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.

Regulation of the Gene Encoding Thrombin-Activable Fibrinolysis Inhibitor

Disequilibrium between coagulation and fibrinolysis can lead to severe haemostatic disorders such as thrombosis and hemophilia. Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. TAFI may also mediate connections between coagulation and inflammation. Studies have associated high plasma TAFI levels with risk for thrombotic diseases. Interestingly, steroid hormones, such as estrogen and progestogens used in hormone replacement therapy or oral contraceptive preparations, have been shown to affect plasma TAFI levels. Regulation of the expression of the gene encoding TAFI, CBP2, is likely an important determinant of the role of the TAFI pathway in vivo; this concept motivated the investigations described in this thesis. In Chapter 2, the results of my research lead to the identification of key transcription factors regulating CPB2. Specifically, we described the binding of NF-Y and HNF-1 to the CPB2 promoter. NF-Y was shown to be an important factor for the basal CPB2 promoter activity. Binding of HNF-1 is essential for the activity of the promoter and is potentially responsible for the liver specific expression of CPB2. In Chapter 3, we set to investigate the effect of female sex hormone on hepatic expression of CPB2. We demonstrated that the levels of TAFI protein secreted from cultured hepatoma cells (HepG2) are decreased by 17beta-estradiol and progesterone. The change in protein expression was paralleled by decreases in CPB2 mRNA abundance and promoter activity. Deletion analysis of the CPB2 promoter indicated that the genomic effects of estrogen and progesterone are likely mediated via a non-classical mechanism. In Chapter 4, we evaluated the effects of various inflammatory mediators on expression of the gene encoding mouse TAFI (Cpb2). Our results showed that Cpb2 mRNA abundance and promoter activity are up-regulated by inflammatory mediators IL-1beta, IL-6, and TNFalpha. We also showed that TNFalpha mediates its effect via the binding of NFkB. Additionally, our results suggest that TNFalpha promotes the binding of NFkB to the promoter by increasing its translocation to the nucleus. The NFkB site is not conserved between human and mouse and may explained the different responses to inflammation observed in vivo.