Data from subjects receiving intrathecal laronidase for cervical spinal stenosis due to mucopolysaccharidosis type I.

Five subjects with mucopolysaccharidosis type I and symptomatic cervical spinal stenosis received intrathecal laronidase in a 4-month pilot study and/or a 12-month extension study [1]. Clinical descriptions of study subjects, nonserious adverse events, individual data tables, and scoring system methods are provided. There were ten nonserious adverse events that occurred in more than one study subject. Somatosensory evoked potentials were absent in two subjects and normal in two subjects, limiting their utility as an endpoint. There were no significant changes in magnetic resonance imaging of cervical spinal cord or brain, pulmonary function tests, or cerebrospinal fluid opening pressure. These data are presented along with the scoring methods used in evaluation of the study subjects.

Continuous infusion of enzyme replacement therapy is inferior to weekly infusions in MPS I dogs.

Intravenous enzyme replacement therapy with recombinant human α-L-iduronidase (rhIDU) is used weekly to treat mucopolysaccharidosis (MPS) I. We tested continuous administration of rhIDU at two dosing levels (0.58 mg/kg per week and 2 mg/kg per week) in MPS I dogs, and compared the efficacy of continuous infusion with the clinically used 0.58 mg/kg weekly three-hour infusion. Peak plasma concentrations of rhIDU were much higher in weekly-treated dogs (mean 256 units/ml) than steady-state concentrations in dogs treated with continuous infusion (mean 1.97 units/ml at 0.58 mg/kg per week; 8.44 units/ml at 2 mg/kg per week). Dogs receiving continuous IV rhIDU, even at a higher (2 mg/kg per week) dose, had consistently lower iduronidase levels in tissues than dogs receiving a weekly (0.58 mg/kg per week) dose. GAG storage was also less improved by continuous intravenous infusion. Adverse events were similar in all dosing groups. We found that continuous administration of 2 mg/kg per week rhIDU to MPS I dogs was insufficient to achieve GAG storage reduction comparable to 0.58 mg/kg weekly dosing.

Penetration, diffusion, and uptake of recombinant human alpha-L-iduronidase after intraventricular injection into the rat brain.

Central nervous system disease can have devastating consequences in the severe or Hurler form of mucopolysaccharisosis I (MPS I). Intravenously administered recombinant human alpha-L-iduronidase (rhIDU) is not expected to reach and treat the brain disease due to the blood-brain barrier. To determine whether administration of rhIDU into the cerebrospinal fluid could successfully treat the brain, we studied intraventricular administration of rhIDU in rats. RhIDU was stereotactically administered directly to the lateral ventricle of the intact rat brain and the brain tissues assessed by enzyme assays, immunofluorescence and confocal microscopy 30 min, 24 h, or 7 days later. Quantitation of activity revealed that rhIDU was widely distributed throughout the brain following injection into the lateral ventricle, with activities increased by a factor of 3.3 higher than control in most samples 30 min-24 h after injection and highest levels on the side of injection. The enzyme crossed the ependymal lining of the ventricle and entered neurons into lysosomal-like vesicles. The enzyme was able to diffuse through brain tissue as demonstrated by a decreasing signal gradient from 0.2 to 4.8 mm from the ventricle surface. The largest amount of rhIDU, as detected by immunostaining, was observed 24 h after injection and decreased approximately 50% during the first 7 days. Although the immunostaining decreased with time, specific vesicular staining was still detectable 28 days after injection. The data suggest that rhIDU given into the ventricle can diffuse, penetrate at least several millimeters of brain tissue and be taken up into neurons and glial cells.

Intrathecal enzyme replacement therapy reduces lysosomal storage in the brain and meninges of the canine model of MPS I.

Enzyme replacement therapy (ERT) has been developed for several lysosomal storage disorders, including mucopolysaccharidosis I (MPS I), and is effective at reducing lysosomal storage in many tissues and in ameliorating clinical disease. However, intravenous ERT does not adequately treat storage disease in the central nervous system (CNS), presumably due to effects of the blood-brain barrier on enzyme distribution. To circumvent this barrier, we studied whether intrathecal (IT) recombinant human alpha-L-iduronidase (rhIDU) could penetrate and treat the brain and meninges. An initial dose-response study showed that doses of 0.46-4.14 mg of IT rhIDU successfully penetrated the brain of normal dogs and reached tissue levels 5.6 to 18.9-fold normal overall and 2.7 to 5.9-fold normal in deep brain sections lacking CSF contact. To assess the efficacy and safety in treating lysosomal storage disease, four weekly doses of approximately 1 mg of IT rhIDU were administered to MPS I-affected dogs resulting in a mean 23- and 300-fold normal levels of iduronidase in total brain and meninges, respectively. Quantitative glycosaminoglycan (GAG) analysis showed that the IT treatment reduced mean total brain GAG to normal levels and achieved a 57% reduction in meningeal GAG levels accompanied by histologic improvement in lysosomal storage in all cell types. The dogs did develop a dose-dependent immune response against the recombinant human protein and a meningeal lymphocytic/plasmacytic infiltrate. The IT route of ERT administration may be an effective way to treat the CNS disease in MPS I and could be applicable to other lysosomal storage disorders.

Successful induction of immune tolerance to enzyme replacement therapy in canine mucopolysaccharidosis I.

Immune responses can interfere with the effective use of therapeutic proteins to treat genetic deficiencies and have been challenging to manage. To address this problem, we adapted and studied methods of immune tolerance used in canine organ transplantation research to soluble protein therapeutics. A tolerization regimen was developed that prevents a strong antibody response to the enzyme alpha-l-iduronidase during enzyme replacement therapy of a canine model of the lysosomal storage disorder mucopolysaccharidosis I. The tolerizing regimen consists of a limited 60-day course of cyclosporin A and azathioprine combined with weekly i.v. infusions of low-dose recombinant human alpha-l-iduronidase. The canines tolerized with this regimen maintain a reduced immune response for up to 6 months despite weekly therapeutic doses of enzyme in the absence of immunosuppressive drugs. Successful tolerization depended on high plasma levels of cyclosporin A combined with azathioprine. In addition, the induction of tolerance may require mannose 6-phosphate receptor-mediated uptake because alpha-l-iduronidase and alpha-glucosidase induced tolerance with the drug regimen whereas ovalbumin and dephosphorylated alpha-l-iduronidase did not. This tolerization method should be applicable to the treatment of other lysosomal storage disorders and provides a strategy to consider for other nontoleragenic therapeutic proteins and autoimmune diseases.

Enzyme-replacement therapy in mucopolysaccharidosis I.

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.

Lysosomal sulphate transport is dependent upon sulphydryl groups.

Using thiol blocking agents, we examined the role of sulphydryl groups for function of the lysosomal sulphate transport system. Monothiol binding reagents, p-hydroxymercuribenzoic acid (p-HMB) and p-chloromercuribenzene sulphonic acid (p-CMBS), dithiol binding reagents such as CuCl2, the alkylating agent, N-ethylmaleimide (NEM), and NADH all inhibited lysosomal sulphate transport. The inhibitory effects of NEM and Cu2+ were not additive, suggesting that they both act upon the same critical sulphydryl group(s). Unlike the case for NEM, the inhibitory effects of Cu2+ were reversed by the reducing agent, dithiothreitol. Exposure to NEM resulted in a seven-fold increase in Km to 867 microM versus a control value of 126 microM and a modest decrease in Vmax to 99 pmolperunit beta-hexosaminidase per 30 s versus a control value of 129 pmolperunit beta-hexosaminidase per 30 s. Similar although somewhat less dramatic results were obtained using Cu2+ with an increase of Km to 448 microM and a Vmax of 77 pmolperunit beta-hexosaminidase per 30 s. The sulphate transport activity of detergent solubilized lysosomal membranes could be bound to a p-chloromercuribenzoic acid (p-CMB)-Sepharose sulphydryl affinity resin and eluted with mercaptoethanol. Sulphydryl groups thus appear to play a role in sulphate transport through effects on substrate affinity. Sulphydryl-binding appears to be a strategy that may be useful for purification of the transporter.

ATP stimulates lysosomal sulphate transport at neutral pH: evidence for phosphorylation of the lysosomal sulphate carrier.

ATP markedly stimulated sulphate uptake by rat liver lysosomes that had been treated with N-ethylmaleimide to block the effects of the lysosomal proton-translocating ATPase (H+-ATPase). Maximal stimulation required millimolar concentrations of ATP and neutral buffer pH. ATP-stimulated transport exhibited saturation kinetics with a Km of 175 microM, identical with the Km for lysosomal sulphate uptake at pH 5.0, a process that does not require ATP. The requirement for ATP was specific: other nucleotides such as AMP, ADP, CTP, GTP, ITP and UTP failed to stimulate transport. Adenosine 5'-[beta,gamma-imido]triphosphate, the non-hydrolysable analogue of ATP, also failed to stimulate sulphate uptake, suggesting a requirement for ATP hydrolysis. Lysosomal pH, membrane potential and glucose transport were unchanged by the presence of ATP under the experimental conditions, consistent with a direct effect of ATP on the sulphate transporter. Exposure of lysosomes to protein kinase A and protein kinase C inhibitors did not alter the stimulation of sulphate transport by ATP. The lysosomal sulphate transport protein might be subject to regulation by a phosphorylation pathway that is not dependent on protein kinase A or protein kinase C.

Generation of sequence-tagged sites from Xp22.3 by isolating common Alu-PCR products of radiation hybrids retaining overlapping human X chromosome fragments.

Several human diseases have been mapped to Xp22.3 on the distal short arm of the human X chromosome, and many genes in this area have been found to be expressed from the inactive X chromosome. To facilitate physical mapping and characterization of this interesting region, we have constructed a battery of radiation hybrids containing human X chromosomal fragments, and isolated two hybrid clones A with overlapping fragments of Xp22.3. Alu-PCR on these hybrids and identification of sequences common to both hybrids allowed the isolation of six sequences-tagged sites (STSs) from Xp22.3. Five of the STSs were mapped+ to individual YACs comprising a recently constructed contig of this region. These novel STSs are useful markers for further physical characterization of this part of the genome.

Regulation of lysosomal sulfate transport by thyroid hormone.

Sulfate transport was examined in rat liver lysosomes that were isolated from thyroid hormone-treated, thyroidectomized, and control animals. Sulfate uptake was significantly decreased in lysosomes from animals that had received intraperitoneal T3 (3,5,3'-triiodothyronine) at a dose of 20 micrograms/100 g body weight. The effect of T3 was maximal by 24 h post-injection and resulted in marked decreases in both Vmax (control: 155 +/- 33 pmol/unit of beta-hexosaminidase/30 s versus T3 treated: 24 +/- 7 pmol/unit of beta-hexosaminidase/30 s) and Km (control: 213 +/- 34 microM versus T3 treated: 92 +/- 6 microM). Thyroidectomy was associated with a significant increase in Vmax (control: 250 pmol/unit of beta-hexosaminidase/30 s versus thyroidectomized: 564 pmol/unit of beta-hexosaminidase/30 s), while Km was not significantly affected. The effect of thyroid hormone on lysosomal sulfate transport appeared to be relatively specific. In contrast to its effect on sulfate transport, T3 treatment had no effect on the uptake of either glucose or N-acetylglucosamine by rat liver lysosomes. Lysosomal pH, acidification in response to Mg/ATP, and the specific activities of alpha-L-iduronidase, beta-hexosaminidase, beta-D-glucosidase, and acid phosphatase were unaffected by T3 administration. Incubation of T3 with lysosomes from control animals had little or no effect on sulfate transport. Treatment of isolated lysosomes with either protein kinase A or alkaline phosphatase resulted in modest stimulation of transport. Thus, T3 does not appear to regulate transport by either direct interaction with the lysosomal transporter or protein kinase A-mediated phosphorylation. The exact mechanism for the inhibitory effect of T3 on lysosomal sulfate transport remains to be determined.

An evaluation of the inactive mouse X chromosome in somatic cell hybrids.

The expression of mouse Zfx, Rps4, Ube1x, and Xist was evaluated in hamster-mouse somatic cell hybrids containing either an active or an inactive mouse X chromosome using polymerase chain reaction of reverse transcribed RNA (RT-PCR). The results showed that Zfx, Rps4, and Ube1x are expressed exclusively from the active mouse X, while Xist is expressed exclusively from the inactive X. These findings confirm the pattern of X inactivation for these mouse genes reported previously based on expression in somatic tissues of F1 females from interspecific crosses. These results demonstrate the existence of differences between human and mouse X inactivation, as the corresponding human genes, ZFX, RPS4X, and UBE1 escape X inactivation.

Role of the pseudoautosomal region in sex-chromosome pairing during male meiosis: meiotic studies in a man with a deletion of distal Xp.

Meiotic studies were undertaken in a 24-year-old male patient with short stature, chondrodysplasia punctata, ichthyosis, steroid sulfatase deficiency, and mild mental retardation with an inherited cytologically visible deletion of distal Xp. Molecular investigations showed that the pseudoautosomal region as well as the steroid sulfatase gene were deleted, but telomeric sequences were present at the pter on the deleted X chromosome. A complete failure of sex-chromosome pairing was observed in the primary spermatocytes of the patient. Telomeric approaches between the sex chromosomes were made at zygotene in some cells, but no XY synaptonemal complex was formed. The sex chromosomes were present as univalents at metaphase I, and germ-cell development was arrested between metaphase I and metaphase II in the vast majority of cells, consistent with the azoospermia observed in the patient. The failure of XY pairing in this individual indicates that the pseudoautosomal sequences play an important role in initiating XY pairing and formation of synaptonemal complex at meiosis.

Directed isolation of human genes that escape X inactivation.

Existing methodologies have been combined to produce a directed approach to the isolation of human genes that escape X inactivation. A mouse-human somatic cell hybrid line was established that has an inactive X as its only human chromosome, and nuclear RNA from this cell line was used to construct a cDNA library. Transcribed human sequences were isolated by screening the library with labeled human DNA. The corresponding genomic sequences were isolated in phage or cosmid clones, and exons were identified by detection of transcripts on northern blots. By these means three human loci have been identified that contain genes expressed from an inactive X chromosome. Fluorescence in situ hybridization has been used to map these genes to Xp21.1-22.1, Xp22.1-22.2, and terminal Xp/Yp. One of the three genes (XE45) corresponds to the ZFX gene, while the other two genes (XE7 and XE59) represent novel cloned sequences. Physical and genetic evidence indicate that XE7 is a newly identified pseudoautosomal gene.

Physical mapping of loci in the distal half of the short arm of the human X chromosome: implications for the spreading of X-chromosome inactivation.

The relative order of 11 loci in the distal half of the short arm of the human X chromosome was examined using a panel of somatic cell hybrids containing structurally rearranged X chromosomes. The results show that the gene for phosphoribosylpyrophosphate synthetase 2 (PRPS2) is located between ZFX (zinc finger protein, X-linked) and STS (steroid sulfatase). The results also confirm the localization of ZFX distal to POLA (alpha-DNA polymerase). Previous studies have shown that STS and ZFX escape X-inactivation whereas POLA undergoes inactivation. Evaluation of PRPS2 expression in somatic cell hybrids containing inactive human X chromosomes showed that PRPS2 undergoes X-inactivation. These results provide further evidence for interspersion of loci that do and do not undergo X-inactivation on the human X chromosome.

Uniparental heterodisomy for chromosome 14 in a phenotypically abnormal familial balanced 13/14 Robertsonian translocation carrier.

A 9-year-old mentally retarded girl with multiple congenital anomalies was found to carry a balanced 13/14 Robertsonian translocation [45,XX,t(13q14q)] which was also present in her father. Her mother carried a balanced reciprocal translocation between chromosomes 1 and 14 [46,XX,t(1;14) (q32;q32)]. Both of her parents were phenotypically normal. Molecular studies were carried out to determine the parental origin of chromosomes 1, 13, and 14 in the patient. Using probes for D14S13 and D14S22, we could show that the patient inherited both chromosomes 14 from her father and none from her mother. Similar studies using probes for chromosomes 1 (D1S76) and 13 (D13S37) loci showed the presence of both maternal and paternal alleles in the patient. Our findings indicate that paternal uniparental heterodisomy for chromosome 14 most likely accounts for the phenotypic abnormalities observed in our patient. It is suggested that uniparental disomy may be the basis for abnormal development in at least some phenotypically abnormal familial balanced-translocation carriers.

Steroid sulfatase gene in XX males.

The human X and Y chromosomes pair and recombine at their distal short arms during male meiosis. Recent studies indicate that the majority of XX males arise as a result of an aberrant exchange between X and Y chromosomes such that the testis-determining factor gene (TDF) is transferred from a Y chromatid to an X chromatid. It has been shown that X-specific loci such as that coding for the red cell surface antigen, Xg, are sometimes lost from the X chromosome in this aberrant exchange. The steroid sulfatase functional gene (STS) maps to the distal short arm of the X chromosome proximal to XG. We have asked whether STS is affected in the aberrant X-Y interchange leading to XX males. DNA extracted from fibroblasts of seven XX males known to contain Y-specific sequences in their genomic DNA was tested for dosage of the STS gene by using a specific genomic probe. Densitometry of the autoradiograms showed that these XX males have two copies of the STS gene, suggesting that the breakpoint on the X chromosome in the aberrant X-Y interchange is distal to STS. To obtain more definitive evidence, cell hybrids were derived from the fusion of mouse cells, deficient in hypoxanthine phosphoribosyltransferase, and fibroblasts of the seven XX males. The X chromosomes in these patients could be distinguished from each other when one of three X-linked restriction-fragment-length polymorphisms was used. Hybrid clones retaining a human X chromosome containing Y-specific sequences in the absence of the normal X chromosome could be identified in six of the seven cases of XX males.(ABSTRACT TRUNCATED AT 250 WORDS)

X-chromosome inactivation in cultured cells from human chorionic villi.

X-chromosome inactivation was investigated in human chorionic villi in the first trimester of pregnancy and cultured cells established from them. Expression of glucose-6-phosphate dehydrogenase (G6PD) was evaluated in these extraembryonic cells from four females heterozygous for the electrophoretic variants (AB) of G6PD. In each case the uncultured villi as well as derived cultured cells expressed the AB phenotype for G6PD with about equal intensity for the A and B bands. Single-cell-derived clones established from two of the four cases expressed either G6PD A or B. One clone expressing G6PD B was fused with mouse cells, and a hybrid clone retaining the inactive human X chromosome was isolated; there was no evidence of human G6PD expression in this clone retaining an inactive human X. DNA methylation in the first intron of the human gene for hypoxanthine phosphoribosyltransferase (HPRT) was evaluated in the four pairs of cultured villi and fetal cells. No differences were detected between the cultured villi and fetal cells as they all showed bands characteristic of an inactive X from somatic cells. These results show that there is no preferential inactivation of an X in the majority of cells that constitute human tertiary chorionic villi or in cultured cells derived from them. Long-term cultures established from chorionic villi appear to be no different from somatic cells with respect to X-chromosome inactivation.

Marker chromosomes: cytogenetic characterization and implications for prenatal diagnosis.

Satellited marker chromosomes were identified in four individuals from unrelated families; one was first encountered in cultured amniotic fluid cells obtained for prenatal diagnostic studies. We present cytogenetic characterization of these marker chromosomes and clinical findings in the individuals carrying them. Identification of a marker chromosome in amniotic fluid cell cultures presents problems in genetic counseling, as it is often difficult to determine the clinical significance of such a finding. Chromosome-banding techniques now allow the precise identification of satellited marker chromosomes originating from chromosome 15. Presence of a supernumerary bisatellited der(15) marker chromosome containing the proximal long arm of 15 has been associated with mental and developmental retardation. Application of chromosome-banding techniques was useful in characterization of the marker chromosomes and providing prenatal genetic counseling.

Late replication studies in a human X/13 translocation: correlation with autosomal gene expression.

Chromosome replication pattern was reevaluated in an unbalanced X/13 translocation carrier, 46,X,der (X),t(X;13)(q27;q12), using the BudR-acridine orange technique. The translocated chromosome, Xpter leads to q27::13q12 leads to 13qter, was late replicating in all analyzed cells. The autosomal segment showed a replication pattern comparable to the two normal, and presumably genetically active, chromosome 13's, with the exception of band 13q22, which was consistently late replicating. Measurements of red-cell levels of esterase D (ESD) (a marker assigned to 13q14) showed a 50% increase in ESD activity, compared to that in normal controls and parents of the patient. This suggests noninactivation of the ESD locus on the der(X) chromosome, a finding consistent with the late-replication data.