| Summary: | Induced pluripotent stem (iPS) cells are valuable for generating patient-specific disease models. Many reprogramming systems have been developed however some technological concerns remain including insertional mutagenesis caused by integrating viruses, low efficiency and low transgene capacity. Herpes simplex virus type-1 (HSV-1) amplicons address the main limitations of current reprogramming methods as they are non-integrating and have a large transgene capacity of 150 Kb. In addition HSV-1 amplicons have a high transduction efficiency in both primary fibroblasts and iPS cells. We have developed three sets of HSV-1 amplicons to reprogram fibroblasts to iPS cells, directly differentiate fibroblasts to dopaminergic neurons and aid dopaminergic neuron differentiation from iPS cells. 1) We constructed and characterised a set of HSV-1 reprogramming amplicons, which express the reprogramming factors Oct-4, Sox-2, Klf-4, C-Myc, Nanog and Lin28. These vectors were capable of generating partially reprogrammed colonies as confirmed by RT-PCR analysis of pluripotency factors. We were unable to generate fully reprogrammed colonies and our results suggest that this is due to an induction of the p53 pathway. 2) We constructed an HSV-1 direct differentiation amplicon containing the polycistronic cassette Mash-1, Nurr-1 and Lmx1a. All elements were functional however we were unable to generate dopaminergic neurons. 3) We constructed and characterised a set of HSV-1 neuronal differentiation amplicons expressing the neuronal transcription factors Nurr-1, Lmx1a, FoxA2 and PitX3. We employed a 96 well differentiation protocol and transduced the differentiating cultures with our neuronal amplicons to try and improve dopaminergic neuron efficiency. Transduction at early time points in the protocol resulted in a significant increase in TH positive neurons.
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