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Human Molecular Genetics Advance Access originally published online on October 11, 2005
Human Molecular Genetics 2005 14(22):3321-3335; doi:10.1093/hmg/ddi364
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Development of MPS IVA mouse (Galnstm(hC79S·mC76S)slu) tolerant to human N-acetylgalactosamine-6-sulfate sulfatase

Shunji Tomatsu1,*,{dagger}, Monica Gutierrez1,{dagger}, Tatsuo Nishioka1,{dagger}, Masamichi Yamada5, Mana Yamada5, Yasuhiro Tosaka5, Jeffrey H. Grubb2, Adriana M. Montaño1, Matheus B. Vieira1, Georgeta G. Trandafirescu1, Olga M. Peña1, Seiji Yamaguchi3, Koji O. Orii4, Tadao Orii4, Akihiko Noguchi1 and Leticia Laybauer1

1Department of Pediatrics and 2Department of Biochemistry and Molecular Biology, Pediatric Research Institute, Saint Louis University, St. Louis, Missouri, USA, 3Department of Pediatrics, Shimane University, Izumo, Japan, 4Department of Pediatrics, Gifu University, Gifu, Japan and 5JCR Pharmaceuticals, Research Center, Asiya, Japan

* To whom correspondence should be addressed at: Department of Pediatrics, Pediatric Research Institute, Saint Louis University, 3662 Park Avenue, St. Louis, MO 63110-2586, USA. Tel: +1 3145775623 ext. 6213; Fax: +1 3145775398; Email: tomatsus{at}slu.edu

Received June 2, 2005; Accepted September 22, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disease caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. In recent studies of enzyme replacement therapy for animal models with lysosomal storage diseases, cellular and humoral immune responses to the injected enzymes have been recognized as major impediments to effective treatment. To study the long-term effectiveness and side effects of therapies in the absence of immune responses, we have developed an MPS IVA mouse model, which has many similarities to human MPS IVA and is tolerant to human GALNS protein. We used a construct containing both a transgene (cDNA) expressing inactive human GALNS in intron 1 and an active site mutation (C76S) in adjacent exon 2 and thereby introduced both the inactive cDNA and the C76S mutation into the murine Galns by targeted mutagenesis. Affected homozygous mice have no detectable GALNS enzyme activity and accumulate glycosaminoglycans in multiple tissues including visceral organs, brain, cornea, bone, ligament and bone marrow. At 3 months, lysosomal storage is marked within hepatocytes, reticuloendothelial Kupffer cells, and cells of the sinusoidal lining of the spleen, neurons and meningeal cells. The bone storage is also obvious, with lysosomal distention in osteoblasts and osteocytes lining the cortical bone, in chondrocytes and in the sinus lining cells in bone marrow. Ubiquitous expression of the inactive human GALNS was also confirmed by western blot using the anti-GALNS monoclonal antibodies newly produced, which resulted in tolerance to immune challenge with human enzyme. The newly generated MPS IVA mouse model should provide a good model to evaluate long-term administration of enzyme replacement.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mucopolysaccharidosis type IVA (MPS IVA: Morquio type A) is a storage disease of undegraded glycosaminoglycans (GAGs) caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS: E.C.3.1.6.4). It is one of a class of diseases due to a deficiency of one of the 11 enzymes involved in the stepwise degradation of GAGs (1Go). GALNS is one of several sulfatases required to degrade GAGs, keratan sulfate (KS) and chondroitin-6-sulfate (C6S). As in other mucopolysaccharidosis, MPS IVA patients show a spectrum of clinical severity. Phenotypes vary from the classical form with severe bone dysplasia, short trunk dwarfism, hearing loss, heart valve involvement, corneal opacity and a life span of 20–30 years, to an attenuated form with fewer manifestations. Patients with an attenuated form can have close to normal quality of life with little bone and visceral organ involvement. Patients with MPS IVA can usually be clinically distinguished from patients with other MPS because they preserve intelligence and have additional skeletal manifestations derived from a unique spondyloepiphyseal dysplasia and ligamentous laxity. The broad range of clinical phenotypes seen in MPS IVA is presumed to be the result of many different GALNS mutations. Over 130 different mutations have been found in the GALNS gene in patients with MPS IVA (2Go–16Go). Around 80% of mutations identified in MPS IVA patients were point mutations expressing an inactive protein.

GALNS is encoded by a member of the sulfatase gene family of which 13 different sulfatase human genes have been cloned. Enzymes of the sulfatase family mediate hydrolysis of sulfate esters such as GAGs, sulfolipids and steroid sulfates in eukaryotic cells. Deficiencies in sulfatase activities are associated with lysosomal storage disorders such as mucopolysaccharidosis (MPSII, MPSIIIA, MPSIIID, MPSIVA and MPSVI) and metachromatic leukodystrophy and to non-lysosomal disorders such as X-linked ichthyosis and chondrodysplasia punctata. Another related disease is multiple sulfatase deficiency (MSD), in which the activity of all the sulfatases is impaired (17Go).

To catalyze the hydrolysis of their natural substrates, the sulfatases must be post-translationally activated. A consensus sequence (CS/TPSRXXXL/MTGR/K/L) in the catalytic domain of sulfatases contains a highly conserved cysteine (C79 in exon 2 of human GALNS) that is modified into a formylglycine (FGly) (18Go). During or shortly after protein translocation and while the sulfatase polypeptides are still largely unfolded, FGly residues are generated (19Go) by lumenal components of the endoplasmic reticulum (20Go). This sequence motif following the cysteine residue to be modified post- translationally directs the FGly generation in sulfatases. This modification is necessary for the catalytic activities of the sulfatases, generated by the protein product of sulfatase- modifying factor 1 (SUMF1), which encodes the formylglycine-generating enzyme (FGE; 21,22). The FGly is essential for the activity of the sulfatases as it contains an aldehyde hydrate that attacks the sulfate ester, which leads to the formation of the enzyme–substrate complex and to the cleavage of the sulfate residue (18Go). Cotransfection of SUMF1 with the sulfatase complementary DNAs greatly enhances the activities of the overexpressed sulfatases (21Go–24Go).

The goal of an experimental therapy for MPS and related disorders was greatly advanced by the discovery of the MPS VII mouse (gusmps/mps) (25Go–27Go), which shows facial dysmorphism, growth retardation, deafness, behavioral defects and shortened lifespan. This is an attractive model to study multiple experimental therapies for lysosomal storage disorders. Thus, natural MPS VII mice have been widely used for evaluating the effectiveness of bone marrow transplantation (28Go–30Go), enzyme replacement (31Go–34Go), and gene therapy with retroviral (35Go–37Go), adenoviral (38Go–40Go) and adeno-associated viral vectors (41Go–43Go). However, cellular and humoral immune responses to the injected enzymes and to enzymes expressed in gene therapy have recently been recognized as major impediments to evaluating experimental enzyme replacement and gene therapy strategies. Antibodies to the gene products in MPS I, MPS VI and MPS VII animal models were detected after multiple injections of enzyme, confounding the interpretation of experiments aimed at evaluating new approaches to therapy (44Go–47Go).

To solve this immunologic problem in the mice models, we attempted to make mice immunotolerant to the human ß-glucuronidase (GUS) enzyme by two approaches. First, a transgene expressing human inactive GUS cDNA with an active site mutation was introduced on the natural MPS VII (gusmps/mps) background. The murine MPS VII model with an inactive human GUS transgene retained the MPS VII phenotype but had the added desirable feature of being immunotolerant to human GUS (48Go). We have also reported a novel approach to creating a new mouse model of MPS VII. It lacked GUS because of a targeted knock-in mutation (E536A) in the mouse Gus gene and was tolerant to human GUS because a human GUS E540A transgene was simultaneously introduced into the mouse Gus gene (49Go).

We have applied this second approach to produce an MPS IVA mouse tolerant to human GALNS enzyme. This new mouse model has substantial storage materials in bones and hepatocytes, making it more similar to human MPS IVA than the original MPS IVA knock-out mouse (50Go). The mouse lacks GALNS activity because of a targeted active site mutation (C76S) in the mouse Galns gene (which corresponds to the active site mutation C79S in the human GALNS gene) and confers tolerance to human GALNS by targeting human inactive GALNS cDNA with C79S mutation in the mouse Galns gene. This mouse is a useful tool for evaluating the long-term benefits of enzyme therapy with human GALNS.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of MPS IVA mice
To introduce the C76S point mutation in the Galns gene and human GALNS cDNA with a C79S mutation in mouse ES cells, we designed a targeting vector with a total of 8.7 kb of homologous genomic sequence flanking the neor cassette and human GALNS cDNA (Fig. 1). After selection with G418 and ganciclovir, doubly resistant clones were screened for homologous recombination by PCR, and the Southern blot hybridized with a 5' external probe. Of 109 clones screened by PCR, the 4.5-kb PCR fragment diagnostic of homologous recombination was amplified in nine homologous clones, whereas no fragment was amplified from the non-homologous clones. Moreover, two out of nine clones contained the C76S point mutation confirmed by a SacI restriction enzyme digestion (Fig. 1B). Targeted ES cells containing one mutant allele were injected into C57BL/6 blastocysts and chimeric males were obtained, followed by germ-line transmission of the mutant allele (F1). Heterozygous F1 offspring were independently intercrossed to generate F2 homozygous mice of 129/SvxC57BL/6 hybrid strain background.




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  		Figure 1. Targeted mutagenesis of the Galns gene. (A) Targeting strategy. The structure of the endogenous gene, the targeting construct, the homologous recombinant allele and the neor-excised allele are presented schematically on successive lines. Filled rectangles represent exons, whereas open rectangles indicate TK, neor and human GALNS cDNA, respectively. The striped bar over the wild-type allele represents the probe used for Southern blots. Abbreviations for restriction enzymes are Eco, EcoRI; Sac, SacI; Xba, XbaI; Hind, HinDIII; Bam, BamHI. The same amino acid change at the mouse C76 and the corresponding human C79 residues were introduced in both the Galns and GALNS genes, respectively. (B) Detection of C76S point mutation at the murine Galns gene by genomic PCR amplification and the successive SacI digestion. The C76S mutation (TGC to AGC transversion) creates a new restriction site, SacI. Restriction enzyme analysis of the mutation introduced at codon 76, C76S, was performed using Galnstm(hC79S·mC76S)slu, Galnstm(hC79S·mC76S)slu/+ and wild-type mice. Unnumbered lanes at left end are DNA ladders of 100 bp markers. Lane 1: PCR product from normal control digested with SacI (549 bp); lane 2: PCR product from a heterozygote digested with SacI (189, 360 and 549 bp); lane 3: PCR product from a C76S homozygote digested with SacI (189 and 360 bp). (C) Expression of human GALNS mRNA. Northern blot analysis of human GALNS from human normal fibroblast (lane 1), spleen (lane 2), liver (lane 3) and kidney (lane 4) of Galnstm(hC79S·mC76S)slu homozygote mice (left panel). Twenty micrograms of total RNA from each tissue were analyzed by northern blotting with human GALNS cDNA probe. The 1.6 kb human GALNS mRNA was expressed in Galnstm(hC79S·mC76S)slu homozygous tissues.

 
Phenotype of Galnstm(hC79S·mC76S)slu MPS IVA mice
Homozygous C76S MPS IVA mice carrying the human GALNS C79S cDNA, herein referred to as Galnstm(hC79S·mC76S)slu, were not distinguishable phenotypically from Galnstm(hC79S·mC76S)slu/+ and Galns+/+ littermates at birth, but could only be identified by genotyping. Both males and females were fertile at least up to 12 months of age as indicated by the normal litter sizes produced from mating homozygous males to heterozygous females or vice versa. No obvious difference in weight or mortality rate was seen in the first 12 months of life. At age 4 months, radiographic analysis of the axial and appendicular skeleton of Galnstm(hC79S·mC76S)slu mice did not reveal any obvious abnormalities of long bones, thorax or calvaria.

Combined data from crosses between these heterozygous Galnstm(hC79S·mC76S)slu/+ mice showed a normal distribution of 25% (wild-type), 53% (heterozygote) and 22% (homozygote), in over 100 offspring analyzed, suggesting that the survival rate of homozygous offspring is not reduced.

The colony of Galnstm(hC79S·mC76S)slu mice was maintained by brother–sister matings, genotyped by PCR analysis of genomic DNA, and confirmed by enzymatic analysis of tail sample extracts for GALNS activity. Homozygous offspring from this colony were analyzed for morphologic, biochemical, and histopathologic phenotypes and tested for tolerance to immune challenge with human GALNS.

Human GALNS mRNA transcript levels
To confirm that Galnstm(hC79S·mC76S)slu mice express the GALNS gene product, we performed northern blot analyses on total RNA isolated from liver, kidney and spleen of Galnstm(hC79S·mC76S)slu littermates (Fig. 1C). A human GALNS transcript of 1.6 kb was present in substantial amounts in homozygous mice.

Biochemical findings
GALNS enzyme activity was determined in homogenates of liver, kidney, brain, spleen, lung, heart, muscle, bone, bone marrow cells from femur and plasma. In wild-type mice, the activity was highest in the kidney, spleen, and liver and lowest in brain and bone. The enzyme activity of homozygous mutant mice was less than 5% or very low compared with normals, and heterozygotes had nearly half the normal activity (Table 1). The gene dosage response was seen in all tissues tested.


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  		Table 1. Tissue levels of GALNS activity (U/mg)
 
Urinary GAG excretion
Urine was collected from Galnstm(hC79S·mC76S)slu (n=16), +/+ and +/– littermates (mixed background; n=21). Total urinary GAG excretion in Galnstm(hC79S·mC76S)slu mice was 540±174 mgGAG/gCre compared with 417±159 mgGAG/gCre in their littermates (P=0.0319). The level of urine GAG in Galnstm(hC79S·mC76S)slu mice was comparable to that of murine MPS IVA knock-out (Galns–/–) models (50Go).

Histopathology of the Galnstm(hC79S·mC76S)slu mouse (MPS IVA tolerant mouse)
Multiple tissues from five homozygous mice of 2–5 months of age were studied morphologically as described (50Go). Tissues were evaluated for the extent of lysosomal storage and alterations were compared with those in the MPS IVA knock-out mouse and wild-type controls from littermates. Widespread lysosomal storage was seen throughout the fixed tissue macrophage system. In the liver (Fig. 2A), lysosomal storage was observed in the sinus lining cells and liver cells. The storage material in liver cells was not observed in MPS IVA knock-out mice. Heart valvular cells and cells at the base of the valves had vacuolar distention (Fig. 2B). In the brain, there was storage in meningeal cells covering the brain (Fig. 2C). The hippocampal neurons and neocortical glial cells showed a greater storage than the original knock-out mouse. The cortical neurons and neuroglia also contained vacuoles, whereas no vacuoles were seen in those particular cells of the knock-out mice (Fig. 2D). In the kidney (Fig. 2E), there was a vacuolar change affecting the visceral epithelial cells of the glomeruli. Tubular epithelial cells had no evidence of storage. In the spleen (Fig. 2F), lysosomal storage was observed in the sinus lining cells.





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  		Figure 2. Morphological alteration in the MPS IVA tolerant Galnstm(hC79S·mC76S)slu mice (Toluidine blue staining). (a) Visceral organs and brain (1000x): (A) Liver from a 4-month-old Galnstm(hC79S·mC76S)slu mouse has Kupffer cells that are distended with lysosomal storage (arrow). (B) In the heart valve of the 4-month-old mouse, cells at the heart valves (arrow) and interstitial cells in the base of the heart valve (data not shown) had vacuolar distention. (C) The meninges covering the brain contain cells distended with lysosomal storage (arrow) in a 4-month-old Galnstm(hC79S·mC76S)slu mouse. (D) Hippocampal neurons and neocortical glial cells had lysosomal distension (arrow). (E) Kidney from a 4-month-old mouse has vacuolar change affecting the visceral epithelial cells of the glomeruli (arrow). (F) Spleen from a 4-month-old Galnstm(hC79S·mC76S)slu mouse has prominent lysosomal storage in the sinus lining cells. (b) Bone: The wild-type mice had no evidence of lysosomal storage in (G and I) chondrocytes in epiphyseal cartilage plate, (K) osteoblasts and bone marrow and (M) periosteum. (H) A 4-month-old MPS IVA tolerant mouse shows the chondrocytes in epiphyseal cartilage plate were vacuolated. (J) A 4-month-old MPS IVA tolerant mouse shows storage material in chondrocytes and the thin disorganized cartilage layer. Vacuolated chondrocytes with lysosomal distension were obvious. (L) The femur of a 4-month-old MPS IVA tolerant mouse shows vacuolated osteoblasts lining the cortical bone (arrow) and osteocytes within the bone with storage material. The sinus lining cells in bone marrow (arrow) also contain a small amount of storage. (N) A 4-month-old MPS IVA tolerant mouse shows the cells in the periosteum contain storage material (arrow). The pathology in bone from original knock-out mice was not shown here as there was no difference from the wild-type mice.

 
Affected mice had distention of the sinus lining cells in the bone marrow. This pathological finding was similar to that seen in the MPS IVA knock-out mice. In addition, the storage materials were observed in osteoblasts and osteocytes lining the cortical and trabecular bone, in chondrocytes and connective tissue in the periosteum apart from the original knock-out mouse (Fig. 2G–N). Ligaments associated with the femur had also vacuoles (data not shown). Growth plate exhibited an irregular structure with ballooned, vacuolated chondrocytes. The cartilage layer, especially in the proliferative layer, was shorter than those in a wild-type mouse. In MPS IVA tolerant mice, the hypertrophic zone was thicker and the cells in this zone were not organized in columns. Resting and proliferating zones were visible, but the cells in the proliferating zone were sometimes shorter than that in a wild-type mouse. Some of the cells in the growth plate appeared hypertrophic and abnormally round.

Immunostaining of collagen X of mouse growth plates showed clear differences between an MPS IVA tolerant mouse and a wild-type mouse. The 2- to 5-month-old tolerant mice increased immunostaining, whereas the wild-type mice at the same age showed little staining color, suggesting higher expression of collagen X in tolerant mice (Fig. 3A and B). The same higher expression of collagen X was seen in the MPS IVA knock-out mice on the pure background (data not shown). Intracellular collagen X is characteristic of non-proliferating, hypertrophic chondrocytes. In wild-type mice, collagen X was localized to the matrix of the hypertrophic zone of the growth plate, which was one or two cells thick (Fig. 3A). In MPS IVA mice, collagen X staining was more intense and was localized to a broad area of the growth plate matrix that was four to six cells thick (Fig. 3B). Collagen X staining was also visible within cells throughout the growth plate.



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  		Figure 3. Immunostaining for cartilage cell layers by anti-collagen X antibody (A and B) and for mouse liver by anti-Lamp-2 antibody (C and D). (A) The 4-month-old wild-type mouse shows a low level of collagen X indicated by the faint brown color. (B) The 4-month-old MPS IVA tolerant mouse shows increased immunostaining (arrow). (C) The 4-month-old wild-type mouse shows a lower level of Lamp-2 indicated by the faint brown color. (D) The 4-month-old MPS IVA tolerant mouse shows a substantial increase of Lamp-2 staining (arrow).

 
The articular chondrocytes did not show marked alteration and storage material, although there was storage in periarticular connective tissue. Joint laxity from the limb of MPS IVA tolerant mouse was not observed.

Expression level of Lamp-2 in visceral organs in wild-type mice showed a lower level of Lamp-2 by immunostaining, whereas the tolerant mouse had a substantial increase of Lamp-2 expression level (Fig. 3C and D).

Fine corneal clouding is frequently observed in MPS IVA patients. However, MPS IVA tolerant mice did not develop the end stage of this condition. The corneal sections of MPS IVA mice showed a little histomorphological alteration. In comparison with wild-type mice, which are characterized by regular arrayed stroma and a four- to six-layered corneal epithelia (Fig. 4A and B), the corneal alteration was limited to mild disarrangement of basal cells of corneal epithelium. Basal cells of the MPS IVA corneal epithelium appeared to be flattened with a distortion of nuclei. In most cases, the corneal stroma in MPS IVA tolerant mice showed the typical cell alignment. GAG accumulation could be demonstrated by Mowry-staining of histological sections of cornea from MPS IVA tolerant mouse. Remarkable staining was observed throughout the corneal layers. GAG storage by Mowry-staining was also observed in other visceral organs such as liver (Fig. 4C and D), spleen and kidney. The accumulation of storage material did not occur in the retina and the optic nerve (data not shown).



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  		Figure 4. Detection of storage material in the liver and cornea by Mowry's colloidal staining. Mowry's colloidal staining reveals GAGs (blue color) in corneal layers. (A) The wild-type mouse with layered corneal epithelium, basal layer of cylindrical cells, regularly arrayed stroma and endothelium shows blue staining with a physiologic amount of GAG. (B) Some cells of the basal layer of the epithelium from a 4-month-old Galnstm(hC79S·mC76S)slu mouse appear flattened with distorted nuclei (arrows). The cornea has marked staining in epithelium, stroma and endothelium with increased GAG. The corneal stroma of MPS IVA mouse does not show distension of cells and loss of typical cell alignment. The corneal epithelium, stroma and endothelium are indicated as (Ep), (St) and (En) in the figure. Neither corneal epithelial nor endothelial cells had any lysosomal distention apparent by light microscopy. Mowry staining for GAG detection in liver from wild-type and Galnstm(hC79S·mC76S)slu mice at 4-months of age shows clear difference. (C) The liver and Kupffer cells in a wild-type mouse exhibit typical compact structure with a physiologic amount of GAG (blue color). (D) The Kupffer cells in MPS IVA tolerant mouse appear markedly stained with high GAG load. The liver cells in MPS IVA tolerant mouse show staining inside the cells.

 
Thus, the pathological alterations in the MPS IVA tolerant mice were different from those seen in the original MPS IVA mouse previously described. Especially, storage was more obvious in the fixed tissues in bone, liver and brain in 2- to 5-month-old mice.

Expression of human GALNS
Tissues of bone, brain, heart, kidney, liver, lung and spleen were obtained from Galnstm(hC79S·mC76S)slu and normal control mice and were homogenized to analyze the expression of the human GALNS protein. Western blots of these tissues are shown in Figure 5. A single band with the expected Mr of the human GALNS protein (55 kDa) was detected by the anti-human GALNS antibody in all tissues of Galnstm(hC79S·mC76S)slu mice. No signal for human GALNS protein was found in the tissues from control mice.



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  		Figure 5. Expression of human GALNS protein. Western blot for human GALNS protein in extracts of tissues from Galnstm(hC79S·mC76S)slu. Lane 1 was from liver of the wild-type mouse. The human GALNS proteins from human purified GALNS, bone, heart, kidney, liver and brain tissues of the Galnstm(hC79S·mC76S)slu mouse were identified on western blot by anti-human GALNS antibody (lanes 2–7). No band was observed in any tissue from the control wild-type mouse.

 
Tolerance of the Galnstm(hC79S·mC76S)slu mice to immune challenge with human GALNS
Having confirmed that the Galnstm(hC79S·mC76S)slu mouse has the MPS IVA phenotype, we tested the hypothesis that the gene products expressed for the human C79S cDNA in intron 1 and the endogenous Galns gene containing the C76S mutation in exon 2 would confer tolerance to human GALNS. To provide an immunogenic challenge, we used weekly intravenous injection of human purified GALNS at 5 mg/kg body weight for 4, 8 and 12 weeks, respectively. As a control, homozygous MPS IVA knock-out mice which do not express human GALNS were used. These received the same purified human GALNS on the same schedule. At the first bleed without any infusion, none of the MPS IVA knock-out and Galnstm(hC79S·mC76S)slu mice showed anti-human GALNS antibodies by ELISA. Figure 6 shows the ELISA plate assay on blood taken 7 days after 8 and 12 weekly infusions. Antibody was not raised in any mouse with 4 weekly infusions. All MPS IVA knock-out control mice with 8 weekly infusions had titers of 104 or greater. In contrast, none of the Galnstm(hC79S·mC76S)slu mice showed any response even after 12 weekly infusions. These data demonstrate two important points: (i) the MPS IVA knock-out mice that do not express human GALNS are capable of mounting a strong antibody response to human GALNS when challenged with weekly injections during enzyme replacement therapy and (ii) the Galnstm(hC79S·mC76S)slu conferred tolerance to human GALNS, when provided with an immunogenic challenge up to 12 weekly infusions.



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  		Figure 6. Humoral immune tolerance of Galnstm(hC79S·mC76S)slu mice to human and GALNS. (A) Lane 1: blank; lane 2: monoclonal antibody (19B2-B); lane 3: Galnstm(hC79S·mC76S)slu without treatment. (B) ELISA plate assay of antibodies to human GALNS in serum of control MPS IVA knock-out (Galns–/–) mice (lane 1) and Galnstm(hC79S·mC76S)slu mice (lanes 2–4) following primary injection with human GALNS and 7 weekly injections with human GALNS (upper panel) and 11 weekly injections (lower panel). Galnstm(hC79S·mC76S)slu mice show no antibody response, whereas control knock-out mouse has an antibody detectable at 104 dilutions or greater.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have found that the MPS IVA knock-out (Galns–/–) mouse generates an immune response to human GALNS by a weekly infusion of the enzyme. Antibodies to the infused enzymes change the targeting and fate of the infused enzymes and may limit the response to therapy as previously described (44Go–47Go). Expression of human gene in experimental gene therapy protocols also led to antibody production to human enzyme (51Go,52Go). To eliminate these immunological obstacles to enzyme and gene therapy in the mouse model, we have developed two approaches. One is a simple transgenic approach, introducing a transgene expressing inactive human gene on the original natural background to create tolerance to human enzyme (48Go). The other is to produce tolerant mice by introducing a construct into ES cells that carries not only an active site mutation in the endogenous murine gene, but also the human inactive cDNA inserted in the adjacent intron of the murine gene (49Go). The homologous recombinant ES cells which contained both the targeted mutation and the inactive cDNA were used to generate mutant mice that expressed an inactive stable human enzyme.

Here we have applied the latter strategy to produce MPS IVA mutant mice tolerant to human GALNS enzyme even with multiple injections as in enzyme replacement therapy. The Galnstm(hC79S·mC76S)slu mice had the MPS IVA phenotype and the storage materials could be observed in tissues by 2 months of age. By that time, the biochemical, clinical and pathologic characterization of Galnstm(hC79S·mC76S)slu mice revealed evidence of lysosomal storage in the bone as well as visceral organs, brain and the cornea, and increased excretion of urinary GAGs as in MPS IVA in humans.

The storage materials in cartilage cells and cortex were not detected in the original MPS IVA knock-out mice on the mixed background at the early generations (50Go). As we could detect low levels of storage materials in bone in MPS IVA knock-out mice on the pure B6 background, it is possible that difference in background may affect the pathological phenotype.

Alternatively, excessive mutant GALNS protein with an active site mutation in Galnstm(hC79S·mC76S)slu mice may induce more GAG storage in bone and brain etc., even in early generations. Sulfatases are enzymes essential for degradation and remodeling of sulfate esters. Sulfatases undergo a common and unique post-translational modification, which is essential for catalytic activity. FGly, the key catalytic residue in the active site, is unique to sulfatases. In higher eukaryotes, FGly is generated from a cysteine precursor in an active site of sulfatases, by SUMF1 (18Go,19Go). Recent studies showed that a paralogue of SUMF1, namely, SUMF2, inhibits the enhancing effects of SUMF1 on sulfatases, suggesting that a stoichiometric equilibrium between the SUMF1–SUMF2–sulfatase complex and SUMF1 and SUMF2 homodimers is the regulatory mechanism that controls the amounts of free SUMF1, and therefore, the amounts of active sulfatases in different cells and tissues (24Go).

Considering recent studies on sulfatases, the following hypotheses can be offered to explain increased storage owing to excessive amounts of mutant GALNS protein with the active site mutation (C79S) in a tolerant mouse: excessive mutant protein (i) will downregulate SUMF1, leading to decrease of sulfatase activities, (ii) and will directly downregulate synthesis of other lysosomal enzymes including sulfatases that degrade GAGs and/or (iii) will inhibit the availability of SUMF 1 to activate other sulfatases.

As shown in Figure 5, the expression level of mutant protein in bone and brain of tolerant mice was higher than that in other tissues. This finding correlates with the higher level of storage in these tissues compared with the knock-out MPS IVA mouse. Analyses of the other lysosomal enzymes showed that the activities of all the sulfatases investigated (iduronate-2-sulfatase, arylsulfatase B and sulfaminidase) were reduced in the bone, bone marrow and brain in tolerant mice (5–30% of enzyme activities in wild-type mice). In contrast, there was no decrease in enzyme activities of other non-sulfatase lysosomal enzymes (ß-galactosidase, ß-glucuronidase and {alpha}-galactosidase; data not shown). When homogenized tissue extracts from tolerant mice were mixed with extracts from wild-type mice there was no inhibition of any of the sulfatase activities (data not shown). This suggested that no inhibitory factors were expressed in tissues of tolerant mice that were responsible for a reduction of the activity of the sulfatases. These findings support hypothesis (iii).

To understand the precise mechanism, a full investigation of the excessive GAG storage in the tolerant mouse awaits a fully congenic line. At that time, detailed analyses of additional pathological findings, the interaction between SUMF 1 or SUMF 2 and the mutant protein, and the extent of elevation of each specific GAG in individual tissues will be required. A simple mouse model with a C76S active site mutation in the mouse galns gene is also under development. This mouse can be compared with the current tolerant mouse to confirm whether the increase in storage derived specifically from an excessive amount of human C79S mutant protein or not.

The majority of the vertebrate skeleton, including the axial and appendicular structures as well as certain cranial bones, forms primarily by endochondral ossification. The distinctive feature of this mechanism comprises the hypertrophic cartilage matrix where endochondral ossification initiates, and where collagen X is the major biosynthetic product. Collagen X has been associated with endochondral ossification by its predominant expression in a subset of cartilage cells, the hypertrophic chondrocytes. By 2 months of age, the expression level of collagen X increased in growth plate cartilage of MPS IVA tolerant mice relative to wild-type controls. The level of collagen X was greater in 4-month-old MPS IVA tolerant mice when compared with 2-month-old mice. MPS VII tolerant and point-mutated mice with a severe or attenuated form also had an increase of collagen X expression in hypertrophic cartilage (data not shown). Collagen X has also been detected in fibrillated cartilage from human osteoarthritis patients (53Go). It is localized to sites of newly formed osteophytic and repair cartilage, and marks areas of endochondral bone formation (53Go). Osteoarthritis mouse model showed an increase of collagen X, and most of the cells in the growth plate appeared hypertrophic and abnormally round (54Go). Accumulation of GAGs or other secondary elevated factors may enhance increased hypertrophic differentiation as measured by an increase in type X collagen. This has not been looked at in MPS IVA patients because of the difficulty in obtaining samples.

The studies performed on articular chondrocytes of MPS VI cat and rat models, which also exhibit bone lesions, revealed that the MPS cells were proliferating faster than normal cells (55Go). The osteopenia observed in MPS VI may be because of a delayed turnover of hypertrophic chondrocytes in the growth plate, resulting in a marked reduction in hypertrophic cells in the epiphyseal plate of MPS VI rats (56Go). Increased TGF-ß expression in MPS VI models inhibits terminal differentiation of immature chondrocytes (54Go) and is in part responsible for reduced hypertrophy with an increased expression of matrix metalloproteinases. Therefore, the MPS VI models showed a defect in bone production because of a reduction in hypertrophic chondrocytes available for mineralization into bone (53Go). Why there is a difference in hypertrophic cells between the present MPS IVA mouse model and MPS VI cat and rat models is unknown.

Further analyses on bone makers (osteonectin, MMP-2 and -9), cytokines (tumor necrosis), factor-alpha (TNF-{alpha} and IL-1 ß, nitric oxide production) and apoptosis of chondrocytes will be needed to understand the mechanism of elevation of collagen X and increase of hypertrophic cartilage in MPS IVA mice compared with MPS VI rat and cat models.

Producing the MPS IVA model tolerant to human GALNS has the following advantages. First, the pathological phenotype is more obvious, especially in bone, than in the MPS IVA knock-out mice. Second, the Galnstm(hC79S·mC76S)slu mouse produces stable, catalytically inactive human GALNS, detectable by immunoblot, which is true of most MPS IVA patients with a point mutation. Absence of immunological reaction for 12 weekly infusions provides the opportunity to evaluate the effectiveness of replacement therapy by human enzyme more precisely.

As gene-targeted mutagenesis is used to introduce the human mutated GALNS cDNA in this method for making tolerant mice, a natural mouse mutant or a gene-targeted knock-out mouse is unnecessary. In addition, by controlling the localization and copy number of the human GALNS cDNA in the mouse genome, we can prevent disruption or change in the expression of other genes by a randomly inserted transgene from influencing the phenotype. The tolerant mouse described here should provide an improved model to evaluate the long-term benefits of experimental attempts to overcome the enzyme deficiency—whether by ERT, bone marrow transplantation or gene therapy. Given the growing interest in producing animal models of human diseases, this method of producing tolerant mouse models of human diseases may have broader application.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of the GALNS targeting vector
The mouse Galns gene was cloned from a 129/Sv mouse genomic library (Stratagene, La Jolla, CA). The C76 residue in exon 2 was selected for making a point mutation because the corresponding residue in the human GALNS gene (C79) was identified as the active site. The C79S point mutation was introduced in exon 2 of the human GALNS cDNA by in vitro mutagenesis with the mutagenic primer (HMO C79S: 5'-TCTGCCAACCCTCTGAGCTTCGCCATCGAGGGCGGCACTG-3') and created a new SacI restriction site.

The human GALNS C79S cDNA and chicken ß-actin promoter were introduced at the 5' end of the neor gene, resulting in the TK-neor-cDNA cassette (Fig. 1). Then, the 4.2-kb fragment of genomic DNA upstream of the BamHI site in intron 1 was introduced between the TK gene and neor-cDNA cassette. Next, the 4.5-kb fragment containing exons 2–4 of the Galns gene were added to the end of human GALNS cDNA to create the complete targeting vector. The C76S substitution in exon 2 of the Galns gene had been introduced into this 4.5-kb fragment by in vitro mutagenesis with the mutagenic primer (GMO C79S: 5'-TCTGCCAACCCTTTGAGCTCACCATGTAATT-3'). The loxp sequences were positioned at both ends of the neor gene, which can be eliminated by mating the heterozygotes with transgenic mice expressing the Cre recombinase enzyme. The final construct contained 4.2 kb and 4.5 kb of 5' and 3' homology of the Galns gene, respectively, and human GALNS cDNA with a C79S mutation (Fig. 1).

Gene targeting in embryonic stem (ES) cells and generation of mutant mice
The targeting was done by minor modifications of previously reported strategy (49Go). Briefly, the linearized targeting vector was introduced into the 129/Sv-derived ES cell line RW4 (Incyte Genome Systems, St. Louis, MO) by electroporation. The cells were placed under selection with G418 (GIBCO BRL, Rockville, MD) and ganciclovir (Syntex Chemicals, Boulder, CO). Genomic DNAs of resistant ES clones were screened by PCR for the homologous recombinant allele. This method utilizes a forward primer in intron 1 (CMO 1: 5'-AAGAAGTGCCTCTCGTCCCACTAG-3') and a reverse primer in exon 4 outside the targeting sequences (MOex4-1R: 5'-CTTGTTGGTATAGCCTTCTTCAGGAGCTC-3'), which produces a 4.5-kb fragment in the mutant allele but not in the wild-type allele. The presence of the C76S point mutation in exon 2 of the Galns gene was detected by PCR of the same ES cell DNA using a forward primer in intron 1 (mGMO1F: 5'-CCTGTGTCATTTGCATGTGACTATT-3') and a reverse primer in intron 2 (mGMO2R: 5'-TTGTCCTGTGACCAGGAAGTGCAG-3'), which amplified a 549 bp fragment of the mouse Galns gene. Digested with SacI, the 189- and 360 bp restriction fragments present in the mutant allele are distinguished from the uncleaved 549 bp PCR fragment from the normal allele on a 1.2% agarose gel (Fig. 2). Furthermore, genomic DNA of resistant clones was digested with KpnI, followed by Southern blot and hybridization with the 5'1.3 kb external probe (Fig. 1). The fragment is larger in the mutant allele than in the wild-type allele (15.7 kb versus 12.0 kb; data not shown).

Chimeric male offspring with targeted ES cells were bred to C57BL/6J females. The resultant F1 mice with the mutant allele were crossed with mice expressing Cre enzyme to remove the neor gene (57Go) and the resultant neo-excised heterozygous mice were mated to produce homozygous mutant mice. The resultant homozygous mice with the C76S mutation on the mouse Galns gene and C79S mutation on human GALNS cDNA were named Galnstm(hC79S·mC76S)slu.

The removal of neor was diagnosed by PCR of tail DNA using a forward primer in intron 1 (mGALNS2: 5'-CCCCTGGAGTGTAGTCACAGACAGTTGCAAGC-3') and a reverse primer in human GALNS cDNA (HG4R: 5'-AATAGAAGTTTGGGAAAAGCAGGCC-3'), which amplified a 1.7 kb fragment, whereas the non-neor-excised allele did not reveal any fragment. The complete excision of neor was confirmed by PCR using a forward primer, mGALNS2 and a reverse primer in neor (NeoR: 5'-AATAGAAGTTTGGGAAAAGCAGGCC-3'), which did not reveal any fragment, whereas the non-neor-excised allele amplified a 2.0 kb fragment.

Northern blot analysis and RT-PCR
Total cellular RNA was isolated from tissues of homozygous MPS IVA (Galnstm(hC79S·mC76S)slu), heterozygous (Galnstm(hC79S·mC76S)slu/+) and wild-type mice using a guanidinium/phenol solution. Twenty micrograms of RNA from each source were denatured in formaldehyde buffer and electrophoresed in 1% agarose, 2.2 M formaldehyde gels. The RNA was transferred to nylon membranes and hybridized overnight at 65°C with 32P-labeled human GALNS cDNA probes.

Monoclonal antibody
Monoclonal antibodies were prepared by a conventional method through immunization of mice with purified GALNS as the antigen. The GALNS was produced and purified from the G418-selected CHO cell line, which expressed high levels of the enzyme. The cells from lymph nodes of BALB/c mice that had been immunized with recombinant human GALNS were fused with the mouse myeloma cell-line P3-X63-Ag8-U1 purchased from ATCC (Manassas, VA). Isolated clones were screened by ELISA and cloned by the limiting-dilution. Epitope mapping was performed using recombinant GALNS fragments as probes for western blotting. These recombinant human GALNS fragments were expressed in Escherichia coli as glutathione-S-transferase (GST) fusion peptide. Briefly, the coding region for human GALNS 1–116, 59–232, 175–348, 291–464 and 407–522 amino acid residues of GALNS were amplified and inserted into the expression vector pGEX (Amersham Biosciences). E. coli cells were transformed with the above recombinant vector, and protein expression was induced with 1 mM isopropyl-D-thiogalactopyranoside for 4 h at 37°C. The GST fusion peptides were affinity-purified from cell lysates using glutathione-Sepharose (Amersham Biosciences).

Seven anti-GALNS antibody-producing clones were identified from 370 clones screened by ELISA. At least one clone was obtained specific for each of the abovementioned peptide sequences. We used clone 19B2-8 specific to GALNS amino acid residues 465–522, for the following western blot and for analyzing the sera from immune challenge.

Western blot analysis
Tissues were dissected and homogenized immediately (by Brinkmann Polytron homogenizer for 30 s at 4°C) in five volumes of homogenization buffer (25 mM Tris–HCl, pH 7.2, 140 mM NaCl, 1 mM PMSF). Samples containing 20 µg of protein were analyzed by SDS-PAGE under reducing conditions. The polypeptides were electronically transferred to Immobilon-P membranes (Millipore, Bedford, MA). After transblotting, the polypeptides were immunostained by using monoclonal mouse anti-human GALNS antibody, followed by incubation with rabbit anti-mouse IgG (Sigma-Aldrich, St. Louis, MO) coupled with peroxidase. The peroxidase activity was visualized using a chemiluminescent substrate.

Enzyme assays
The enzyme assay of GALNS and analysis of urinary GAGs was done as previously described (49Go,50Go).

Immunization method and analysis of sera from immunized mice by ELISA
Four MPS IVA (Galnstm(hC79S·mC76S)slu) and four MPS IVA (Galns–/–) mice were immunized with purified human GALNS derived from CHO cell lines beginning at 2 months of age. Each mouse received 250 U/g (1 mg/kg) body weight of human GALNS intravenously, and 3, 7 or 12 subsequent weekly infusions with the same dose of human GALNS intravenously. Blood was collected by eye bleed to measure antibodies to human GALNS by ELISA before each injection. The last blood collection was done 7 days after the last infusion.

Analysis of sera from immunized mice was done by ELISA assay as described previously (49Go).

Pathology
Four MPS IVA tolerant mice from 2–4 months of age were examined for morphological evidence of lysosomal storage. Multiple tissues, including limbs, liver, spleen, kidney, heart, rib, eye and brain were studied. Tissues fixed in 4% paraformaldehyde/2% glutaraldehyde, and embedded in Spurr's resin were sectioned, stained with toluidine blue, evaluated by light microscopy and compared to age-matched control mice. The tissues were also fixed in 10% neutral buffered formalin or in methacarn (60% methanol v/v, 30% chloroform v/v and 10% glacial acetic acid v/v), embedded in paraffin, sectioned for immunohistochemistry. Skeletons from 2–4-month-old Galnstm(hC79S·mC76S)slu mice were radiographed and compared with those of normal mice, as previously described (50Go,58Go). Tissues were evaluated for the extent of lysosomal storage and for comparison with the amount of storage seen in the previously described MPS IVA knock-out mouse model.

Immunohistochemistry
Anti-Collagen X antibody
Immunohistochemical detection of collagen X in tibiae or femurs of 2- to 5-month-old Galnstm(hC79S·mC76S)slu mice as well as the age-matched wild-type mice was carried out using rabbit anti-mouse collagen X antibody, RDI-COLL10abr (Research Diagnostics) at formalin-fixed sections as previously described by minor modifications (53Go).

Anti-Lamp-2 antibody
The mouse anti-rat Lamp-2 rat antibody ABL-93 was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Methacarn-fixed sections were treated with xylene to remove paraffin, rehydrated, treated with hydrogen peroxide in methanol to eliminate endogenous peroxidase activity and the antigen was unmasked by proteinase K. The following procedure was done as described previously (59Go). After counterstaining by Harris-hematoxylin solution (Sigma-Aldrich), sections were mounted.

Mowry iron colloidal staining
The 10% formalin-fixed sections of visceral organs and cornea were stained with Mowry's colloidal iron solution as described previously with modifications (60Go,61Go). The procedure involved the use of fresh colloidal ferric oxide. The sections were counterstained with nuclear fast red. Stained sections were dehydrated and mounted.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Austrian Research Society for Mucopolysaccharidosis and Related Diseases, German MPS, International Morquio Organization, Italian MPS Society, the Jacob Randall Foundation, the Bennett Foundation and Japan Chemical Research.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors. Back


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S. Tomatsu, A. M. Montano, A. Ohashi, M. A. Gutierrez, H. Oikawa, T. Oguma, V. C. Dung, T. Nishioka, T. Orii, and W. S. Sly
Enzyme replacement therapy in a murine model of Morquio A syndrome
Hum. Mol. Genet., March 15, 2008; 17(6): 815 - 824.
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