Enbar T, Hickmott J W, Siu R, Gao D, Garcia-Flores E, Smart J, Casabuenas D L, Faiz M, Morshead C M
Institute of Medical Sciences, University of Toronto, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada.
Department of Surgery, Division of Anatomy, University of Toronto, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada.
Sci Rep. 2024 Dec 30;14(1):31583. doi: 10.1038/s41598-024-79124-5.
Astrocyte to neuron reprogramming has been performed using viral delivery of neurogenic transcription factors in GFAP expressing cells. Recent reports of off-target expression in cortical neurons following adeno-associated virus (AAV) transduction to deliver the neurogenic factors have confounded our understanding of the efficacy of direct cellular reprogramming. To shed light on potential mechanisms that may underlie the neuronal off-target expression of GFAP promoter driven expression of neurogenic factors in neurons, two regionally distinct cortices were compared-the motor cortex (MC) and medial prefrontal cortex (mPFC)-and investigated: (1) the regional tropism and astrocyte transduction with an AAV5-GFAP vector, (2) the expression of Gfap in MC and mPFC neurons; and (3) material transfer between astrocytes and neurons. Using a Cre-based system (AAV5-hGFAP-Cre; Rosa26R-tdTomato reporter mice), regional differences were observed in tdTomato expression between the MC and mPFC. Interestingly, this correlated with the presence of a greater expression of Gfap mRNA in neurons in the mPFC. Additionally, intercellular material transfer of Cre and tdTomato was observed between astrocytes and neurons in both regions, albeit at very low frequencies. Our study highlights regionally distinct variation in neurons that warrants consideration when designing genetic constructs for gene therapies targeting astrocytes including astrocyte to neuron reprogramming.
在表达胶质纤维酸性蛋白（GFAP）的细胞中，通过病毒递送神经源性转录因子实现了星形胶质细胞向神经元的重编程。最近有报道称，在腺相关病毒（AAV）转导以递送神经源性因子后，皮质神经元中出现了脱靶表达，这使我们对直接细胞重编程的功效的理解变得复杂。为了阐明神经元中GFAP启动子驱动的神经源性因子表达的神经元脱靶表达可能潜在的机制，比较了两个区域不同的皮质——运动皮质（MC）和内侧前额叶皮质（mPFC）——并进行了以下研究：（1）用AAV5-GFAP载体进行区域嗜性和星形胶质细胞转导；（2）MC和mPFC神经元中Gfap的表达；以及（3）星形胶质细胞和神经元之间的物质转移。使用基于Cre的系统（AAV5-hGFAP-Cre；Rosa26R-tdTomato报告小鼠），在MC和mPFC之间观察到tdTomato表达的区域差异。有趣的是，这与mPFC神经元中Gfap mRNA表达量更高相关。此外，在两个区域的星形胶质细胞和神经元之间均观察到了Cre和tdTomato的细胞间物质转移，尽管频率非常低。我们的研究强调了神经元中区域不同的差异，在设计针对星形胶质细胞的基因治疗（包括星形胶质细胞向神经元的重编程）的基因构建体时，这一点值得考虑。
