Prevalence of anti-adeno-associated virus serotype 8 neutralizing antibodies and arylsulfatase B cross-reactive immunologic material in mucopolysaccharidosis VI patient candidates for a gene therapy trial.

Recombinant vectors based on adeno-associated virus serotype 8 (AAV8) have been successfully used in the clinic and hold great promise for liver-directed gene therapy. Preexisting immunity against AAV8 or the development of antibodies against the therapeutic transgene product might negatively affect the outcomes of gene therapy. In the prospect of an AAV8-mediated, liver-directed gene therapy clinical trial for mucopolysaccharidosis VI (MPS VI), a lysosomal storage disorder caused by arylsulfatase B (ARSB) deficiency, we investigated in a multiethnic cohort of MPS VI patients the prevalence of neutralizing antibodies (Nab) to AAV8 and the presence of ARSB cross-reactive immunologic material (CRIM), which will either affect the efficacy of gene transfer or the duration of phenotypic correction. Thirty-six MPS VI subjects included in the study harbored 45 (62.5%) missense, 13 (18%) nonsense, 9 (12.5%) frameshift (2 insertions and 7 deletions), and 5 (7%) splicing ARSB mutations. The detection of ARSB protein in 24 patients out of 34 (71%) was predicted by the type of mutations. Preexisting Nab to AAV8 were undetectable in 19/33 (58%) analyzed patients. Twelve out of 31 patients (39%) tested were both negative for Nab to AAV8 and CRIM-positive. In conclusion, this study allows estimating the number of MPS VI patients eligible for a gene therapy trial by intravenous injections of AAV8.

Osteoimmunology in mucopolysaccharidoses type I, II, VI and VII. Immunological regulation of the osteoarticular system in the course of metabolic inflammation.

BACKGROUND: Mucopolysaccharidoses (MPSs) are rare genetic diseases caused by a deficient activity of one of the lysosomal enzymes involved in the glycosaminoglycan (GAG) breakdown pathway. These metabolic blocks lead to the accumulation of GAGs in various organs and tissues, resulting in a multisystemic clinical picture. The pathological GAG accumulation begins a cascade of interrelated responses: metabolic, inflammatory and immunological with systemic effects. Metabolic inflammation, secondary to GAG storage, is a significant cause of osteoarticular symptoms in MPS disorders. OBJECTIVE AND METHOD: The aim of this review is to present recent progress in the understanding of the role of inflammatory and immune processes in the pathophysiology of osteoarticular symptoms in MPS disorders and potential therapeutic interventions based on published reports in MPS patients and studies in animal models. RESULTS AND CONCLUSIONS: The immune and skeletal systems have a number of shared regulatory molecules and many relationships between bone disorders and aberrant immune responses in MPS can be explained by osteoimmunology. The treatment options currently available are not sufficiently effective in the prevention, inhibition and treatment of osteoarticular symptoms in MPS disease. A lot can be learnt from interactions between skeletal and immune systems in autoimmune diseases such as rheumatoid arthritis (RA) and similarities between RA and MPS point to the possibility of using the experience with RA in the treatment of MPS in the future. The use of different anti-inflammatory drugs requires further study, but it seems to be an important direction for new therapeutic options for MPS patients.

Mucopolysaccharidosis type VI phenotypes-genotypes and antibody response to galsulfase.

BACKGROUND: Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome; MPS VI) is an autosomal recessive lysosomal storage disorder in which deficiency of N-acetylgalactosamine 4-sulfatase (arylsulfatase B; ARSB) leads to the storage of glycosaminoglycans (GAGs) in connective tissue. The genotype-phenotype correlation has been addressed in several publications but the picture is not complete. Since 2007, enzyme-replacement therapy (ERT) has been available for patients with MPS VI in the Netherlands. The purpose of our study was to learn more about the genotype-phenotype correlations in MPS VI and the antibody response to ERT with galsulfase (recombinant human arylsulfatase B). METHODS: We identified ARSB mutations in 12 patients and used site-directed mutagenesis to study their effect. Antibody levels to galsulfase were measured using ELISA and a semi-quantitative immunoprecipitation method. We assessed the in vitro inhibitory effect of antibodies on galsulfase uptake and their effect on clinical outcome. RESULTS: Five patients had a rapidly progressive phenotype and seven a slowly progressive phenotype. In total 9 pathogenic mutations were identified including 4 novel mutations (N301K, V332G, A237D, and c.1142 + 2 T > C) together composing 8 pathogenic genotypes. Most mutations appeared not to affect the synthesis of ARSB (66 kD precursor), but to hamper its maturation (43 kD ARSB). Disease severity was correlated with urinary GAG excretion. All patients developed antibodies to galsulfase within 26 weeks of treatment. It was demonstrated that these antibodies can inhibit the uptake of galsulfase in vitro. CONCLUSIONS: The clinical phenotypes and the observed defects in the biosynthesis of ARSB show that some of the mutations that we identified are clearly more severe than others. Patients receiving galsulfase as enzyme-replacement therapy can develop antibodies towards the therapeutic protein. Though most titers are modest, they can exceed a level at which they potentially affect the clinical outcome of enzyme-replacement therapy.

Gene therapy for mucopolysaccharidosis type VI is effective in cats without pre-existing immunity to AAV8.

Liver gene transfer with adeno-associated viral (AAV) 2/8 vectors is being considered for therapy of systemic diseases like mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disease due to deficiency of arylsulfatase B (ARSB). We have previously reported that liver gene transfer with AAV2/8 results in sustained yet variable expression of ARSB. We hypothesized that the variability we observed could be due to pre-existing immunity to wild-type AAV8. To test this, we compared the levels of AAV2/8-mediated transduction in MPS VI cats with and without pre-existing immunity to AAV8. In addition, since levels of lysosomal enzymes as low as 5% of normal are expected to be therapeutic, we evaluated the impact of pre-existing immunity on MPS VI phenotypic rescue. AAV2/8 administration to MPS VI cats without pre-existing neutralizing antibodies to AAV8 resulted in consistent and dose-dependent expression of ARSB, urinary glycosaminoglycan (GAG) reduction, and femur length amelioration. Conversely, animals with pre-existing immunity to AAV8 showed low levels of ARSB expression and limited phenotypic improvement. Our data support the use of AAV2/8-mediated gene transfer for MPS VI and other systemic diseases, and highlight that pre-existing immunity to AAV8 should be considered in determining subject eligibility for therapy.

Repeated intrathecal injections of recombinant human 4-sulphatase remove dural storage in mature mucopolysaccharidosis VI cats primed with a short-course tolerisation regimen.

All MPS-VI cats treated thus far with weekly intravenous enzyme replacement therapy (IV ERT) with recombinant human N-acetylgalactosamine-4-sulphatase (rhASB) from 3 months of age onwards developed circulating anti-rhASB antibodies. In view of this, the possibility of inducing immune tolerance by using a short-course tolerisation regimen was tested. Starting at 4 months of age, MPS-VI (n=5) and unaffected cats (n=2) received cyclosporine and azathioprine over a 22-day period plus weekly IV ERT with 0.1mg/kg rhASB. After a 4-week resting period, these cats were administered weekly IV ERT with 1mg/kg rhASB until 11 or 17 months of age. Four unaffected cats (n=4) received weekly IV ERT only. Health, growth and seroconversion were regularly monitored. Four out of five MPS-VI cats tolerated rhASB well, as indicated by negligible or low antibody titres and absence of hypersensitivity reactions. One MPS-VI cat exhibited elevated antibody titres and hypersensitivity reactions during some IV treatments. The two unaffected cats that received the tolerisation regimen remained seronegative, however, only half of the unaffected cats not submitted to this regimen seroconverted. Only minor side-effects were attributed to the short-course of cyclosporine and azathioprine. Two MPS-VI cats also well-tolerated four weekly intrathecal injections of rhASB and consequently exhibited less oligosaccharide fragments in cerebrospinal fluid and less vacuolation within their dura mater. These data indicate that a relatively high rate of immunotolerance towards rhASB can be achieved in MPS-VI cats with a short-course tolerisation regimen ultimately permitting removal of lysosomal storage within the dura mater with the use of intrathecal therapy.

Long-term follow-up of a girl with Maroteaux-Lamy syndrome after bone marrow transplantation.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI or Maroteaux-Lamy syndrome) is a rare autosomal recessive genetic disorder. We treated a 10-year-old girl with Maroteaux-Lamy syndrome successfully with bone marrow transplantation (BMT). METHODS: The patient had reconstitution with bone marrow from her HLA-matched brother. One month after BMT, arylsulfatase activity of the recipient's leukocytes became normal. No graft-versus-host disease (GVHD) was observed. Arylsulfatase B activity was maintained and the urinary excretion of glycosaminoglycans (GAGs) became normal. RESULTS: The clinical response of the patient was slow but persistent during 12 years after BMT. Improved motor function included walking alone for a long distance without aid, riding a bicycle, taking a bath by herself, etc. Besides, few infections occurred. Exertional dyspnea, severe snoring, and vertigo were much improved. CONCLUSIONS: Early intervention is recommended for BMT. Allogeneic BMT may provide a better life quality as illustrated in the present case.

Development, validation, and clinical implementation of an assay to measure total antibody response to naglazyme (galsulfase).

Naglazyme (galsulfase, rhASB) was developed as enzyme replacement therapy for mucopolysaccharidosis type VI. Naglazyme generated an IgG antibody response in most patients. To better characterize Naglazyme immunogenicity, a solution phase bridged immunoassay was developed to measure total antibody response regardless of isotype. Overnight incubation of serum dilutions with rhASB labeled with biotin and ruthenium-based tags allowed antibody-antigen complexes to form prior to capture on a streptavidin plate. Neat serum was tolerated in the assay, with a 1:10 screening dilution implemented for testing. At this dilution, the assay was sensitive to 75 ng/ml anti-rhASB. Titers were reported as the highest dilution factor with signal above a 95% confidence interval from naïve individual sera. Precise measurement of titers, within two consecutive dilution factors, was observed across analysts and days. Clinical samples showed similar positive/negative results between the IgG ELISA and the total antibody ECLA, although with an imperfect correlation. Improvements in assay performance and implementation strategy altered some positive clinical samples to negative and vice versa. Comparison of the titer readout for clinical samples with the screening signal illustrates a range of relationships for signal versus sample dilution factor, confirming that signal from a screening dilution cannot directly predict the reported titer.

Molecular cloning of feline CD34.

In humans, baboons, dogs and mice CD34 is a cell surface molecule that is expressed on primitive hematopoietic cells and in all these species CD34 positive cells can be used to effect long-term haematopoietic reconstitution. CD34 positive haematopoietic cells therefore provide a convenient and relatively small cell population to target when attempting gene therapy via the haematopoietic system. In order to develop the mucopolysaccharidosis type VI (MPS VI) cat as a model for haematopoietic cell-mediated gene therapy we have isolated the feline CD34 gene as a first step in the generation of antibodies for purification of feline CD34 positive cells. The coding sequence for feline CD34 was isolated from brain cDNA using the polymerase chain reaction (PCR) with oligonucleotides designed to conserved regions of known CD34 gene sequences as primers. Sequence analysis of PCR products revealed the complete amino acid sequence of feline CD34 and allowed analysis of sequence conservation with CD34 from other species. Northern blot analysis showed a 2.6 kb CD34 transcript was present in feline brain, spleen, heart, testis and thymus, and to a lesser extent, in liver. A full-length cDNA clone of the feline CD34 coding sequence was assembled and expressed in CHO-K1 cells. The isolation and expression of the feline CD34 cDNA should facilitate the production of antibodies suitable for the purification of CD34 positive cells.

Replacement therapy in Mucopolysaccharidosis type VI: advantages of early onset of therapy.

This study evaluates the immunological response following weekly 2h infusions of recombinant human N-acetylgalactosamine 4-sulfatase (rh4S) in Mucopolysaccharidosis VI (MPS VI) cats. The results of three trials (Trial "A": 9 month duration with onset at 3-5 months of age, n = 5; and Trials "B" and "C": 6 month duration starting at birth, n = 9) were compared. No detrimental effects were noted throughout Trials B and C. Temporary hypersensitivity reactions (e.g., vomiting, diarrhoea) occurred in four cats in Trial A and were alleviated by increasing the dose of antihistamine premedication and the duration of infusion. All cats in Trial A developed antibodies to rh4S (range of final titres: 1041-134,931). All cats treated from birth showed negligible titres (range: < 50-598). In vitro inhibition of rh4S activity (up to 47%) was demonstrated with plasma from four cats with elevated titres. Significant reduction of urinary glycosaminoglycan concentration in all cats indicated the ability of rh4S to metabolize stored substrates regardless of the presence of circulating antibodies. Similarly, lysosomal storage in reticuloendothelial cells and fibroblasts of kidney interstistium, dura and skin was reduced in all cats irrespective of their antibody titre although cats with elevated titre had less beneficial effect on cardiovascular tissues (aorta smooth muscle cells, heart valve fibroblasts). Overall improvement in the disease condition (at physical, neurological, and skeletal levels) was most pronounced for cats treated from birth compared with cats treated at a later age.

Immune response to enzyme replacement therapy: 4-sulfatase epitope reactivity of plasma antibodies from MPS VI cats.

The mucopolysaccharidoses (MPS) are a group of multiple pathology disorders which are part of a larger group of genetic diseases known as lysosomal storage disorders. Enzyme replacement therapy (ERT) has been developed as a therapy for MPS patients. However, immune responses to ERT have been reported in MPS animal models and in human Gaucher patients. Antibodies can have adverse effects during ERT, which include hypersensitivity/anaphylactic reactions, enzyme inactivation, and enzyme degradation. This study aimed to characterize the immune response to ERT in a feline model of MPS VI, by defining the epitope reactivity of cat plasma antibody against human recombinant N-acetylgalactosamine 4-sulfatase (4-sulfatase) replacement protein. For MPS VI cat plasma, antibody reactivity was observed prior to ERT, with distinct regions of 4-sulfatase linear sequence displaying low affinity antibody reactivity. There was an increase in antibody titer to 4-sulfatase for MPS VI cats post-ERT, with the majority of the immune response detected to linear sequence epitopes. One cat displayed a high titer and high affinity epitope reactivity following prolonged exposure (>/=9 months) to the replacement protein. MPS VI cats on shorter term ERT (3 months) showed high titers to 4-sulfatase and similar patterns of epitope reactivity, but lower affinity antibody reactivity, when compared to the latter cat. This study reports the linear amino acid sequence reactivity and nature of the immune response produced to 4-sulfatase before and after ERT. The monitoring of antibody production during replacement therapy is an important consideration for patient management, as high titer antibodies can affect the efficacy of therapy.

The monocyte-macrophage system is affected in lysosomal storage diseases: an immunoelectron microscopic study.

Studying peripheral blood mononuclear cells (PBMCs) has become an important diagnostic tool in lysosomal storage diseases. Previous studies revealed that B and subclasses of T lymphocytes participate in the storage process, whereas the role of circulating monocytes was not clear. In this study, the involvement of CD14+ monocytes in lysosomal diseases was investigated. Blood samples from six patients with different lysosomal storage disorders were studied, including one with late--infantile and three with juvenile neuronal ceroid--lipofuscinoses, and two with mucopolysaccharidosis type VI. CD14+ cells were separated immunomagnetically from PBMCs and studied by light and electron microscopy. In all investigated disorders, disease-specific lysosomal storage material could be found in monocytes. The ratio of affected to non-affected cells did not differ from previously reported data on lymphocytes and their subforms in these diseases. Our data were obtained by studying a small number of different lysosomal storage disorders. Nevertheless, they suggest that lysosomal storage in the monocyte-macrophage system might also be found in other forms of lysosomal diseases.

Enzyme replacement therapy in Mucopolysaccharidosis VI: evidence for immune responses and altered efficacy of treatment in animal models.

Enzyme replacement therapy (ERT) can potentially result in an immunological response to the introduced protein. The immunological response by Mucopolysaccharidosis type VI (MPS VI) cats to recombinant human N-acetylgalactosamine 4-sulfatase (rh4S) ERT has been investigated. Plasma antibody titres to rh4S were detected in untreated MPS VI and normal control cats, but the antibody titres to rh4S were higher in ERT treated MPS VI cats. The reactivity by cats to rh4S did not appear to be just due to species cross reactivity, as plasma antibodies from normal control, MPS VI and MPS VI ERT cats reacted equally with feline and human 4-sulfatase. Normal control and MPS VI human plasma also had antibody titres to rh4S. Plasma antibodies to rh4S, from an ERT treated cat, could be temporarily removed from circulation by enzyme infusion, confirming specificity for rh4S and indicating a possible window for ERT in the absence of antibody. In enzyme distribution studies with 3H-rh4S, evidence of altered targeting, and enzyme inactivation and degradation were observed in high compared to low titre rats. In high titre rats, the observed loss of 3H-label from vacuolar organelles of the liver may represent either degradation of antibody bound 3H-rh4S for reutilisation within the liver, or antigen presentation. The development of high titre antibody may have a detrimental effect on the efficacy of ERT.

Maroteaux-Lamy syndrome in a large consanguineous kindred: biochemical and immunological studies.

We describe a large consanguineous German-Acadian ("Cajun") family from a rural area in Louisiana in which 11 persons in two generations had the Maroteaux-Lamy syndrome. The mutant arylsulfatase B enzyme in this family was similar to the mutant enzyme in previously studied families in its cross-reactivity with specific antibodies to the enzyme, but it differed in both its electrophoretic mobility and its residual enzymatic activity. These findings indicate that a different mutational event leading to Maroteaux-Lamy syndrome occurred in this family.