Differential Prevalence of Antibodies Against Adeno-Associated Virus in Healthy Children and Patients with Mucopolysaccharidosis III: Perspective for AAV-Mediated Gene Therapy.

Recombinant adeno-associated virus (AAV) vectors are promising gene therapy tools. However, pre-existing antibodies (Abs) to many useful AAV serotypes pose a critical challenge for the translation of gene therapies. As part of AAV gene therapy program for treating mucopolysaccharidosis (MPS) III patients, the seroprevalence profiles of AAV1-9 and rh74 were investigated in MPS IIIA/IIIB patients and in healthy children. Using enzyme-linked immunosorbent assay for αAAV-IgG, significantly higher seroprevalence was observed for AAV1 and AAVrh74 in 2- to 7-year-old MPS III patients than in healthy controls. Seroprevalence for the majority of tested AAV serotypes appears to peak before 8 years of age in MPS III subjects, with the exception of increases in αAAV8 and αAAV9 Abs in 8- to 19-year-old MPS IIIA patients. In contrast, significant increases in seroprevalence were observed for virtually all tested AAV serotypes in 8- to 15-year-old healthy children compared to 2- to 7-year-olds. Co-prevalence and Ab level correlation results followed the previously established divergence-based clade positions of AAV1-9. Interestingly, the individuals positive for αAAVrh74-Abs showed the lowest co-prevalence with Abs for AAV1-9 (22-40%). However, all or nearly all (77-100%) of subjects who were seropositive for any of serotypes 1-9 were also positive for αAAVrh74-IgG. Notably, the majority (78%) of αAAV seropositive individuals were also Ab-positive for one to five of the tested AAV serotypes, mostly with low levels of αAAV-Abs (1:50-100), while a minority (22%) were seropositive for six or more AAV serotypes, mostly with high levels of αAAV-IgG for multiple serotypes. In general, the highest IgG levels were reactive to AAV2, AAV3, and AAVrh74. The data illustrate the complex seroprevalence profiles of AAV1-9 and rh74 in MPS patients and healthy children, indicating the potential association of AAV seroprevalence with age and disease conditions. The broad co-prevalence of Abs for different AAV serotypes reinforces the challenge of pre-existing αAAV-Abs for translating AAV gene therapy to clinical applications, regardless of the vector serotype.

Mucopolysaccharidosis IIIB, a lysosomal storage disease, triggers a pathogenic CNS autoimmune response.

BACKGROUND: Recently, using a mouse model of mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease with severe neurological deterioration, we showed that MPS IIIB neuropathology is accompanied by a robust neuroinflammatory response of unknown consequence. This study was to assess whether MPS IIIB lymphocytes are pathogenic. METHODS: Lymphocytes from MPS IIIB mice were adoptively transferred to naïve wild-type mice. The recipient animals were then evaluated for signs of disease and inflammation in the central nervous system. RESULTS: Our results show for the first time, that lymphocytes isolated from MPS IIIB mice caused a mild paralytic disease when they were injected systemically into naïve wild-type mice. This disease is characterized by mild tail and lower trunk weakness with delayed weight gain. The MPS IIIB lymphocytes also trigger neuroinflammation within the CNS of recipient mice characterized by an increase in transcripts of IL2, IL4, IL5, IL17, TNFalpha, IFNalpha and Ifi30, and intraparenchymal lymphocyte infiltration. CONCLUSIONS: Our data suggest that an autoimmune response directed at CNS components contributes to MPS IIIB neuropathology independent of lysosomal storage pathology. Adoptive transfer of purified T-cells will be needed in future studies to identify specific effector T-cells in MPS IIIB neuroimmune pathogenesis.

Effect of cisternal sulfamidase delivery in MPS IIIA Huntaway dogs--a proof of principle study.

Mucopolysaccharidosis type IIIA (MPS IIIA) results from lack of functional sulfamidase (SGSH), a lysosomal enzyme. Its substrate, heparan sulfate, and other secondarily-stored compounds subsequently accumulate primarily within the central nervous system (CNS), resulting in progressive mental deterioration and early death. Presently there is no treatment. As a potential therapeutic strategy, recombinant human sulfamidase (rhSGSH) was administered into the CSF (via the cerebellomedullary cistern) of three adult MPS IIIA dogs either twice with a 4 day interval, or weekly for up to 4 weeks. The dogs were euthanased 24 h post-injection along with one untreated unaffected and two MPS IIIA controls. We have examined the three dimensional pattern of distribution of enzyme in the CNS and its ability to reduce primary substrate storage. High concentrations of rhSGSH protein, with up to 39-fold normal enzyme activity levels were detected within widespread areas of the CNS. RhSGSH protein was also detectable by immunohistochemistry in neurons and glia in all three enzyme-treated dogs. In both weekly-treated dogs, relative levels of a heparan sulfate-derived disaccharide, measured using tandem mass spectrometry, were lower in many brain regions when compared to untreated MPS IIIA controls. A moderately severe meningitis was also present as well as antibodies to rhSGSH in CSF/plasma. These findings demonstrate proof of principle that MPS IIIA can be treated by intracisternal enzyme replacement warranting further experiments in animals tolerant to rhSGSH. This enzyme delivery method may represent a means of treating neuropathology in MPS IIIA and other lysosomal storage disorders affecting the CNS.

Innate and adaptive immune activation in the brain of MPS IIIB mouse model.

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to alpha-N-acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and real-time quantitative reverse transcriptase-polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect.

Sanfilippo disease type B: presence of material cross reacting with antibodies against alpha-N-acetylglucosaminidase.

1. alpha-N-Acetylglucosaminidase, the enzyme deficient in Sanfilippo disease type B (mucopolysaccharidosis III B) was purified from normal human urine. An antiserum was raised in rabbits against the purified enzyme. Preincubation of the antiserum with crude alpha-N-acetylglucosaminidase from normal human urine, followed by centrifugation, led to a marked reduction of the enzyme activity in the supernatant. Formation of the antibody-enzyme complex had no influence on the activity. The thermal stability of the enzyme was markedly enhanced by complex formation with the antiserum. 2. In the urine from three patients with Sanfilippo disease type B the presence of cross-reacting material could be demonstrated by incubating the antiserum with alpha-N-acetylglucosaminidase in the presence of Sanfilippo B urine or by pretreatment of the antiserum with Sanfilippo B urine. 3. Immunodiffusion and immunoelectrophoresis of crude normal or Sanfilippo B urine gave rise to up to four precipitation lines, only one of which exhibited alpha-N-acetylglucosaminidase activity in the case of normal urine. Purified alpha-N-acetylglucosaminidase yielded only a single precipitation line. After adsorption with the purified enzyme the antiserum did not cross react with any of the urinary proteins. 4. On a quantitative determination of cross-reacting material using Sepharose immobilized antibodies in the urine from two Sanfilippo B patients the amount of cross-reacting material appeared to be less than one fourth of the amount of alpha-N-acetylglucosaminidase protein in an age-matched control urine. The cross-reacting material present in the urine of Sanfilippo B patients had a significant lower binding affinity for antibodies against alpha-N-acetylglucosaminidase than preparations from normal human urine. Taking into account this lower binding affinity, it can be calculated that the amount of cross-reacting material in the urine of Salfilippo B patients exceeds that of normal controls. 5. It is concluded that Sanfilippo disease type B is due to a mutation of a structural gene coding for alpha-N-acetylglucosaminidase. The mutation affects the catalytical and immunological properties of the enzyme protein.