzyme possible. high levels of the c-glucosidase was established. In 48 hr, the Recombinant human acid a-glucosidase made in bacteria cells cultured in Iscove's medium with 5 mM butyrate secreted was not catalytically active (14), and that made in baculovirus- 11O-kDa precursor enzyme that accumulated to 91 ,ug ml-t in infected insect cells was active but was not taken up efficiently the medium (activity, >22.6 ,umol hr-l ml-"). This enzyme by a Pompe patient's fibroblasts (15). Enzyme secreted by has a pH optimum similar to that of the mature form, but a transiently transfected COS cells was active and taken up lower Vmax and Km for 4-methylumbelliferyl-ca-D-glucoside. It efficiently by a Pompe patient's fibroblasts (3). Thus, mam- is efficiently taken up by fibroblasts from Pompe patients, malian cells are required for correct posttranslational modi- restoring normal levels of acid c-glucosidase and glycogen. fications for use in enzyme replacement. Chinese hamster The uptake is blocked by mannose 6-phosphate. Following ovary (CHO) cells are most frequently used for recombinant intravenous injection, high enzyme levels are seen in heart protein production in a mammalian system. Here we describe and liver. An efficient production system now exists for a system for high-level production of recombinant human acid recombinant human acid ai-glucosidase targeted to heart and a-glucosidase in CHO cells. capable of correcting fibroblasts from patients with Pompe disease. MATERIALS AND METHODS Lysosomal acid a-glucosidase (1,4-a-D-glucan glucohydrolase; Materials. Glycogen (rabbit liver type III), Aspergillus niger EC 3.2.1.20) is an exo-1,4-a-D-glucosidase that hydrolyzes both amyloglucosidase, 4-methylumbelliferone and its derivatives, a-1,4 and a-1,6 linkages of oligosaccharides, liberating glu- the lactate diagnostic kit, and dialyzed fetal bovine serum cose. It catalyzes the complete degradation of glycogen, with (FBS) (Mr cutoff, 20,000) were from Sigma. Con A-Sepharose slowing at branching points. The 28-kb acid a-glucosidase gene 4B and Sephadex G-200 were from Pharmacia. FBS and on human chromosome 17 encodes a 3.6-kb mRNA which Geneticin were from Life Technologies (Gaithersburg, MD). produces a 951-aa polypeptide (1, 2). The enzyme receives Tissue culture media were from BioWhittaker. Restriction cotranslational N-linked glycosylation in the endoplasmic re- enzymes were from New England Biolabs. T4 DNA ligase was ticulum. It is synthesized as a 110-kDa precursor form, which from Life Technologies, and Epicurian coli TOPP competent matures by extensive modification of its glycosylation, phos- bacteria, from Stratagene. phorylation, and proteolytic processing through a 90-kDa Plasmid Constructs. The full-length acid a-glucosidase endosomal intermediate into the final lysosomal 76- and cDNA (1) was excised with EcoRI from the pSHAG2 vector 67-kDa forms (1, 3-5). (3), a gift from A. Reuser (Erasmus University, Rotterdam), In Pompe disease, a deficiency of acid a-glucosidase causes and ligated into the multicloning site of the mammalian expres- massive accumulation of glycogen in lysosomes, disrupting sion vector pcDNA3 (Invitrogen) to give pcDNA3-GAA. The cellular function (6). In the most common infantile form, cDNA was also cloned as a polycistronic construct in the EcoRI patients exhibit progressive muscle degeneration and cardio- restriction site of the pMT2 vector (16), a gift from R. J. Kaufman myopathy and die before 2 years of age. Intravenous injection (Genetics Institute, Cambridge, MA), and called pMT2-GAA. of enzyme obtained from human placenta or Aspergillus niger The dihydrofolate reductase (DHFR) gene and its adenovirus corrected enzyme and glycogen levels in liver but not in muscle major late promoter were excised from the pMT2 vector with or heart in patients with Pompe disease (7-9). Acid at-gluco- BamHI and ligated in the Bgl II site of pcDNA3-GAA. The sidase is targeted to lysosomes via the mannose-6-phosphate resultant plasmid, pJW-GAA, had the DHFR gene in the same receptor as well as by sequences associated with delayed cleavage orientation as the acid a-glucosidase gene. A 547-bp fragment, of the signal peptide (6). Mannose 6-phosphate-containing en- starting 18 bp 5' of the ATG start codon and ending 529 bp into zyme from bovine testes, human urine, or medium of transiently the coding sequence of acid a-glucosidase, was prepared by PCR The publication costs of this article were defrayed in part by page charge Abbreviations: CMV, cytomegalovirus; DHFR, dihydrofolate reduc- payment. This article must therefore be hereby marked "advertisement" in tase; FBS, fetal bovine serum; MTX, methotrexate. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 65 Downloaded by guest on September 29, 2021 66 Genetics: Van Hove et al. Proc. Natl. Acad. Sci. USA 93 (1996) amplification using the primers GAA224(+) (5'-TCC-AGG- the medium to pH 6.6) in 25-cm2 confluent flasks with 0.2 ml CCA-TCT-CCA-ACC-AT-3') and GAA751(-) (5'-TCT-CAG- of medium per cm2. TCT-CCA-TCA-TCA-TCA-CG-3') and cloned into pCRII (In- Enzyme Purification. Acid a-glucosidase was purified from vitrogen). The resultant plasmid, pCRII-1, contained the Hindlll human placenta (18) and human urine (10). Antiserum to the site 5' relative to the ATG start codon and a Sac II site 320 bp placental enzyme was raised in rabbits. For purification of into the coding sequence of acid a-glucosidase. pJW-5' sGAA, recombinant acid a-glucosidase, the method used for urinary containing the 5'-shortened version of the acid a-glucosidase enzyme was employed but with a 2.6 cm x 100 cm Sephadex cDNA, was then constructed by replacing the HindIII-Sac II G-200 column. After elution, enzyme-containing fractions fragment of pJW-GAA with the HindIII-Sac II fragment of were combined into four fractions. Following the addition of pCRII-1. 0.1 volume of 0.65 M NaCl/0.5 M citrate, pH 6.50, they were Cell Culture, Transfection, and Selection. Plasmids were concentrated in 10-kDa Centriprep cartridges (Amicon). To screened by transient transfection of COS-7 cells with Lipo- examine purity, 10 ,ug of total protein was analyzed by SDS/ fectin (Life Technologies) and assay of enzymatic activity in PAGE and stained with Coumassie blue R. Denatured frac- cells and medium. CHO cells (CHO-Kl) (ATCC CCL 61) and tions (2 gl of enzyme boiled with 1% SDS) were deglycosylated DHFR- CHO cells (ATCC CRL 9096) were maintained in overnight at 37°C with N-glycosidase F (0.5 unit; Boehringer Mannheim). The molecular weight of the enzyme in the CHO minimal essential medium a (MEM-a), supplemented with ribonucleotides and deoxyribonucleotides for the DHFR- cells and culture medium under various culture conditions was and detection with cells. pJW-5'sGAA was transfected into regular CHO-Kl cells. analyzed by SDS/PAGE, Western blotting, a-glucosidase antiserum and chemolumines- pMT2-GAA, pcDNA3-GAA, and pJW-5'sGAA were trans- rabbit anti-acid fected into DHFR- CHO cells. Transfections used 50 ,ug of cence (ECL; Amersham). Enzyme Assays. Acid and neutral a-glucosidase were as- linearized plasmid DNA and 200 ,ug of sheared salmon sperm DNA in 800 ,ul of Hepes-buffered saline, with electroporation sayed using 4-methylumbelliferyl a-D-glucoside in a 96-well plate (19). Acid fB-galactosidase, ,3-glucuronidase, and total at 320 V and 960 ,uF in a Bio-Rad Gene Pulser (17). After 2 hexosaminidase were similarly assayed with the respective days, the cells were subcultured and selection was initiated 4-methylumbelliferyl derivatives (20). a-Glucosidase activity with Geneticin at 700 for plasmids pcDNA3-GAA and jig/ml was also measured by following the release of glucose from pJW-5'sGAA. For pMT2-GAA and pJW-5'sGAA, selection maltose or glycogen (21). The glycogen content of fibroblast in nucleotide-free medium, with 10% dialyzed FBS, was lysates was assayed by the release of glucose after digestion of started after 4 days. For some clones 5 or 25 nM methotrexate 25 ,ll of boiled supernatant with 0.5 unit of Aspergillus niger (MTX) was added.