Enhancement of a novel gene therapy approach for Sandhoff disease through complimentary drug therapy

GM2 gangliosidoses is a family of severe, neurodegenerative disorders resulting from a deficiency in the β-hexosaminidase A (Hex A) enzyme. This disorder is typically caused by a mutation to either the HEXA gene, causing Tay Sachs disease, or a mutation to the HEXB gene, causing Sandhoff disease. The HEXA and HEXB genes are required to produce the α and β subunits of the Hex A enzyme respectively. Using a Sandhoff disease (SD) mouse model (Hexb-/-) we tested the potential of a low dose of systemically delivered single stranded adeno-associated virus 9 (ssAAV9) expressing human HEXB and human HEXA cDNA under the control of a single promoter through the use of a bicistronic vector design with a P2A linker to correct the neurological phenotype. Neonatal mice were injected with either this ssAAV9-HexB-P2A-HexA vector (HexB-HexA) or a vehicle solution via the superficial temporal vein. HexB-HexA treatment alone conferred an increase in survival of 56% compared to vehicle-injected controls and biochemical analysis of the brain tissue and serum revealed an increase in HexA activity and a decrease in brain GM2 ganglioside buildup. Additionally, treatments with the non-steroidal anti-inflammatory drug indomethacin (Indo), the histone deactylase inhibitor ITF2357 (ITF) and the pharmacological chaperone pyrimethamine (Pyr) were tested. The anti-inflammatory treatments of Indo and ITF conferred an increase in survival of 12% and 8% respectively while causing no alteration in the HexA activity or GM2 ganglioside buildup. Pyr had no observable effect on disease progression. Lastly HexB-HexA treatment was tested in conjunction with Indo, ITF and Pyr individually. Additive increases in survival and behavioural testing results were observed with Indo and ITF treatments while no additional benefit to HexA activity or GM2 ganglioside levels in the brain tissue was observed. This indicates the two treatments slowed the progression of the disease through a different mechanism than the reduction of the GM2 ganglioside substrate. Pyr treatment was shown to have no effect when combined with HexB-HexA treatment. This study demonstrates the potential amelioration of SD with a novel AAV9 gene therapy approach as well as helped to identify the additive potential of anti-inflammatory treatments in gene therapy of GM2 gangliosidoses.

ROLE OF MULTIPLE TOLL-LIKE RECEPTOR ACTIVATION IN REGULATING CYTOTOXIC T CELL PRIMING TO LYMPOCYTIC CHORIOMENINGITIS VIRUS INFECTIONS

Foreign pathogens are recognized by toll-like receptors (TLR), present on various immune cells such as professional antigen-presenting cells (pAPCs). On recognition of its ligand, these receptors activate pAPCs, which may in turn influence naïve CD8+ T cell activation and affect their abilities to clear viral infection. However, how TLR ligands (TLR-L) can regulate CD8+ T cell responses have not been fully elucidated. This thesis will focus on examining how the presence of components from foreign pathogens, e.g. viral or bacterial infection, can contribute to shaping host immunity during concurrent viral infections. Since nitric oxide (NO), an innate effector immune molecule, was recently suggested to regulate proteasome activity; we sought to examine if NO can influence MHC-I antigen presentation during viral infections. The data in this section of the thesis provides evidence that combined TLR engagement can alter the presentation of certain CD8+ epitopes due to NO-induced inhibition in proteasome activity. Taken together, the data demonstrate that TLR ligation can influence the adaptive immune response due to induction of specific innate effector molecules such as NO. Next, the influence of combined TLR engagement on CD8+ T cell immunodominance hierarchies during viral infections was examined. In this section, we established that dual TLR2 and TLR3 stimulation alters immunodominance hierarchies of LCMV epitopes as a result of reduced uptake of cell-associated antigens and reduced cross-presentation of NP396 consequently suppressing NP396-specific CD8+ T cell responses. These findings are significant as they highlight a new role for TLR ligands in regulating anti-viral CD8+ T cell responses through impairing cross-presentation of cell-associated antigens depending on the type of TLR present in the environment during infections. Finally, we addressed TLR ligand induced type I interferon production and the signalling pathways that regulate them in two different mouse macrophage populations – those derived from the spleen or bone marrow. In this study, we observed that concomitant TLR2 stimulation blocked the induction of type I IFN induced by TLR4 in bone marrow-derived macrophages, but not spleen-derived macrophages in SOCS3-dependent manner. Taken together, the data presented in this thesis have defined new facets of how anti-viral responses are regulated by TLR activation, especially if multiple receptors are engaged simultaneously.

The Influence of 1,25-Dihydroxyvitamin D3 on the Cross-Priming of Lymphocytic Choriomeningitis Virus Nucleoprotein

Biologically active 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) binds the vitamin D receptor (VDR) to exert its effect on target cells. VDR expression is found in a number of immune cells including professional antigen-presenting cells such as dendritic cells. It has been found that the actions of 1,25-(OH)2D3 on the immune system are mainly immunosuppressive. The cross-presentation pathway allows for exogenously derived antigens to be presented by pAPCs on MHC-I molecules to CD8+ T cells. CD8+ T cell activation results in the expansion of epitope-specific T cell populations that confer host protection. These epitopes can be organized into an immunodominance hierarchy. Previous work demonstrated that introducing LCMV-NP via the cross-priming pathway significantly alters the immunodominance hierarchy of a subsequent LCMV infection. Building upon these observations, our study assessed the effects of LCMV-NP cross priming in the presence of a single dose of 1,25-(OH)2D3. Treatment with 1,25-(OH)2D3 was found to have biological effects in our model system. In vitro pAPCs were demonstrated to up-regulate IL-10 and CYP24A1 mRNA, in addition to the transactivation of cellular VDR, as demonstrated by a relocalization to the nuclear region. Mice treated with 1,25-(OH)2D3 were found to produce up-regulated IL-10 and CYP24A1 transcripts. Expression of VDR was increased at both the transcript and protein level. Our results demonstrate that a single dose of 1,25-(OH)2D3 does not affect the cross-priming pathway in this system. Treatment with 1,25-(OH)2D3 did not influence the ability of differentiated pAPCs to phagocytose or cross-present exogenous antigen to epitope-specific CD8+ T cells. Furthermore, 1,25-(OH)2D3 did not alter cross-priming or the establishment of the LCMV immunodominance hierarchy in vivo. By confirming that 1,25-(OH)2D3 does not suppress cross-priming in our model, our study helps to expand the understanding of the immunomodulatory role of exogenous 1,25-(OH)2D3 on the outcome of virus infection. Collectively, our data supports the observation that the role of 1,25-(OH)2D3 in the immune system is not always associated with suppressive effects.