Neuroinflammation and defining gene therapy approaches for ovine CLN6 Batten disease

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of fatal inherited childhood diseases which result in severe cortical atrophy, blindness, seizures, and the accumulation of fluorescent lysosome derived organelles in neurons and most other cells throughout the body. A number of naturally occurring animal models of NCL have been found, the most informative being the CLN6 form in New Zealand South Hampshire sheep. Previous studies in ovine CLN6 have shown a strong correlation between glial activation and subsequent neuronal loss, suggesting that it has a primary role in the development of disease pathology. This thesis describes changes in the expression of inflammatory mediators in affected animals, neuropathological changes that take place in chimeric animals and the implications that these findings have for future therapeutic options. Quantitative real-time PCR revealed significant increases in the expression of pro- and anti-inflammatory cytokines, TNF-α, IL-1β, TGF-β and IL-10, in the brains of affected animals compared to normal controls. Expression of all four cytokines was significantly increased in affected animals at all ages analysed, including 6 months of age, prior to clinical disease manifestation. These results validate the central role that neuroinflammation is proposed to play in disease pathogenesis and indicates that an atypical, dysregulated inflammatory response is ongoing in affected animals. Chimeric animals were generated by mixing homozygous affected and homozygous normal blastomeres. Genotypic and histological examination of the resulting chimeric animals indicated a good degree of colonisation of both cell types in the brain and evidence of cross-cell communication. CAT scans revealed that the brain volumes of two chimeras were within the normal brain volume range, whilst three animals had progressively recovering brain volumes. All normal and recovering-like chimeras presented with reduced or absent disease associated glial activation, no evidence of neurodegeneration, normal cortical thickness and laminar organisation of cells, and no loss of vision, long after these symptoms had progressed to terminal disease in affected animals. PSA-NCAM staining indicated extended neurogenesis in chimeric animals. Individual PSA-NCAM positive cells were present throughout all cortical layers in chimeric animals, in contrast to affected animals in which newly generated cells are largely confined to cellular aggregates in upper cortical layers. Genotyping brain regions of these animals indicated up to 75% of cells were genotypically affected. Despite this storage bodies were rarely observed indicating that storage had been cleared from most cells. These studies indicate that given the correct environmental milieu newly generated and affected cells can survive and are amenable to correction by normal cells in CLN6 NCL, resulting in an amelioration of disease pathology. GFP lentiviral vectors injected into the sheep brain resulted in stable cell transductions evident up to 80 days post-injection. Incidental leakage of the vector into the lateral ventricles resulted in GFP expression in ependymal and subependymal cells along the extent of the ventricular surface. Transduced cells included those likely to be type B astrocytic cells, thought to be the bona fide adult NSCs. Future therapies targeting this zone of extended neurogenesis in affected animals could lead to the widespread distribution of transduced cells and cross-correction of affected cells in the brain.