Direct cellular reprogramming to drive lineage switching from one differentiated cell type to another can be exploited to develop cell-based therapies for neurodegenerative diseases. In vivo reprogramming provides an attractive therapeutic strategy to circumvent the hurdles of immune rejection and ethical constraints associated with transplant-based therapy. Supporting glial cells of the CNS can be reprogrammed to neurons by targeted viral delivery of transcription factors and small molecules. Previous studies have demonstrated the effectiveness of a single proneural transcription factor NeuroD1 to drive reprogramming in the reactive glial lesions of Alzheimer’s disease and stroke. However, the ability of NeuroD1 (ND1) to promote a similar benefit in models of Parkinson’s disease (PD) has yet to be demonstrated. The current study aims to test the hypothesis that NeuroD1 delivered via an Adeno-Associated Virus (AAV) can promote reprogramming in striatal astrocytes to neurons in an in vivo PD model. The FDA-approved, clinically employed AAV-9 gene delivery platform was used to transduce non-dividing cells with minimal off-target effects. A two-part AAV-9 viral system was designed to express ND1 in astrocytes, driven by the GFAP promoter. First, ND1-mediated direct neuronal reprogramming was tested using a simple and scalable in vitro culture system. Primary astrocytes in vitro transduced with the AAV9-ND1 dual virus system display characteristics of immature neuroglial precursor stage, suggesting successful reprogramming. Second, the potential of ND1 to drive reprogramming was assessed in vivo in mice. Intracranial and intravenously delivered AAV-9 dual virus system driven by the GFAP promoter targets astrocytes and surprisingly, mature resident neurons in vivo. Finally, ND1-mediated in vivo reprogramming was assessed in a well-established chemically-induced 6-hydroxydopamine (6-OHDA) Parkinson’s disease mouse model. The 6-OHDA injury model provides insight into the novel application of the AAV-9 dual virus system to target astrocyte-to-neuron reprogramming as well as to target resident neurons for potential neuronal repair in Parkinson’s disease.
University of Minnesota M.S. thesis.December 2019. Major: Stem Cell Biology. Advisor: Walter Low. 1 computer file (PDF); vi, 63 pages.
Proneural transcription factor NeuroD1-mediated direct neuronal reprogramming - an AAV approach.
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