Investigating Immune Responses to the scAAV9-HEXM Gene Therapy Treatment in Tay-Sachs Disease and Sandhoff Disease Mouse Models.

GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme ß-hexosaminidase A (HexA). HexA consists of an α- and ß-subunit; a deficiency in either subunit results in Tay-Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and "non-self"-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed "non-self" proteins, and potentially improve treatment efficacy.

FcRγ-dependent immune activation initiates astrogliosis during the asymptomatic phase of Sandhoff disease model mice.

Sandhoff disease (SD) is caused by the loss of ß-hexosaminidase (Hex) enzymatic activity in lysosomes resulting from Hexb mutations. In SD patients, the Hex substrate GM2 ganglioside accumulates abnormally in neuronal cells, resulting in neuronal loss, microglial activation, and astrogliosis. Hexb-/- mice, which manifest a phenotype similar to SD, serve as animal models for examining the pathophysiology of SD. Hexb-/- mice reach ~8 weeks without obvious neurological defects; however, trembling begins at 12 weeks and is accompanied by startle reactions and increased limb tone. These symptoms gradually become severe by 16-18 weeks. Immune reactions caused by autoantibodies have been recently associated with the pathology of SD. The inhibition of immune activation may represent a novel therapeutic target for SD. Herein, SD mice (Hexb-/-) were crossed to mice lacking an activating immune receptor (FcRγ-/-) to elucidate the potential relationship between immune responses activated through SD autoantibodies and astrogliosis. Microglial activation and astrogliosis were observed in cortices of Hexb-/- mice during the asymptomatic phase, and were inhibited in Hexb-/- FcRγ-/- mice. Moreover, early astrogliosis and impaired motor coordination in Hexb-/- mice could be ameliorated by immunosuppressants, such as FTY720. Our findings demonstrate the importance of early treatment and the therapeutic effectiveness of immunosuppression in SD.

AAV-mediated gene delivery attenuates neuroinflammation in feline Sandhoff disease.

Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme ß-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity.

Thymic alterations in GM2 gangliosidoses model mice.

BACKGROUND: Sandhoff disease is a lysosomal storage disorder characterized by the absence of ß-hexosaminidase and storage of GM2 ganglioside and related glycolipids. We have previously found that the progressive neurologic disease induced in Hexb(-/-) mice, an animal model for Sandhoff disease, is associated with the production of pathogenic anti-glycolipid autoantibodies. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we report on the alterations in the thymus during the development of mild to severe progressive neurologic disease. The thymus from Hexb(-/-) mice of greater than 15 weeks of age showed a marked decrease in the percentage of immature CD4(+)/CD8(+) T cells and a significantly increased number of CD4(+)/CD8(-) T cells. During involution, the levels of both apoptotic thymic cells and IgG deposits to T cells were found to have increased, whilst swollen macrophages were prominently observed, particularly in the cortex. We employed cDNA microarray analysis to monitor gene expression during the involution process and found that genes associated with the immune responses were upregulated, particularly those expressed in macrophages. CXCL13 was one of these upregulated genes and is expressed specifically in the thymus. B1 cells were also found to have increased in the thy mus. It is significant that these alterations in the thymus were reduced in FcRγ additionally disrupted Hexb(-/-) mice. CONCLUSIONS/SIGNIFICANCE: These results suggest that the FcRγ chain may render the usually poorly immunogenic thymus into an organ prone to autoimmune responses, including the chemotaxis of B1 cells toward CXCL13.

Development and maturation of invariant NKT cells in the presence of lysosomal engulfment.

A defect in invariant NKT (iNKT) cell selection was hypothesized in lysosomal storage disorders (LSD). Accumulation of glycosphingolipids (GSL) in LSD could influence lipid loading and/or presentation causing entrapment of endogenous ligand(s) within storage bodies or competition of the selecting ligand(s) by stored lipids for CD1d binding. However, when we analyzed the iNKT cell compartment in newly tested LSD animal models that accumulate GSL, glycoaminoglycans or both, we observed a defective iNKT cell selection only in animals affected by multiple sulfatase deficiency, in which a generalized aberrant T-cell development, rather than a pure iNKT defect, was present. Mice with single lysosomal enzyme deficiencies had normal iNKT cell development. Thus, GSL/glycoaminoglycans storage and lysosomal engulfment are not sufficient for affecting iNKT cell development. Rather, lipid ligand(s) or storage compounds, which are affected in those LSD lacking mature iNKT cells, might indeed be relevant for iNKT cell selection.

Peripheral blood mononuclear cell infiltration and neuroinflammation in the HexB-/- mouse model of neurodegeneration.

Myeloid-derived immune cells, including microglia, macrophages and monocytes, have been previously implicated in neurodegeneration. We investigated the role of infiltrating peripheral blood mononuclear cells (PBMC) in neuroinflammation and neurodegeneration in the HexB-/- mouse model of Sandhoff disease. Ablation of the chemokine receptor CCR2 in the HexB-/- mouse resulted in significant inhibition of PBMC infiltration into the brain, decrease in TNFalpha and MHC-II mRNA abundance and retardation in clinical disease development. There was no change in the level of GM2 storage and pro-apoptotic activity or astrocyte activation in HexB-/-; Ccr2-/- double knockout mice, which eventually succumbed secondary to GM2 gangliosidosis.

Intraperitoneal inoculation of Sandhoff mouse neonates with an HIV-1 based lentiviral vector exacerbates the attendant neuroinflammation and disease phenotype.

We aimed to evaluate the efficacy of VSV-G pseudotyped, defective HIV-1 based lentiviral vectors for the neonatal transfer of therapeutic genes following systemic administration in Sandhoff mouse pups. Despite transgene expression in mouse brains, these animals presented with significant exacerbation and acceleration of the disease neurological phenotype. We observed an increase and acceleration in the presence of MHC-II and CD45+ cells in their brains, along with neuroinflammation, but not in control heterozygous or wild type littermates that also received the same treatment.

Possible role of autoantibodies in the pathophysiology of GM2 gangliosidoses.

Mice containing a disruption of the Hexb gene have provided a useful model system for the study of the human lysosomal storage disorder known as Sandhoff disease (SD). Hexb(-/-) mice rapidly develop a progressive neurologic disease of ganglioside GM2 and GA2 storage. Our study revealed that the disease states in this model are associated with the appearance of antiganglioside autoantibodies. Both elevation of serum antiganglioside autoantibodies and IgG deposition to CNS neurons were found in the advanced stages of the disease in Hexb(-/-) mice; serum transfer from these mice showed IgG binding to neurons. To determine the role of these autoantibodies, the Fc receptor gamma gene (FcR gamma) was additionally disrupted in Hexb(-/-) mice, as it plays a key role in immune complex-mediated autoimmune diseases. Clinical symptoms were improved and life spans were extended in the Hexb(-/-)FcR gamma(-/-) mice; the number of apoptotic cells was also decreased. The level of ganglioside accumulation, however, did not change. IgG deposition was also confirmed in the brain of an autopsied SD patient. Taken together, these findings suggest that the production of autoantibodies plays an important role in the pathogenesis of neuropathy in SD and therefore provides a target for novel therapies.

Antigenic homology of feline and human beta-hexosaminidase.

The immunological characteristics of feline beta-hexosaminidase (beta-D-N-acetylglucosaminidase, EC 3.2.1.30) isoenzymes, Hex A and Hex B, were studied. Immunization of rabbits and goats with either cat Hex A or Hex B produced antibodies which reacted with a common antigenic marker shared by both Hex A and Hex B. With properly absorbed antisera, a unique antigenic marker was demonstrated on cat Hex A, but not on Hex B. This antigenic profile is comparable to that of the human beta-hexosaminidase isozymes, in which both Hex A and Hex B share the antigenic determinant, beta, while only Hex A possesses the antigenic determinant, alpha. Ho cross-reactivity between the two species could be demonstrated using goat or rabbit antisera to either feline of human beta-hexosaminidase. These immunological data validate feline Gm2 gangliosidosis as a model for human Gm2 gangliosidosis type II, and facilitate the investigation of enzyme replacement therapy.