Long-term and high-dose trials of enzyme replacement therapy in the canine model of mucopolysaccharidosis I.

Enzyme replacement is a potential therapy for mucopolysaccharidosis I (MPS I), a lysosomal storage disorder caused by alpha-L-iduronidase deficiency. Previous work showed improvement in the tissues of MPS I dogs treated intravenously for 3 months with recombinant human alpha-L-iduronidase (25,000 units or approximately 0.1 mg/kg/week). We have now treated an MPS I-affected dog for 13 months to assess the clinical effects of enzyme replacement. The treated dog gained more weight, was more active, and had less joint stiffness than the untreated littermate. Biochemical and histologic studies demonstrated uptake of alpha-L-iduronidase and decreased lysosomal storage in the liver, kidney, spleen, lymph nodes, synovium, adrenals, and lungs. The brain had detectable enzyme activity and decreased glycosaminoglycan storage although histologic improvement was not evident. Cartilage and heart valve did not show any detectable improvement. A fivefold higher dose (approximately 0.5 mg/kg) administered five times over 10 days to two other dogs resulted in higher tissue enzyme activity and similarly decreased glycosaminoglycan storage and excretion. Antibodies to human alpha-L-iduronidase were induced in all treated dogs and may be associated with immune complex deposition and proteinuria. Recombinant canine alpha-L-iduronidase also induced antibody formation to a similar degree. The results support the conclusion that enzyme replacement is a promising therapy for MPS I though immunologic complications may occur.

Myoblast gene therapy in canine mucopolysaccharidosis. I: Abrogation by an immune response to alpha-L-iduronidase.

Three dogs with deficiency of the lysosomal enzyme alpha-L-iduronidase were treated by gene replacement therapy targeted at muscle. Direct intramuscular injections of plasmid encoding the alpha-L-iduronidase gene cDNA resulted in no detectable enzyme production, but may have resulted in immunologic sensitization to iduronidase protein, which the dogs lack totally. Myoblasts were grown from skeletal muscle biopsies and transduced with a retroviral vector containing the canine gene under control of the muscle creatine kinase enhancer. Several hundred-fold overexpression of enzyme production occurred in cultured cells; however, following reintroduction of the cultured cells into dogs, enzyme production declined rapidly. Concurrent with the falling enzyme levels, there was production of specific immunoglobulin G (IgG) antibody against iduronidase that was further associated with cellular infiltration of the myoblast injection sites. Most inflammatory cells were lymphocytes and plasma cells, suggesting local humoral and cellular immune responses to the enzyme-producing muscle cells. PCR analysis of tissues collected 2-22 weeks after the final treatment showed the persistence of Neo and canine alpha-L-iduronidase sequences in a progressively decreasing percentage of myoblasts. Results from this study in a canine model of mucopolysaccharidosis I underscore the fact that immunologic reactions to cells producing desirable, normal, but foreign, proteins may be as much an impediment to gene therapy as reactions to the viral vectors used to introduce the foreign gene.

Enzyme replacement in a canine model of Hurler syndrome.

The Hurler syndrome (alpha-L-iduronidase deficiency disease) is a severe lysosomal storage disorder that is potentially amenable to enzyme-replacement therapy. Availability of a canine model of the disease and a sufficient supply of corrective enzyme have permitted a therapeutic trial lasting 3 mo. Recombinant human alpha-L-iduronidase, purified to apparent homogeneity from secretions of a stably transfected Chinese hamster ovary cell line, was administered i.v. to homozygous affected animals in doses of approximately 1 mg. The enzyme rapidly disappeared from the circulation in a biphasic manner, with t1/2 of 0.9 and 19 min, respectively, and was taken up primarily by the liver. Biopsy of the liver before and after a very short trial (seven doses administered over 12 days) showed remarkable resolution of lysosomal storage in both hepatocytes and Kupffer cells. After weekly administration of enzyme to three affected animals over a period of 3 mo, the level of enzyme was about normal in liver and spleen, lower but significant in kidney and lung, and barely detectable (0-5% of normal) in brain, heart valves, myocardium, cartilage, and cornea. Light and electron microscopic examination of numerous tissues showed normalization of lysosomal storage in liver, spleen, and kidney glomeruli, but there was no improvement in brain, heart valves, or cornea. Even though the treated dogs developed complement-activating antibodies against alpha-L-iduronidase, clinical symptoms could be prevented by slow infusion of enzyme and premedication.

Canine stem cell factor (c-kit ligand) supports the survival of hematopoietic progenitors in long-term canine marrow culture.

The cDNA for canine stem cell factor (cSCF, c-kit ligand) was cloned and expressed in Escherichia coli. The recombinant protein (rcSCF), 165 amino acids in length, is very similar structurally to the soluble form of previously cloned and sequenced rodent and human SCFs. The biological effects of rcSCF were studied in a day-10 granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay and in long-term liquid bone marrow culture of non-adherent hematopoietic cells in the absence of a stromal underlayer. Synergism in the stimulation of growth of CFU-GM was demonstrated between rcSCF and both recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and naturally occurring colony-stimulating activity present in the serum of a neutropenic dog. Alone, rcSCF was nonstimulatory for committed marrow precursors in methylcellulose cultures and had minimal effect on hematopoietic progenitor cell survival in stromaless, liquid cultures. When rcSCF was combined with phytohemagglutinin-stimulated canine lymphocyte-conditioned medium (PHA-LCM) or rh interleukin 6 (IL-6), with or without rhGM-CSF, CFU-GM survived for up to 5 weeks. The combination of rcSCF and rhGM-CSF, without rhIL-6, led to an early increase in CFU-GM in liquid cultures that declined more rapidly than in flasks that included rhIL-6. Survival of progenitor cells was negligible beyond 1 week in flasks with growth factor combinations lacking rcSCF. Sustained production of nonadherent cells in long-term cultures also was dependent on rcSCF in combination with canine PHA-LCM or recombinant human growth factors. It appears that rcSCF, like that from rodent and primate species, has the ability to influence the survival and proliferation of CFU-GM, and perhaps earlier progenitor cells, in hematopoietic tissues. In a long-term liquid culture system in which growth factor production by stromal cells is limited, rcSCF possesses a unique ability to maintain the viability of progenitor cells for up to 5 weeks.

Cloning and characterization of cDNA encoding canine alpha-L-iduronidase. mRNA deficiency in mucopolysaccharidosis I dog.

alpha-L-Iduronidase is a lysosomal enzyme, the deficiency of which causes mucopolysaccharidosis I (MPS I); a canine MPS I colony has been bred to test therapeutic intervention. The enzyme was purified to apparent homogeneity from canine testis and found to consist of two electrophoretically separable proteins that had common internal peptides but differed at their amino termini. A 57-base oligonucleotide, corresponding to the most probable codons of the longest peptide, was used to screen a canine testis cDNA library. Three cDNAs were isolated, two of which lacked the 5'-end whereas the third was full-length except for a small internal deletion. The composite sequence encodes an open reading frame of 655 amino acids that includes all sequenced peptides. The amino terminus of the larger protein, glutamic acid 26, is at the predicted signal peptide cleavage site, whereas the amino terminus of the smaller protein is leucine 106. There are six potential N-glycosylation sites and a non-canonical polyadenylation signal, CTTAAA. A search of GenBank showed that the amino acid sequence of alpha-L-iduronidase has similarity to that of a bacterial beta-xylosidase. A full-length cDNA corresponding to the composite sequence was constructed (pcIdu) and inserted into the pSVL expression vector (pSVcIdu). Two days after Cos-1 cells were transfected with pSVcIdu, their intracellular and secreted level of alpha-L-iduronidase activity has increased 8- and 22-fold, respectively, over the endogenous activity. Fibroblasts of MPS I dogs, which have no alpha-L-iduronidase activity, lacked the normal alpha-L-iduronidase mRNA of 2.2 kilobases and contained instead a trace amount of a 2.8-kilobase species. Isolation and characterization of an expressible alpha-L-iduronidase cDNA represents the first step toward mutation analysis and replacement therapy.

Cardiovascular changes after bone marrow transplantation in dogs with mucopolysaccharidosis I.

Five dogs with mucopolysaccharidosis I, 3 of which had been treated with bone marrow transplantation (BMT), were evaluated for 20 months with electrocardiography, thoracic radiography, and M-mode and 2-dimensional echocardiography. Treated and untreated (control) dogs had widened P waves on ECG. Thoracic radiographs remained normal for all dogs throughout the study. Thickening of the mitral valve was observed on echocardiograms of dogs in both groups, but the untreated dogs appeared to have thicker valves. Concentrations of glycosaminoglycans in the mitral valves and myocardium were higher in control dogs than in treated dogs. Markedly large aortic root diameters were observed on echocardiograms in both untreated dogs, but aortic root diameters remained normal in treated dogs. Echocardiography, but not electrocardiography, was useful in monitoring heart enlargement in each dog. Dogs treated with BMT generally had less severe cardiac changes and slower disease progression than control dogs.

Corneal opacity in canine MPS I. Changes after bone marrow transplantation.

Corneal opacification associated with glycosaminoglycan (GAG) deposition occurs in canine mucopolysaccharidosis I (MPS I), a deficiency of the lysosomal enzyme alpha-L-iduronidase. In affected dogs corneal lesions appear similar to those in children with the same disease. Transplantation of bone marrow from unaffected littermates was performed in 5 MPS I affected dogs at 5 months of age. In three recipients that became long-term survivors corneal clouding was largely alleviated compared to affected control dogs. In no case, however, did the corneas remain totally clear throughout the course of the study (594, 628 and 1425 days). Light and electron microscopic findings correlated with the clinical impression of partial improvement. Glycosaminoglycan analysis of corneal tissue from two transplant recipients, one normal littermate, and one MPS I-affected, untransplanted dog showed quantitative and qualitative changes in stored GAG following bone marrow transplantation.

Effect of canine parvovirus on erythroid progenitors in phenylhydrazine-induced regenerative hemolytic anemia in dogs.

The effects of canine parvovirus (CPV) infection in dogs with hemolytic anemia was compared with the clinical effects of human parvovirus-induced aplastic anemia in human beings with chronic regenerative anemias. Phenylhydrazine was used to induce a transient, severe, hemolytic anemia in dogs to evaluate the effects of CPV infection on rapidly dividing bone marrow precursors. Erythrocyte colony-forming unit bone marrow cultures and cytologic examination of bone marrow were used to determine the effects of CPV infection on erythroid bone marrow precursors. The induced hemolytic anemia regenerated rapidly and although the bone marrow was infected, it was determined that CPV infection did not induce a detectable decrease in erythroid progenitors in dogs with severe hemolytic anemia.

Long-term effects of bone marrow transplantation in dogs with mucopolysaccharidosis I.

The therapeutic effects of allogeneic bone marrow transplantation (BMT) in a canine model of mucopolysaccharidosis I (MPS I) were investigated. Long-term post-BMT pathologic and biochemical studies were performed on three groups of dogs: 1) MPS I-affected dogs that did not receive BMT, 2) MPS I-affected dogs that received total body irradiation followed by an allogeneic BMT, and 3) normal, unaffected dogs that served as BMT donors. All dogs were necropsied at approximately 20 months after BMT. The severity of MPS I-related lesions in the dogs receiving BMT was greatly diminished. These dogs had only slight cardiac valvular thickening, no meningeal thickening, no renal tubular epithelial vacuolation, decreased neuronal vacuolation, decreased corneal stromal vacuolation, and greatly diminished arterial medial thickening. The severity and incidence of degenerative arthropathy also were decreased in BMT dogs, however, vertebral lesions were similar to nontransplanted, affected dogs. Chondrocytes of both MPS I-BMT and MPS I-no BMT groups had similar marked cytoplasmic vacuolation, except for MPS I-BMT chondrocytes near the articular surface, which had more normal morphology. Ultrastructurally, the liver and kidney tissue in BMT recipients had no appreciable lysosomal accumulation of GAGs. These morphologic findings were supported by near normal levels and electrophoretic patterns of glycosaminoglycans (GAG) in most tissues of BMT recipient dogs. This study demonstrates that BMT is capable of substantially diminishing the severity of MPS I-related lesions in this canine model.

Dexamethasone-induced serum biochemical changes in goats.

Dexamethasone was administered at sequential dosages of 0.1 mg/kg/day for 3 days and 1 mg/kg/day for 3 days to 6 adult female goats. The greatest effects induced were hypokalemia, hypophosphatemia, and hyperglycemia. At low dosages of dexamethasone, transient increases (P less than 0.05) developed in serum sodium and chloride concentrations. An effect on hepatic physiology or iron metabolism was not observed.

Long-term neurological effects of bone marrow transplantation in a canine lysosomal storage disease.

A naturally occurring disease in Plott hound dogs, caused by deficiency of the lysosomal enzyme alpha-L-iduronidase, was used to study the feasibility of bone marrow transplantation therapy in a neurodegenerative storage disease. Three long-term survivors of transplantation with littermate marrow at 5 months of age (before clinical signs) had CNS enzyme activity, glycosaminoglycan storage, and light microscopic and ultrastructural changes evaluated 594, 628, and 740 days after treatment. Iduronidase activity in small amounts (1-3% of donor values) was detectable in brain tissue. Cerebrospinal fluid had higher iduronidase activity after transplantation (7-15% of donor values). Enzyme activity within the CNS resulted in significant reductions in stored glycosaminoglycans and resolution, to a large extent, of light microscopic and ultrastructural lesions observed in affected, untreated littermate control dogs.

Radiographic findings in a canine model of mucopolysaccharidosis I. Changes associated with bone marrow transplantation.

Eight dogs with mucopolysaccharidosis I (MPS I) were studied and their radiographic lesions compared with those reported in human MPS I. Three of the dogs received bone marrow transplants from unaffected littermates at 5 months of age. These dogs, and two affected control littermates were radiographed at 3, 6, 9, 12, 15, 18 and 20 months post-transplantation to evaluate the effects in chondro-osseous tissues. Although bone marrow transplantation did not alleviate all radiographically detectable changes, there was delayed onset of some lesions and reduced severity of most lesions in the dogs receiving transplants. Those lesions most closely associated with clinical lameness or gait abnormalities in untreated canine MPS I were most improved (or eliminated), and this was reflected in marked clinical improvement.

Alterations in neuron morphology in mucopolysaccharidosis type I. A Golgi study.

Morphological changes in neurons with inborn defects of the lysosomal hydrolase, alpha-L-iduronidase, and with concomitant storage of glycosaminoglycans, were evaluated by Golgi staining in two animal models and compared to a similar study of a child with the same disease. Cortical pyramidal neurons in feline mucopolysaccharidosis type I often displayed axon hillock enlargements (meganeurites) and/or ectopic, secondary neuritic processes sprouting from this same region of the cell. The latter structures were prominent and often appeared longer than similar neurites reported in other neuronal storage diseases. Although most meganeurites were aspiny, a few were observed which possessed spine-like processes or neurites. Other than these morphological changes in cortical pyramidal neurons, few other cell types displayed abnormalities demonstrable by Golgi impregnation. In the canine model of this disorder, abnormal Golgi-impregnated cortical neurons resembled more closely those seen in human mucopolysaccharidosis. That is, they possessed meganeurites which typically were aspiny in appearance. Ectopic neurite growth was not observed on any Golgi-impregnated neurons in the cases of canine or human mucopolysaccharidosis used in this study. The latter finding, given the advanced ages of these cases, is consistent with the view that ectopic neuritogenesis seen in neuronal storage diseases may be subject to a developmental window, albeit one open well beyond the period of early postnatal maturation.

Bone marrow transplantation in canine mucopolysaccharidosis I. Effects within the central nervous system.

Five dogs with mucopolysaccharidosis I, a model of human Hurler/Scheie syndrome, were transplanted with marrow from phenotypically normal littermates at 5 mo of age. At 3 and 9 mo posttransplantation, biopsies of cerebral cortex, liver, and cerebrospinal fluid were obtained. The alpha-L-iduronidase levels in these tissues were 0.8-7.4, 26-45, and 6.3-14.9% of the paired donor tissues, respectively. Although iduronidase was present in relatively low levels in the recipients' brains and cerebrospinal fluid at both biopsy times, reduction in brain glycosaminoglycan (GAG) was comparable to that observed in liver. Ultrastructural studies of cells within the transplanted dogs' brains showed less lysosomal distension and storage product than in affected, nontransplanted, littermate controls. The most marked clearing of stored GAG was in cells surrounding blood vessels, but decreased lysosomal storage in neurons and glial cells was also observed. Urinary GAG excretion also decreased to near normal levels by 5 mo posttransplantation.

Ehrlichia canis-related polyarthritis in a dog.

Ehrlichia canis-related polyarthritis was diagnosed in a 7-month-old Boxer. The diagnosis was based on intraneutrophilic morulae found in synovial fluid specimens, thrombocytopenia, a positive result for indirect fluorescent antibody testing for E canis, the presence on the dog of the known vector of E canis infection (Rhipicephalus sanguineus), and a favorable response to treatment with tetracycline hydrochloride. The dog has had no recurrence of lameness for 18 months after cessation of treatment.

Neurochemical characterization of canine alpha-L-iduronidase deficiency disease (model of human mucopolysaccharidosis I).

This report presents the neurochemical findings on the first dog to die with deficiency of alpha-L-iduronidase (mucopolysaccharide alpha-L-iduronohydrolase; EC 3.2.1.76). The principal findings were (a) markedly increased glycosaminoglycan content in all neural tissues examined (from threefold in sciatic nerve to 15-fold in brainstem), (b) a modest increase in levels of gangliosides GM2, GM3, and GD3, particularly in gray matter, (c) excessive accumulation of glycosaminoglycans in the CSF, (d) the increased glycosaminoglycans were dermatan sulfate and heparan sulfate, and (e) the molecular weights of the liver glycosaminoglycans were shifted toward smaller sizes, indicating partial degradation. The canine disorder thus resembles mucopolysaccharidosis I in all aspects.

Fluorometric assay of alpha-L-iduronidase in serum for detection of affected and carrier animals in a canine model of mucopolysaccharidosis I.

We investigated measuring serum alpha-L-iduronidase (EC 3.2.1.76) by a sensitive fluorometric assay in 28 members of a canine family with mucopolysaccharidosis I. If assayed on the day of collection, serum was an acceptable specimen for identifying affected, carrier, and normal dogs. The overall correlation (r) between iduronidase activity in serum and mononuclear leukocytes was 0.966. However, iduronidase was extremely labile in serum refrigerated or frozen for 48 h.

Morphologic and biochemical studies of canine mucopolysaccharidosis I.

This report presents the necropsy and biochemical findings on the first dog to die with alpha-L-iduronidase deficiency (mucopolysaccharidosis I, MPS I). Gross pathologic features, light- and electron-microscopic findings, and tissue enzyme, glycosaminoglycan (GAG), and sphingolipid levels are compared with the human disease counterpart and the previously described feline model. Results lend further support for the similarities of the canine disease and human MPS I.

Investigation of the nature and specificity of antinuclear antibody in dogs.

A liquid phase radioimmunoassay was used to test sera from 34 dogs in 3 categories, determined by presence of antinuclear antibody (ANA) and disease, for antibodies against native canine DNA and dAdT, a synthetic double-stranded DNA analog. Antibodies to dAdT were absent in healthy dogs which, as a group, had levels of DNA antibodies consistent with those reported for healthy persons. Canine patients with a variety of illnesses, but which remained ANA negative, had slightly increased levels of binding of DNA and dAdT. As a group, ANA-positive dogs had significantly increased binding of dAdT and native DNA, which was shown to be mainly, but not entirely, double stranded. In the ANA-positive group, no correlation was found among ANA titer, % DNA binding, and % dAdT binding, indicating that these 3 procedures detect antibodies with differing specificities. In dogs, ANA are heterogeneous in antigenic specificity. Antibodies to double-stranded nucleic acid in dogs do not appear to be as specific for systemic lupus erythematosus as they are reported to be in persons with autoimmune disease.