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  • Evaluating the Use of Adeno-Associated Virus as a Vehicle for Reprogramming of Reactive Glial Cells into Neurons After Middle Cerebral Artery Stroke in Mice

    Final Number:

    Joshua Lim MD; Hao Yin MD; Holly Hewitt MS; Andrew W. Grande MD

    Study Design:
    Laboratory Investigation

    Subject Category:

    Meeting: Congress of Neurological Surgeons 2016 Annual Meeting

    Introduction: Recent studies have shown that astrocytes can be directly converted into neurons by transcription factors such as Ascl1 and Neurog2 for the treatment of stroke. However, most effective transcription factors have not been clearly defined. In addition, while various viral vectors have been used translation to human clinical trials will necessitate safer vectors such as adeno-associated virus. In our study we compare transduction efficiency of various AAV serotypes in vivo in a mouse model of stroke. Using the most efficient transcription factor in future studies we will compare reprogramming efficacy of Ngn2, Ascl1, Sox2 and NeuroD1 in vivo in our mouse stroke model.

    Methods: Permanent distal MCA occlusion was generated in C57/B6 wild type mice through a small craniectomy and coagulation of a distal MCA branch over the frontal lobe. Transduction efficiency was then compared 5 days later between AAV serotypes 8, 9, and rh10. Specifically, 1ul of virus was injected each into two sites around the stroke. One week later brains were compared using immunohistochemistry for astrocytes (GFAP+) and transduced cells (GFP+).

    Results: All mice had a clinically silent stroke. After surgery, they were able to resume normal activities. Infection rates of AAV 8, 9, and 10 for astrocytes were 0.5%, 6.9% and 9.1%, respectively and for neurons were 5.0%, 11.6%, and 7.6%, respectively.

    Conclusions: We were able to create a stroke model in wild type mice that gave clinically silent strokes. AAV 10 had the highest infection rate for astrocytes and a relatively lower infection rate for neurons, when compared to AAVs 8 and 9. Therefore, we conclude that AAV 10 is the best vehicle for delivering transcription factors to glial cells in stroke mice. Future studies using AAV rh10 will be used for comparing reprogramming efficacy with the neurogenic transcription factors Ngn2, Ascl1, Sox2, and NeuroD1.

    Patient Care: This will lead to better understanding of reactive glial cells after stroke and possibility of converting reactive glial cells into neurons. Eventually better patient care.

    Learning Objectives: By the conclusion of this session, participants should be able to: 1) understand that AAV 10 has highest infection rate for reactive glial cells when compared to AAV 8 and 9. 2) understand that transcription factors can successfully convert glial cells into neuron in cortical stroke area in brain

    References: Melek Chouchane, M. R. C. Cell therapy for stroke: use of local astrocytes. Frontiers in Cellular Neuroscience 6, (2012). Niu, W. et al. SOX2 reprograms resident astrocytes into neural progenitors in the adult brain. Stem Cell Reports 4, 780–794 (2015). Liu, Y. et al. Ascl1 Converts Dorsal Midbrain Astrocytes into Functional Neurons In Vivo. J Neurosci 35, 9336–9355 (2015). Guo, Z. et al. In Vivo Direct Reprogramming of Reactive Glial Cells into Functional Neurons after Injury and in an Alzheimer’s Disease Model. Cell Stem Cell 14, 188–202 (2014). Torper, O. et al. In Vivo Reprogramming of Striatal NG2 Glia into Functional Neurons that Integrate Local Host Circuitry. Cell Reports 12, 474–481 (2015). Grande, A. et al. Environmental impact on direct neuronal reprogramming in vivo in the adult brain. Nature Communications 4, 2373 (2013). Lee, Y., Messing, A., Su, M. & Brenner, M. GFAP promoter elements required for region-specific and astrocyte-specific expression. Glia 56, 481–493 (2008). Lentz, T. B., Gray, S. J. & Samulski, R. J. Viral vectors for gene delivery to the central nervous system. Neurobiology of Disease 48, 179–188 (2012). Weinberg, M. S., Samulski, R. J. & McCown, T. J. Adeno-associated virus (AAV) gene therapy for neurological disease. Neuropharmacology 69, 82–88 (2013). Shen, F. et al. Intravenous delivery of adeno-associated viral vector serotype 9 mediates effective gene expression in ischemic stroke lesion and brain angiogenic foci. Stroke 44, 252–254 (2013).

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