Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs

Abstract

Generation of functional dopamine (DA) neurons is an essential step for the development of effective cell therapy for Parkinson's disease (PD). The generation of DA neurons can be accomplished by overexpression of DA-inducible genes using virus- or DNA-based gene delivery methods. However, these gene delivery methods often cause chromosomal anomalies. In contrast, mRNA-based gene delivery avoids this problem and therefore is considered safe to use in the development of cell-based therapy. Thus, we used mRNA-based gene delivery method to generate safe DA neurons. In this study, we generated transformation-free DA neurons by transfection of mRNA encoding DA-inducible genes Nurr1 and FoxA2. The delivery of mRNA encoding dopaminergic fate inducing genes proved sufficient to induce naive rat forebrain precursor cells to differentiate into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neurons in vitro. Additionally, the generation efficiency of DA neurons was improved by the addition of small molecules, db-cAMP, and the adjustment of transfection timing. The successful generation of DA neurons using an mRNA-based method offers the possibility of developing clinical-grade cell sources for neuronal cell replacement treatment for PD.

Keywords: Parkinson’s disease; dopamine neuron; genomic integration free; in vitro transcription; mRNA; neural precursor cell.