|dc.description.abstract||The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of fatal inherited lysosomal storage diseases characterised by progressive neurodegeneration, cortical atrophy, and blindness. Currently there are no effective treatments. Naturally occurring animal models exist, including two forms in sheep which are representative of the different NCL protein defects. A lesion in a soluble lysosomal protein (CLN5) causes NCL in Borderdale sheep, whilst South Hampshire sheep have a defective intracellular endoplasmic reticulum membrane-bound protein (CLN6). This subdivision has consequences for the planning of therapies. This thesis compares the progressive neuropathological changes, and examines the efficacy of viral-mediated in vivo gene therapy, in these two NCL sheep models.
An immunohistochemical study revealed that, despite very different gene products and subcellular localisations, the pathogenic cascade was remarkably similar for CLN5 and CLN6 affected sheep. Dysregulated glial activation preceded regionally specific neurodegeneration in both disease models and both occurred well before clinical onset. Neuropathological changes were more advanced in the CLN5 model, which correlated with the earlier onset of clinical symptoms in these sheep, but by end-stage disease CLN5 and CLN6 brains were similarly affected. Windows for best therapeutic efficacy were established and these data highlight the translational utility of the sheep brain for testing human gene therapies.
Lentiviral vectors have been shown to mediate successful gene delivery to the ovine brain yet the efficacy of adeno-associated viral (AAV) vectors has not been tested in sheep. Stable, predominantly neurotropic, transgene expression was evident one month after intracerebroventricular (ICV) and intraparenchymal (IP) delivery of AAV9 to the normal sheep brain. However with a greater spread and no evidence of vector or procedural neuroinflammation or toxicity, the ICV approach proved most efficacious in sheep.
Deficiencies in soluble lysosomal proteins are deemed particularly amenable to in vivo gene therapy via the normal lysosomal enzyme trafficking system and the phenomenon of ‘cross-correction’. To test this paradigm in sheep, six pre-clinical CLN5 deficient lambs were treated with combinatorial ICV and IP injections of either lentiviral or AAV9 vectors expressing ovine CLN5. Both vector platforms afforded sustained protection against stereotypical disease in these sheep. Cognitive and neurological function was preserved, whilst longitudinal neuroimaging revealed normalisation of intracranial volume and structural brain integrity. Quality of life was profoundly improved for the treated sheep and, apart from delayed-onset visual deficits, treated sheep well exceeded the typical lifespan of untreated animals.
Defects in membrane-bound proteins are generally considered harder therapeutic targets. However the current study indicates that gene therapy is also possible for these NCL forms. Whilst five similarly injected pre-clinical CLN6 deficient sheep developed stereotypical CLN6 disease, one AAV9-CLN6 treated animal was clinically indistinguishable from age-matched control animals through the 24-month follow up period. Post mortem neuropathological studies revealed significant correction of neurodegeneration, normal cortical laminar organisation and a reversal in disease-associated glial activation.
These encouraging results of sustained therapeutic and functional efficacy in large animal models of NCL provide a strong rationale for the clinical translation of viral-mediated gene transfer to human patients with CLN5 and CLN6 disease. In fact, the findings of this thesis have encouraged a Phase I/II CLN6 clinical trial which has just begun.||en