Reprograming Resident Oligodendrocytes into Neurons in Non-human Primate Brain

Ischemic strokes comprise the majority of all strokes and are a leading cause of serious long-term disability (Tsao et al., 2023). The implementation of intravenous thrombolysis using agents such as alteplase has made significant improvements in stroke therapy, while a more recent therapy, endovascular thrombectomy, has become the standard of care for anterior and posterior circulation strokes (Sharma and Lee, 2025). Because the underlying basis for long-term disability arises from the loss of functional neurons, significant therapeutic improvement could be achieved if those neurons lost to the ischemic insult could be replaced. Viral vector gene therapy provides a means to manipulate CNS function, and a series of studies with adeno-associated virus (AAV) vectors support the possibility of in vivo neuronal replacement. We utilized the Olig001 AAV vector to express an miRNA against PTBP1 in oligodendrocytes in the rat striatum where the miRNA expression was driven by a constitutive promoter (Weinberg et al., 2017). At 6 weeks post-vector infusion the vast majority of the GFP positive cells exhibited mature neuronal properties including electrophysiological properties such as spontaneous action potentials and spontaneous inhibitory postsynaptic currents. To advance this approach to the clinical trials, it will be necessary to demonstrate oligodendrocyte to neuron reprograming using an miRNA targeting human PTBP1 in a non-human primate. Therefore the first Specific Aim will determine the most efficient siRNA against human PTBP1 in vitro by assessing 10 different validated siRNAs against human PTBP1. Then the best 4 siRNAs will be converted to miRNA, packaged into AAV2 virus and again tested for the ability to reduce human PTBP1 expression in HEK293 cells. The best construct will then be packaged in an Olig001 capsid AAV virus. The second specific aim test the Olig001 AAV in non-human primates to determine if resident oligodendrocytes in the motor cortex or caudate can be transdifferentiated into neurons 6 months after vector infusion. Measures will include immunohistochemical cellular identification and the presence of neuronal terminals in areas of projection. Certainly, this approach would prove readily applicable to current methods of endovascular thrombectomy.