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Lysosomal storage diseases (LSDs) are a group of over 70 diseases that are characterized by lysosomal dysfunction, most of which are inherited as autosomal recessive traits. These disorders are individually rare but collectively affect 1 in 5,000 live births. LSDs typically present in infancy and childhood, although adult-onset forms also occur. Most LSDs have a progressive neurodegenerative clinical course, although symptoms in other organ systems are frequent. LSD-associated genes encode different lysosomal proteins, including lysosomal enzymes and lysosomal membrane proteins. The lysosome is the key cellular hub for macromolecule catabolism, recycling and signalling, and defects that impair any of these functions cause the accumulation of undigested or partially digested macromolecules in lysosomes (that is, 'storage') or impair the transport of molecules, which can result in cellular damage. Consequently, the cellular pathogenesis of these diseases is complex and is currently incompletely understood. Several LSDs can be treated with approved, disease-specific therapies that are mostly based on enzyme replacement. However, small-molecule therapies, including substrate reduction and chaperone therapies, have also been developed and are approved for some LSDs, whereas gene therapy and genome editing are at advanced preclinical stages and, for a few disorders, have already progressed to the clinic.

Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disease characterized by profound intellectual disability, dementia, and a lifespan of about two decades. The cause is mutation in the gene encoding α-N-acetylglucosaminidase (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. Impediments to enzyme replacement therapy are the absence of mannose 6-phosphate on recombinant human NAGLU and the blood-brain barrier. To overcome the first impediment, a fusion protein of recombinant NAGLU and a fragment of insulin-like growth factor II (IGFII) was prepared for endocytosis by the mannose 6-phosphate/IGFII receptor. To bypass the blood-brain barrier, the fusion protein ("enzyme") in artificial cerebrospinal fluid ("vehicle") was administered intracerebroventricularly to the brain of adult MPS IIIB mice, four times over 2 wk. The brains were analyzed 1-28 d later and compared with brains of MPS IIIB mice that received vehicle alone or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the brain, where it persisted with a half-life of approximately 10 d. Heparan sulfate, and especially disease-specific heparan sulfate, was reduced to control level. A number of secondary accumulations in neurons [β-hexosaminidase, LAMP1(lysosome-associated membrane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), glypican 5, β-amyloid, P-tau] were reduced almost to control level. CD68, a microglial protein, was reduced halfway. A large amount of enzyme also appeared in liver cells, where it reduced heparan sulfate and β-hexosaminidase accumulation to control levels. These results suggest the feasibility of enzyme replacement therapy for MPS IIIB.

Sanfilippo syndrome type B (MPS IIIB) is characterized by profound mental retardation in childhood, dementia and death in late adolescence; it is caused by deficiency of α-N-acetylglucosaminidase and resulting lysosomal storage of heparan sulfate. A mouse model, generated by homologous recombination of the Naglu gene, was used to study pathological changes in the brain. We found earlier that neurons in the medial entorhinal cortex (MEC) and the dentate gyrus showed a number of secondary defects, including the presence of hyperphosphorylated tau (Ptau) detected with antibodies raised against Ptau in Alzheimer disease brain. By further use of immunohistochemistry, we now show staining in neurons of the same area for beta amyloid, extending the resemblance to Alzheimer disease. Ptau inclusions in the dentate gyrus of MPS IIIB mice were reduced in number when the mice were administered LiCl, a specific inhibitor of Gsk3β. Additional proteins found elevated in MEC include proteins involved in autophagy and the heparan sulfate proteoglycans, glypicans 1 and 5, the latter closely related to the primary defect. The level of secondary accumulations was associated with elevation of glypican, as seen by comparing brains of mice at different ages or with different mucopolysaccharide storage diseases. The MEC of an MPS IIIA mouse had the same intense immunostaining for glypican 1 and other markers as MPS IIIB, while MEC of MPS I and MPS II mice had weak staining, and MEC of an MPS VI mouse had no staining at all for the same proteins. A considerable amount of glypican was found in MEC of MPS IIIB mice outside of lysosomes. We propose that it is the extralysosomal glypican that would be harmful to neurons, because its heparan sulfate branches could potentiate the formation of Ptau and beta amyloid aggregates, which would be toxic as well as difficult to degrade.

My postdoctoral training in the biosynthesis of plant polysaccharides at the University of California, Berkeley, led me, rather improbably, to study mucopolysaccharide storage disorders in the intramural program of the National Institutes of Health (NIH). I have traced the path from studies of mucopolysaccharide turnover in cultured cells to the development of therapy for patients. The key experiment started as an accident, i.e., the mixing of cells of different genotypes, resulting in correction of their biochemical defect. This serendipitous experiment led to identification of the enzyme deficiencies in the Hurler and Hunter syndromes, to an understanding of the biochemistry of lysosomal enzymes in general, and to the cell biology of receptor-mediated endocytosis and targeting to lysosomes. It paved the way for the development of enzyme replacement therapy with recombinant enzymes. I have also included studies performed after I moved to the University of California, Los Angeles (UCLA), including a recent unexpected finding in a neurodegenerative mucopolysaccharide storage disease, the Sanfilippo syndrome, with implications for therapy.

Sanfilippo syndrome type B (mucopolysaccharidosis III B, MPS III B) is an autosomal recessive, neurodegenerative disease of children, characterized by profound mental retardation and dementia. The primary cause is mutation in the NAGLU gene, resulting in deficiency of alpha-N-acetylglucosaminidase and lysosomal accumulation of heparan sulfate. In the mouse model of MPS III B, neurons and microglia display the characteristic vacuolation of lysosomal storage of undegraded substrate, but neurons in the medial entorhinal cortex (MEC) display accumulation of several additional substances. We used whole genome microarray analysis to examine differential gene expression in MEC neurons isolated by laser capture microdissection from Naglu(-/-) and Naglu(+/-) mice. Neurons from the lateral entorhinal cortex (LEC) were used as tissue controls. The highest increase in gene expression (6- to 7-fold between mutant and control) in MEC and LEC neurons was that of Lyzs, which encodes lysozyme, but accumulation of lysozyme protein was seen in MEC neurons only. Because of a report that lysozyme induced the formation of hyperphosphorylated tau (P-tau) in cultured neurons, we searched for P-tau by immunohistochemistry. P-tau was found in MEC of Naglu(-/-) mice, in the same neurons as lysozyme. In older mutant mice, it was also seen in the dentate gyrus, an area important for memory. Electron microscopy of dentate gyrus neurons showed cytoplasmic inclusions of paired helical filaments, P-tau aggregates characteristic of tauopathies-a group of age-related dementias that include Alzheimer disease. Our findings indicate that the Sanfilippo syndrome type B should also be considered a tauopathy.

Enzyme replacement therapy for lysosomal storage diseases is currently based on endocytosis of lysosomal enzymes via the mannose or mannose 6-phosphate receptors. We are developing a technology for endocytosis of lysosomal enzymes that depends on generic, chemically conjugated reagents. These reagents are aptamers (single-stranded nucleic acid molecules) selected to bind to the extracellular domain of the mouse transferrin receptor. After selection, an RNA aptamer and a DNA aptamer were modified with biotin and linked to dye-labeled streptavidin for detection by confocal microscopy. Aptamer-streptavidin conjugates showed saturable uptake into mouse fibroblasts (Ltk(-) cells), which could be inhibited by an excess of free aptamer but not by tRNA, calf thymus DNA, or transferrin. The RNA aptamer-streptavidin conjugate was mouse-specific, as human cells (293T) did not take it up unless first transfected with the mouse transferrin receptor. Some streptavidin separated from the recycling pathway of transferrin and colocalized with lysosomes. After characterization in the model system, the DNA aptamer was conjugated to a lysosomal enzyme, alpha-l-iduronidase, from which mannose 6-phosphate had been removed. The aptamer had been modified by attachment of terminal glycerol for oxidation by periodate and reaction of the resulting aldehyde with amino groups on the protein. Dephospho-alpha-L-iduronidase-aptamer conjugate was taken up in saturable manner by alpha-L-iduronidase-deficient mouse fibroblasts, with half-maximal uptake estimated as 1.6 nM. Endocytosed enzyme-aptamer conjugate corrected glycosaminoglycan accumulation, indicating that it reached lysosomes and was functional in those organelles. Both uptake and correction were inhibited by unconjugated aptamer, confirming the role of the aptamer in receptor-mediated endocytosis.

The neurodegenerative disease MPS III B (Sanfilippo syndrome type B) is caused by mutations in the gene encoding the lysosomal enzyme alpha-N-acetylglucosaminidase, with a resulting block in heparan sulfate degradation. A mouse model with disruption of the Naglu gene allows detailed study of brain pathology. In contrast to somatic cells, which accumulate primarily heparan sulfate, neurons accumulate a number of apparently unrelated metabolites, including subunit c of mitochondrial ATP synthase (SCMAS). SCMAS accumulated from 1 month of age, primarily in the medial entorhinal cortex and layer V of the somatosensory cortex. Its accumulation was not due to the absence of specific proteases. Light microscopy of brain sections of 6-months-old mice showed SCMAS to accumulate in the same areas as glycosaminoglycan and unesterified cholesterol, in the same cells as ubiquitin and GM3 ganglioside, and in the same organelles as Lamp 1 and Lamp 2. Cryo-immuno electron microscopy showed SCMAS to be present in Lamp positive vesicles bounded by a single membrane (lysosomes), in fingerprint-like layered arrays. GM3 ganglioside was found in the same lysosomes, but was not associated with the SCMAS arrays. GM3 ganglioside was also seen in lysosomes of microglia, suggesting phagocytosis of neuronal membranes. Samples used for cryo-EM and further processed by standard EM procedures (osmium tetroxide fixation and plastic embedding) showed the disappearance of the SCMAS fingerprint arrays and appearance in the same location of "zebra bodies", well known but little understood inclusions in the brain of patients with mucopolysaccharidoses.

Mucopolysaccharidosis I (MPS I, alpha-l-iduronidase deficiency disease) is a heritable lysosomal storage disorder involving multiple organs, including the heart. Malfunction of the heart is also a major manifestation in the mouse model of MPS I, progressing in severity from 6 to 10 months (of a 1 year life span). In comparisons of MPS I with wild-type mice, the heart was found enlarged, with thickened septal and posterior walls, primarily because of infiltration of the muscle by storage-laden cells. Heart valves were enlarged and misshapen, and contained large numbers of highly vacuolated interstitial cells. The thickened aortic wall contained vacuolated smooth muscle cells and interrupted elastic fibers. Hemodynamic measurements and echocardiography revealed reduced left ventricular function as well as mitral and aortic regurgitation. But despite these abnormalities, free-roaming MPS I mice implanted with radio telemetry devices showed surprisingly normal heart rate and blood pressure, though their electrocardiograms were abnormal. An incidental finding of the telemetry studies was a disturbed circadian rhythm in the MPS I mice. Restoration of enzyme activity in the heart of one mouse, by transplantation of retrovirally modified bone marrow, resulted in normalization of left ventricular function as well as loss of storage vacuoles in myocytes and endothelial cells, though not in valvular interstitial cells. This study demonstrates the usefulness of the mouse model for in-depth studies of the cardiovascular component of MPS I.

Mucopolysaccharidosis IIIB (MPS IIIB) is a lysosomal storage disorder caused by mutations in NAGLU, the gene encoding alpha-N-acetylglucosaminidase. The disease is characterized by profound mental retardation and eventual neurodegeneration, but relatively mild somatic manifestations. There is no available therapy. We have used a mouse knockout model of the disease to test therapy by genetically modified bone marrow. Bone marrow from Naglu -/- male mice was transduced with human NAGLU cDNA in an MND-MFG vector, and transplanted into 6- to 8-week-old lethally irradiated female -/- mice. Sham-treated mice received bone marrow transduced with eGFP cDNA in an MND vector. alpha-N-Acetylglucosaminidase activity in plasma and leukocytes, measured 3 and 6 months after transplantation, varied from marginal to nearly 30 times wild-type. A low level of alpha-N-acetylglucosaminidase activity, as little as provided by transplantation of unmodified Naglu +/+ bone marrow, could normalize biochemical defects (glycosaminoglycan storage and beta-hexosaminidase elevation) in liver and spleen, but a very high level was required for an effect on kidney. Effects on the brain were best seen by examination of cellular morphology using light and electron microcopy. Mice that expressed very high levels of alpha-N-acetylglucosaminidase in blood had an increased number of normal-appearing neurons in the cortex and other parts of the brain, while microglia with engorged lysosomes had almost completely disappeared. Immunohistochemistry showed a marked decrease of staining for subunit c of mitochondrial ATP synthase and for Lamp1, markers of neuronal and microglial pathology, respectively, as well as a decrease in staining for glial fibrillary acid protein, a marker of activated astrocytes. These results show that genetically modified cells of hematopoietic origin can reduce the pathologic manifestations of MPS IIIB in the Naglu -/- mouse brain.

Mucopolysaccharidosis I is a lysosomal storage disorder caused by mutations in the IDUA gene, resulting in deficiency of alpha-L-iduronidase and accumulation of glycosaminoglycans. Bone marrow transplantation has been the only available therapy, soon to be joined by enzyme replacement. We have tested retroviral gene therapy in a knockout mouse model of the disease. Bone marrow from Idua-/- male donor mice was transduced with human IDUA cDNA in an MND vector and transplanted into 6-8-week-old, lethally irradiated female Idua-/- mice. Sham-treated mice received Idua-/- bone marrow that was either unmodified or transduced with eGFP. Unmodified Idua+/+ (wild type) bone marrow was transplanted for comparison. Recipient mice were sacrificed 2-6 months after transplantation. Three biochemical parameters were used to gauge therapeutic success: appearance of alpha-L-iduronidase activity, reduction of beta-hexosaminidase activity and reduction of soluble glycosaminoglycan accumulation. Transplantation of unmodified +/+ bone marrow was effective in reducing storage in liver and spleen, but not in kidney or brain. The level of alpha-L-iduronidase activity achieved by transplantation of IDUA-transduced bone marrow varied greatly between experiments. But even modest activity resulted in correction of pathology of kidney, bladder epithelium, fibrocartilage, choroid plexus, and thalamus, as seen by light microscopy, while electron microscopy showed the presence of some normal neurons in the cortex. The partial correction of brain pathology is attributed to migration of donor hematopoietic cells, demonstrated by the presence of the Y chromosome and of normal microglia in the brain of mice receiving IDUA cDNA.

Alpha-N-acetylglucosaminidase deficiency (mucopolysaccharidosis IIIB, MPS IIIB) and alpha-l-iduronidase deficiency (MPS I) are heritable lysosomal storage diseases; neurodegeneration is prominent in MPS IIIB and in severe cases of MPS I. We have obtained morphologic and molecular evidence for the involvement of microglia in brain pathology of mouse models of the two diseases. In the cortex, a subset of microglia (sometimes perineuronal) consists of cells that are probably phagocytic; they have large storage vacuoles, react with MOMA-2 (monoclonal antibody against macrophages) and Griffonia simplicifolia isolectin IB(4), and stain intensely for the lysosomal proteins Lamp-1, Lamp-2, and cathepsin D as well as for G(M3) ganglioside. MOMA-2-positive cells appear at 1 and 6 months in MPS IIIB and MPS I mice, respectively, but though their number increases with age, they remain sparse. However, a profusion of cells carrying the macrophage CD68/macrosialin antigen appear in the cortex of both mouse models at 1 month. mRNA encoding CD68/macrosialin also increases at that time, as shown by microarray and Northern blot analyses. Ten other transcripts elevated in both mouse models are associated with macrophage functions, including complement C4, the three subunits of complement C1q, lysozyme M, cathepsins S and Z, cytochrome b558 small subunit, macrophage-specific protein 1, and DAP12. An increase in IFN-gamma and IFN-gamma receptor was observed by immunohistochemistry. These functional increases may represent activation of resident microglia, an influx and activation of blood monocytes, or both. They show an inflammatory component of brain disease in the two MPS, as is known for many neurodegenerative disorders.

Sanfilippo syndrome type B (MPS III B) is a neurodegenerative disorder characterized by profound mental retardation and early death. It is caused by deficiency of a lysosomal enzyme involved in heparan sulfate (HS) degradation. Because HS accumulation can be a major feature of this disease, we have examined crucial molecular systems associated with HS function. Using a knockout mouse with disruption of the gene responsible for HS degradation, we evaluated the effects of possible HS accumulation on neuroplasticity that are within the spectrum of action of fibroblast growth factors (FGFs) and their receptor (FGFR). We found that levels of mRNA for the FGFR-1 were attenuated in the mutant mice by the age of 6 months, whereas the mRNAs for FGF-1 and FGF-2 were reduced or unchanged in the brain regions tested. Neurogenesis, in which FGF-2 is involved, was inhibited in the MPS III B mouse brain at both young and adult ages. We also examined the expression of the glial fibrillary acidic protein (GFAP) gene and GFAP-positive cell density in both normal and injured conditions to study the functional response of astrocytes to insult. We found that, although the mutation alone caused drastic induction of reactive astrocytes, acute injury to the mutant brains failed to induce additional reactive astrocytes. Our results showed important alterations in the expression of several genes involved in the maintenance of neuroplasticity in the MPS III B. This in turn may result in reduction of neuronal health and brain function., (Copyright 2002 Wiley-Liss, Inc.)

Nonsense-mediated mRNA decay (NMD), the loss of mRNAs carrying premature stop codons, is a process by which cells recognize and degrade nonsense mRNAs to prevent possibly toxic effects of truncated peptides. Most mammalian nonsense mRNAs are degraded while associated with the nucleus, but a few are degraded in the cytoplasm; at either site, there is a requirement for translation and for an intron downstream of the early stop codon. We have examined the NMD of a mutant HEXA message in lymphoblasts derived from a Tay-Sachs disease patient homozygous for the common frameshift mutation 1278ins4. The mutant mRNA was nearly undetectable in these cells and increased to approximately 40% of normal in the presence of the translation inhibitor cycloheximide. The stabilized transcript was found in the cytoplasm in association with polysomes. Within 5 h of cycloheximide removal, the polysome-associated nonsense message was completely degraded, while the normal message was stable. The increased lability of the polysome-associated mutant HEXA mRNA shows that NMD of this endogenous mRNA occurred in the cytoplasm. Transfection of Chinese hamster ovary cells showed that expression of an intronless HEXA minigene harboring the frameshift mutation or a closely located nonsense codon resulted in half the normal mRNA level. Inclusion of multiple downstream introns decreased the abundance further, to about 20% of normal. Thus, in contrast to other systems, introns are not absolutely required for NMD of HEXA mRNA, although they enhance the low-HEXA-mRNA phenotype.

Background: Mucopolysaccharidosis I is a lysosomal storage disease caused by a deficiency of the enzyme alpha-L-iduronidase. We evaluated the effect of enzyme-replacement therapy with recombinant human alpha-L-iduronidase in patients with this disorder., Methods: We treated 10 patients with mucopolysaccharidosis I (age, 5 to 22 years) with recombinant human alpha-L-iduronidase at a dose of 125,000 U per kilogram of body weight given intravenously once weekly for 52 weeks. The patients were evaluated at base line and at 6, 12, 26, and 52 weeks by detailed clinical examinations, magnetic resonance imaging of the abdomen and brain, echocardiography, range-of-motion measurements, polysomnography, clinical laboratory evaluations, measurements of leukocyte alpha-L-iduronidase activity, and urinary glycosaminoglycan excretion., Results: Hepatosplenomegaly decreased significantly in all patients, and the size of the liver was normal for body weight and age in eight patients by 26 weeks. The rate of growth in height and weight increased by a mean of 85 and 131 percent, respectively, in the six prepubertal patients. The mean maximal range of motion of shoulder flexion and elbow extension increased significantly. The number of episodes of apnea and hypopnea during sleep decreased 61 percent. New York Heart Association functional class improved by one or two classes in all patients. Urinary glycosaminoglycan excretion decreased after 3 to 4 weeks of treatment; the mean reduction was 63 percent of base-line values. Five patients had transient urticaria during infusions. Serum antibodies to alpha-L-iduronidase were detected in four patients., Conclusions: In patients with mucopolysaccharidosis I, treatment with recombinant human alpha-L-iduronidase reduces lysosomal storage in the liver and ameliorates some clinical manifestations of the disease.

The Sanfilippo syndrome type B (MPS III B) is an autosomal recessive disease caused by deficiency of alpha-N-acetylglucosaminidase (EC 3. 2.1.50), one of the lysosomal enzymes required for the degradation of heparan sulfate. The disease is characterized by profound neurodegeneration but relatively mild somatic manifestations, and is usually fatal in the second decade. A mouse model had been generated by disruption of the Naglu gene in order to facilitate the study of pathogenesis and the development of therapy for this currently untreatable disease. Recombinant human alpha-N-acetylglucosaminidase (rhNAGLU) was prepared from secretions of Lec1 mutant Chinese hamster ovary cells. The enzyme, which has only unphosphorylated high-mannose carbohydrate chains, was endocytosed by mouse peritoneal macrophages via mannose receptors, with half-maximal uptake at ca. 10(-7) M. When administered intravenously to 3 month-old mice, rhNAGLU was taken up avidly by liver and spleen but marginally if at all by thymus, lung, kidney, heart, and brain (in order of diminishing uptake). The half-life of the enzyme was 2.5 days in liver and spleen. Immunohistochemistry and electron microscopy showed that only macrophages were involved in enzyme uptake and correction in these two organs, yet the storage of glycosaminoglycan was reduced to almost normal levels. The results show that the macrophage-targeted rhNAGLU can substantially reduce the body burden of glycosaminoglycan storage in the mouse model of Sanfilippo syndrome III B., (Copyright 2000 Academic Press.)

alpha-N-Acetylglucosaminidase (EC 3.2.1.50) is a lysosomal enzyme that is deficient in the genetic disorder Sanfilippo syndrome type B. To study the human enzyme, we expressed its cDNA in Lec1 mutant Chinese hamster ovary (CHO) cells, which do not synthesize complex oligosaccharides. The enzyme was purified to apparent homogeneity from culture medium by chromatography on concanavalin A-Sepharose, Poros 20-heparin, and aminooctyl-agarose. The purified enzyme migrated as a single band of 83 kDa on SDS-PAGE and as two peaks corresponding to monomeric and dimeric forms on Sephacryl-300. It had an apparent K(m) of 0.22 mM toward 4-methylumbelliferyl-alpha-N-acetylglucosaminide and was competitively inhibited by two potential transition analogs, 2-acetamido-1,2-dideoxynojirimycin (K(i) = 0.45 microM) and 6-acetamido-6-deoxycastanospermine (K(i) = 0.087 microM). Activity was also inhibited by mercurials but not by N-ethylmaleimide or iodoacetamide, suggesting the presence of essential sulfhydryl residues that are buried. The purified enzyme preparation corrected the abnormal [(35)S]glycosaminoglycan catabolism of Sanfilippo B fibroblasts in a mannose 6-phosphate-inhibitable manner, but its effectiveness was surprisingly low. Metabolic labeling experiments showed that the recombinant alpha-N-acetylglucosaminidase secreted by CHO cells had only a trace of mannose 6-phosphate, probably derived from contaminating endogenous CHO enzyme. This contrasts with the presence of mannose 6-phosphate on naturally occurring alpha-N-acetylglucosaminidase secreted by diploid human fibroblasts and on recombinant human alpha-l-iduronidase secreted by the same CHO cells. Thus contrary to current belief, overexpressing CHO cells do not necessarily secrete recombinant lysosomal enzyme with the mannose 6-phosphate-targeting signal; this finding has implications for the preparation of such enzymes for therapeutic purposes., (Copyright 2000 Academic Press.)

While seeking conditions for single crystals of human alpha-L-iduronidase, solutions were discovered (pH 3.0-8.5 containing calcium or zinc salts) that transform soluble alpha-L-iduronidase to a solid aggregate. This aggregate is a spherulite of semi-crystalline protein. The X-ray diffraction pattern and ability to bind Congo red characterize the alpha-L-iduronidase spherulite as 'amyloid-like', in that it displays two of the characteristics of amyloidogenic proteins. In addition, alpha-L-iduronidase also interacts with heparin, as do some amyloid-forming proteins.

The Sanfilippo syndrome type B is an autosomal recessive disorder caused by mutation in the gene (NAGLU) encoding alpha-N-acetylglucosaminidase, a lysosomal enzyme required for the stepwise degradation of heparan sulfate. The most serious manifestations are profound mental retardation, intractable behavior problems, and death in the second decade. To generate a model for studies of pathophysiology and of potential therapy, we disrupted exon 6 of Naglu, the homologous mouse gene. Naglu-/- mice were healthy and fertile while young and could survive for 8-12 mo. They were totally deficient in alpha-N-acetylglucosaminidase and had massive accumulation of heparan sulfate in liver and kidney as well as secondary changes in activity of several other lysosomal enzymes in liver and brain and elevation of gangliosides G(M2) and G(M3) in brain. Vacuolation was seen in many cells, including macrophages, epithelial cells, and neurons, and became more prominent with age. Although most vacuoles contained finely granular material characteristic of glycosaminoglycan accumulation, large pleiomorphic inclusions were seen in some neurons and pericytes in the brain. Abnormal hypoactive behavior was manifested by 4.5-mo-old Naglu-/- mice in an open field test; the hyperactivity that is characteristic of affected children was not observed even in younger mice. In a Pavlovian fear conditioning test, the 4.5-mo-old mutant mice showed normal response to context, indicating intact hippocampal-dependent learning, but reduced response to a conditioning tone, perhaps attributable to hearing impairment. The phenotype of the alpha-N-acetylglucosaminidase-deficient mice is sufficiently similar to that of patients with the Sanfilippo syndrome type B to make these mice a good model for study of pathophysiology and for development of therapy.

Sanfilippo syndrome type B (mucopolysaccharidosis III B) is a rare autosomal recessive disease caused by deficiency of alpha-N-acetylglucosaminidase, one of the enzymes required for the lysosomal degradation of heparan sulfate. The gene for this enzyme, NAGLU, recently was isolated, and several mutations were characterized. We have identified, in amplified exons from nine fibroblast cell lines derived from Sanfilippo syndrome type B patients, 10 additional mutations: Y92H, P115S, Y140C, E153K, R203X, 650insC, 901delAA, P358L, A664V, and L682R. Four of these mutations were found in homozygosity, and only two were seen in more than one cell line. Thus, Sanfilippo syndrome type B shows extensive molecular heterogeneity. Stable transfection of Chinese hamster ovary cells, by cDNA mutagenized to correspond to the NAGLU missense mutations, did not yield active enzyme, demonstrating the deleterious nature of the mutations. Nine of the 10 amino acid substitutions identified to date are clustered near the amino or the carboxyl end of alpha-N-acetylglucosaminidase, suggesting a role for these regions in the transport or function of the enzyme.

alpha-L-Iduronidase is a lysosomal hydrolase that is deficient in Hurler syndrome and clinically milder variants. Recombinant human alpha-L-iduronidase, isolated from secretions of an overexpressing Chinese hamster ovary cell line, is potentially useful for replacement therapy of these disorders. Because of the importance of carbohydrate residues for endocytosis and lysosomal targeting, we examined the oligosaccharides of recombinant alpha-L-iduronidase at each of its six N-glycosylation sites. Biosynthetic radiolabeling showed that three or four of the six oligosaccharides of the secreted enzyme were cleaved by endo-beta-N-acetylglucosaminidase H, with phosphate present on the sensitive oligosaccharides and L-fucose on the resistant ones. For structural analysis, tryptic and chymotryptic glycopeptides were isolated by lectin binding and reverse phase high pressure liquid chromatography; their molecular mass was determined by matrix-assisted laser desorption-time of flight mass spectrometry before and after treatment with endo- or exoglycosidases or with alkaline phosphatase. Identification of the peptides was assisted by amino- or carboxyl-terminal sequence analysis. The major oligosaccharide structures found at each site were as follows: Asn-110, complex; Asn-190, complex; Asn-336, bisphosphorylated (P2Man7GlcNAc2); Asn-372, high mannose (mainly Man9GlcNAc2, some of which was monoglucosylated); Asn-415, mixed high mannose and complex; Asn-451, bisphosphorylated (P2Man7GlcNAc2). The Asn-451 glycopeptide was unexpectedly resistant to digestion by N-glycanase unless first dephosphorylated, but it was sensitive to endo-beta-N-acetylglucosaminidase H and to glycopeptidase A. The heterogeneity of carbohydrate structures must represent the accessibility of the glycosylation sites as well as the processing capability of the overexpressing Chinese hamster ovary cells.

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.

The Sanfilippo syndrome type B is a lysosomal storage disorder caused by deficiency of alpha-N-acetylglucosaminidase; it is characterized by profound mental deterioration in childhood and death in the second decade. For understanding the molecular genetics of the disease and for future development of DNA-based therapy, we have cloned the cDNA and gene encoding alpha-N-acetylglucosaminidase. Cloning started with purification of the bovine enzyme and use of a conserved oligonucleotide sequence to probe a human cDNA library. The cDNA sequence was found to encode a protein of 743 amino acids, with a 20- to 23-aa signal peptide immediately preceding the amino terminus of the tissue enzyme and with six potential N-glycosylation sites. The 8.5-kb gene (NAGLU), interrupted by 5 introns, was localized to the 5'-flanking sequence of a known gene, EDH17B, on chromosome 17q21. Five mutations were identified in cells of patients with Sanfilippo syndrome type B: 503del10, R297X, R626X, R643H, and R674H. The occurrence of a frameshift and a nonsense mutation in homozygous form confirms the identity of the NAGLU gene.

The alpha-L-iduronidase deficiency diseases (Mucopolysaccharidosis I) cover a spectrum of clinical severity ranging from the very severe (Hurler syndrome, MPS IH) through an intermediate (Hurler/Scheie syndrome, MPS IH/S) to a relatively mild form (Scheie syndrome, MPS IS). Numerous mutations of the gene encoding alpha-L-iduronidase (IDUA) are known in Hurler syndrome, but only three in the other disorders. We report on novel mutations of the IDUA gene in one patient with the Scheie syndrome and in three patients with the Hurler/Scheie syndrome. The novel mutations, all single base changes, encoded the substitutions R492P (Scheie), and X654G, P496L, and L490P (Hurler/Scheie). The L490P mutation was apparently homozygous, whereas each of the others was found in compound heterozygosity with a Hurler mutation. The deleterious nature of the mutations was confirmed by absence of enzyme activity upon transfection of the corresponding mutagenized cDNAs into Cos-1 cells. These results provide additional information for genotype-phenotype correlations.

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.

Mutations in the gene encoding alpha-L-iduronidase (IDUA) are the cause of Hurler syndrome. Fibroblasts from patients homozygous for nonsense IDUA alleles have much reduced mRNA detectable by Northern analysis, as has been observed in many other instances of premature translation termination. Yet RT-PCR (reverse transcription followed by PCR amplification) showed a normal level of a segment covering exons 1 and 2 in Hurler cells homozygous for alleles bearing the nonsense mutations, Q70X or W402X. The 3' end of the segment was between exons 2 and 4. The results indicate that the nonsense RNA was degraded to fragment(s), independent of the position of the mutation (exon 2 or exon 9, respectively). Treatment of the cells with cycloheximide resulted in some increase of intact mRNA, suggesting that translation is required for mRNA degradation.

We developed a Chinese hamster ovary (CHO) cell line that produces and secretes large quantities of recombinant human alpha-L-iduronidase, the lysosomal hydrolase deficient in mucopolysaccharidosis I (Hurler, Hurler-Scheie, and Scheie syndromes). The alpha-L-iduronidase cDNA was introduced into a vector containing the cytomegalovirus immediate early gene promoter/enhancer, a murine immunoglobulin C alpha region intron, and the bovine growth hormone polyadenylation signal. Following cotransfection with a plasmid containing the neomycin resistance gene, stably transfected lines were selected with G-418. The highest expressing CHO cell line contained 1400-6000 units of alpha-L-iduronidase per milligram of protein, or 0.6-2.4% of total cell protein. Secreted alpha-L-iduronidase was 3000- to 7000 fold increased, with about 5000 units accumulating in 24 h per 10(7) cells. The activity and distribution of five other lysosomal glycosidases were not significantly affected. Metabolic labeling showed that half of the newly synthesized alpha-L-iduronidase was secreted, but generally less was recovered due to its instability in the medium. It was post-translationally processed as previously shown for alpha-L-iduronidase of human fibroblasts. Recombinant alpha-L-iduronidase was efficiently endocytosed by Hurler fibroblasts utilizing a mannose 6-phosphate-dependent mechanism (half maximal uptake at 0.7 nM) and was "corrective" for abnormal glycosaminoglycan accumulation (half-maximal correction at 0.7 pM). The half-life of the recombinant enzyme was 5 days following uptake into Hurler fibroblasts. Production in a 5-liter microcarrier culture system permitted the collection of 15 mg or more per day.(ABSTRACT TRUNCATED AT 250 WORDS)

The mutations underlying Hurler syndrome (mucopolysaccharidosis IH) in Druze and Muslim Israeli Arab patients have been characterized. Four alleles were identified, using a combination of (a) PCR amplification of reverse-transcribed RNA or genomic DNA segments, (b) cycle sequencing of PCR products, and (c) restriction-enzyme analysis. One allele has two amino acid substitutions, Gly409-->Arg in exon 9 and Ter-->Cys in exon 14. The other three alleles have mutations in exon 2 (Tyr64-->Ter), exon 7 (Gln310-->Ter), or exon 8 (Thr366-->Pro). Transfection of mutagenized cDNAs into Cos-1 cells showed that two missense mutations, Thr366-->Pro and Ter-->Cys, permitted the expression of only trace amounts of alpha-L-iduronidase activity, whereas Gly409-->Arg permitted the expression of 60% as much enzyme as did the normal cDNA. The nonsense mutations were associated with abnormalities of RNA processing: (1) both a very low level of mRNA and skipping of exon 2 for Tyr64-->Ter and (2) utilization of a cryptic splice site for Gln310-->Ter. In all instances, the probands were found homozygous, and the parents heterozygous, for the mutant alleles, as anticipated from the consanguinity in each family. The two-mutation allele was identified in a family from Gaza; the other three alleles were found in seven families, five of them Druze, residing in a very small area of northern Israel. Since such clustering suggests a classic founder effect, the presence of three mutant alleles of the IDUA gene was unexpected.

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease caused by deficiency of alpha-L-iduronidase. In addition to the well-known human forms (Hurler, Hurler/Scheie, and Scheie syndromes), there exists a canine model of the disease. By using previously described canine cDNA encoding alpha-L-iduronidase as a probe, the canine IDUA gene has been cloned and characterized. It contains 14 exons spread over 13 kb. An unusual GC dinucleotide was found at the donor splice site of intron 11. A transcriptional start site was identified by primer extension 177 bp upstream of the initiator AUG codon. The upstream region was found to be similar to the promoter region of many housekeeping genes: it is GC rich and has seven potential Sp1 binding sites but no TATA box or CAAT motif. The mutation in canine MPS I was localized to the area of intron 1 by RT-PCR, identified by sequence analysis of amplified genomic DNA, and confirmed by restriction analysis; it is a G-->A transition in the donor splice site of intron 1. The mutation causes retention of intron 1 in the RNA and creates a premature termination codon at the exon-intron junction.

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.

Amnion membrane implantation has been proposed as an approach to enzyme replacement in mucopolysaccharidoses. Human amnion membranes have been subcutaneously implanted in the abdominal wall in 19 patients with mucopolysaccharidoses (MPS I, II and III). A protocol was developed for the objective evaluation of experimental treatments of these patients. Systematic evaluation of the clinical status before and 6 months after amnion membrane implantation reveals no change in function except improvement in joint mobility. The sum of all joint movements showed improvement from baseline values to 6 months after implantation by ANOVA followed by post-hoc analysis (p less than 0.056). The only specific joint movements to significantly improve after 6 months were shoulder extension (p less than 0.01) and hip internal rotation (p less than 0.05). Serial measurements of the deficient lysosomal enzyme activity in serum and white blood cells did not increase in any patient after amnion membrane implantation. Urinary glycosaminoglycan excretion decreased transiently in 2 of 10 patients after implantation, but a second amnion membrane implantation did not result in any change. Biopsy of the implantation site in 10 patients 6 months after amnion membrane implantation revealed a foreign-body reaction with giant cell formation and fibrosis and no recognizable amnion membrane tissue. We conclude that human amnion membrane implantation is not an effective therapy in mucopolysaccharidoses.

Two CpG mutations at codon 504 of the gene encoding the alpha-subunit of beta-hexosaminidase (the HEX A gene) have been identified previously: (1) a C deletion resulting in premature termination of the alpha-subunit and (2) a G----A transition resulting in 504Arg----His substitution, in patients with infantile Tay-Sachs disease and juvenile GM2 gangliosidosis, respectively. This prompted a search for a C----T transition in the same dinucleotide, as would be expected from the mechanism of CpG mutagenesis. Such a mutation, which results in a substitution of cysteine for arginine, was found in a patient with chronic GM2 gangliosidosis, in compound heterozygosity with the known 269Gly----Ser allele. The biochemical phenotype of the 504Arg----Cys mutation was examined by site-directed mutagenesis of the alpha-subunit cDNA and transfection of Cos-1 cells. The expression of the mutagenized cDNA with the cysteine substitution gave rise to an alpha-subunit with the same defects as those resulting from expression of mutagenized cDNA with the histidine substitution--i.e., secretion primarily as the alpha-monomer rather than as the alpha alpha dimer, along with absence of enzymatic activity. The 504Arg----Cys/269Gly----Ser genotype of the chronic GM2 gangliosidosis patient is shared by her sibling, who has mild adult-onset GM2 gangliosidosis, implying that the clinical differences between them must be attributed to other factors. The family is unique in yet another respect--namely, that the normal allele of the mother and of a 504Arg----Cys heterozygous sibling has a silent mutation, a G----A transition in the wobble position of the glutamic acid codon at position 506.(ABSTRACT TRUNCATED AT 250 WORDS)

An elongated beta-subunit of the lysosomal enzyme beta-hexosaminidase was found in fibroblast strains derived from two patients with juvenile Sandhoff disease and two asymptomatic individuals sharing an unusual isoenzyme pattern: a low level of residual A (alpha beta) isoenzyme activity (3-6% of normal for the juvenile Sandhoff and 9-10% for the asymptomatic strains) without B (beta beta) isoenzyme activity. The elongated beta-subunit was abnormal in other ways: It reacted with antiserum against the unfolded polypeptide, it was not phosphorylated on mannose residues, it was not processed to the mature form, and it was degraded rapidly. The increased length of the beta-subunit was caused by two different mutations. Cells from two juvenile Sandhoff and one asymptomatic individuals had the previously described G----A transition in intron 12 that creates a splice site, causing an in-frame insertion of 24 intronic nucleotides into mRNA (Nakano, T., and Suzuki, K. (1989) J. Biol. Chem. 264, 5155-5158). The second mutation was found in cells from the asymptomatic girl whose A+B- isoenzyme pattern had been designated "Hexosaminidase Paris" (Dreyfus, J. C., Poenaru, L., Vibert, M., Ravise, N., and Boue, J. (1977) Am. J. Hum. Genet. 29, 287-293); duplication of a region straddling the junction of intron 13 and exon 14 generates an alternate splice site, causing an in-frame insertion of 18 nucleotides into mRNA. Although the two new splice sites are used preferentially, the normal sites may be used to some extent, accounting for the residual A isoenzyme activity.

Mutations in the HEX A gene, encoding the alpha-subunit of beta-hexosaminidase A (Hex A), are the cause of Tay-Sachs disease as well as of juvenile, chronic, and adult GM2 gangliosidoses. We have examined the distribution of three mutations--a 4-nucleotide insertion in exon 11, a G----C transversion at a 5' splice site in intron 12, and a 269Gly----Ser amino acid substitution in exon 7--among individuals enzymatically diagnosed as carriers of Hex A deficiency. Mutation analysis included polymerase chain reaction (PCR) amplification of the relevant regions of genomic DNA, followed by allele-specific oligonucleotide hybridization; another test for heterozygosity of the exon 11 insertion was based on the formation of heteroduplex PCR fragments of low electrophoretic mobility. The percentage distribution of the exon 11, intron 12, exon 7, and unidentified mutant alleles was 73:15:4:8 among 156 Jewish carriers of Hex A deficiency and 16:0:3:81 among 51 non-Jewish carriers. Regardless of the mutation, the ancestral origin of the Jewish carriers was primarily eastern and (somewhat less often) central Europe, whereas for the non-Jewish carriers it was western Europe. Because a twelfth of the Jewish carriers and four-fifths of the non-Jewish carriers of Hex A deficiency had mutant alleles other than the three common ones tested, enzyme-based tests cannot be replaced by DNA-based tests at the present time. However, DNA-based tests for two-carrier couples could identify those at risk for the chronic/adult GM2 gangliosidoses rather than for infantile Tay-Sachs disease.

As a step toward understanding the metabolic consequences of the cystic fibrosis (CF) mutation, we have examined the kinetics of 36Cl efflux in cells cultured from sweat glands, a tissue that is affected in the disease. Epithelial cells, derived from the secretory coil of sweat glands of CF and control individuals, were cultured in serum-free medium, and primary cultures used for efflux experiments. Cell layers were equilibrated with Na36Cl in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered balanced salt solution for 45 min at 37 degrees C, washed in 0.25 M sucrose, and incubated in nonradioactive buffer for measurement of 36Cl efflux. Efflux from CF and control cells followed biphasic kinetics and was described by the equation Y = Ae-kat + Be-kbt. All efflux was inhibited at 6 degrees C. The fast component of efflux, Ae-kat, of both control and CF cells was inhibited by the anion channel blockers 4,4'-diisothiocyanato-2,2'-stilbene disulfonic acid, 9-anthracene carboxylate, and diphenylamine 2-carboxylate, implicating release through chloride channels. At 23 degrees C, the kinetics of 36Cl efflux from CF and control cells were indistinguishable, but efflux from control cells could be accelerated by cAMP analogs and isoproterenol. At 37 degrees C, 36Cl efflux was more rapid from control cells than from CF cells, but could not be stimulated further by beta-adrenergic agents. In both cases, the increased rate of efflux was due to a severalfold increase in the A parameter of the fast component. These differential responses constitute a "CF phenotype" of secretory sweat gland cells in culture that may be useful for further investigation of the metabolic defect in cystic fibrosis.

Juvenile GM2 gangliosidosis is a rare neurodegenerative disorder closely related to Tay-Sachs disease but of later onset and more protracted course. The biochemical defect lies in the alpha-subunit of the lysosomal enzyme beta-hexosaminidase. Cultured fibroblasts derived from patient A synthesized an alpha-subunit which could acquire mannose 6-phosphate and be secreted, but which failed to associate with the beta-subunit to form the enzymatically active heterodimer. By contrast, fibroblasts from patient B synthesized an alpha-subunit that was retained in the endoplasmic reticulum. To identify the molecular basis of the disorder, RNA from fibroblasts of these two patients was reverse-transcribed, and the cDNA encoding the alpha-subunit of beta-hexosaminidase was amplified by the polymerase chain reaction (PCR) in four overlapping fragments. The PCR fragments were subcloned and shown by sequence analysis to contain a G to A transition corresponding to substitution of histidine for arginine at position 504 in the case of patient A and at position 499 in the case of patient B. The mutations were confirmed by hybridization of allele-specific oligonucleotides to PCR-amplified fragments of DNA corresponding to exon 13 of the alpha-subunit gene. The Arg504----His mutation was found on both alleles of patient A as well as of another unrelated patient; the homozygosity of this mutant allele is attributable to consanguinity in the two families. The Arg499----His mutation was found in patient B in compound heterozygosity with a common infantile Tay-Sachs allele. There is additional heterogeneity in juvenile GM2 gangliosidosis, as neither mutation was found in the DNA of a fourth patient. The Arg----His mutations at positions 499 and 504 are located at CpG dinucleotides, which are known to be mutagenic "hot spots."

A disease discovered in three Plott Hound littermates was found to be associated with a profound and specific deficiency of alpha-L-iduronidase (mucopolysaccharide alpha-L-iduronohydrolase; EC 3.2.1.76) in fibroblasts and leukocytes. The pedigree was consistent with autosomal recessive inheritance. A markedly increased amount of dermatan sulfate and heparan sulfate was excreted in urine. Fibroblasts cultured from the skin of the affected dogs accumulated excessive 35S-labeled mucopolysaccharide; this accumulation could be decreased to a normal level by exogenous human high-uptake alpha-L-iduronidase (Hurler corrective factor) as well as by secretions of normal human or canine fibroblasts. The correction was inhibited by mannose 6-phosphate. Maturation of alpha-L-iduronidase in normal canine fibroblasts followed the pathway previously observed in human fibroblasts; no cross-reactive material was observed in the cells or in secretions from the fibroblasts of the affected dogs. The canine disorder thus resembles mucopolysaccharidosis I in all biochemical parameters tested; the clinical appearance of the animals is closest to Hurler-Scheie syndrome, a form of alpha-L-iduronidase deficiency of intermediate severity. The animal model should prove valuable for therapeutic experiments.