Neuroprotective potential of AAV-mediated NURR1 expression in a rat model of Parkinson’s disease

Abstract

Parkinson’s disease (PD) is characterised by the progressive neurodegeneration of the nigrostriatal dopaminergic neurons that results in motor deficits including bradykinesia, rigidity and tremor. Currently available pharmacological therapies for PD provide only symptomatic relief, and hence lead to therapeutic failure in the advanced stages of the disease. Gene therapy that targets the underlying disease mechanisms is an appealing alternative strategy to ameliorate disease progression. Adeno-associated viral (AAV) vectors are currently the preferred gene delivery vehicles in clinical gene therapy applications. To date, clinically-approved AAV vectors for CNS gene therapy almost exclusively target neurons, and astrocytes that regulate CNS physiology and contribute to disease pathogenesis represent a largely unexplored therapeutic target. Therefore the aims of this thesis were to develop an AAV vector that selectively and efficiently target nigral astrocytes by coupling astrocyte-tropic AAV serotypes and DNA promoter elements, and subsequently utilise this vector to overexpress the therapeutic transcription factor NURR1in a rat model of PD. Recent evidence suggests that astrocytic molecular diversity may heterogeneously regulate transcriptional activity of the commonly used astrocyte-specific glial-fibrillary acidic protein (GFAP) promoter, prompting us to isolate putative promoter sequences from the recently characterised pan-astrocytic marker aldehyde dehydrogenase family 1, member L1 (ALDH1L1), to determine the ability of these promoters to efficiently regulate transgene expression in astrocytes. Four promoter sequences were generated and tested in the context of the astrocyte tropic AAV serotypes 5, 8 and 9 in the rat substantia nigra pars compacts (SNpc). Unexpectedly, these promoters mediated exclusive and efficient neuronal transgene expression, indicating the potential applicability of these promoters in neuronal-targeted gene therapy. In a subsequent comparative analysis of AAV5 and AAV9, AAV5 that predominantly targeted astrocytes in the nigra with minimal dissemination of transgene expression to neighbouring midbrain regions was selected for further studies. Moreover, comparative analysis of the promoters, constitutively active cytomegalovirus (CMV), GFAP and shorter GFAP variant gfaABC1D in the context of AAV5 indicated that the GFAP promoter mediated the most efficient transgene expression predominantly in nigral astrocytes. Therefore, the GFAP promoter in the context of AAV5 was selected to mediate efficient transgene expression in astrocytes. Using this vector construct, the multifaceted transcription factor NURR1 that regulates midbrain DA neuronal phenotype, and downregulates glia-driven inflammation was selectively expressed in astrocytes to determine the neuroprotective potential of astrocyte-targeted gene therapy. NURR1 overexpression promoted nigral DA neuronal survival, ameliorated astrocyte reactivity, and improved contralateral forepaw deficit, indicating therapeutic potential of astrocyte-targeted gene transfer. These results support the development of a dual cell targeting AAV vector in future studies for the expression of NURR1 in both neurons and astrocytes to target DA phenotypic dysfunction, chronic inflammation and neurodegeneration in PD.