Transcription factor EB (TFEB)-based gene therapy is a promising therapeutic strategy in treating neurodegenerative diseases by promoting autophagy/lysosome-mediated degradation and clearance of misfolded proteins that contribute to the pathogenesis of these diseases. However, recent findings have shown that TFEB has proinflammatory properties, raising the safety concerns about its clinical application. To investigate whether TFEB induces significant inflammatory responses in the brain, male C57BL/6 mice were injected with phosphate-buffered saline (PBS), adeno-associated virus serotype 8 (AAV8) vectors overexpressing mouse TFEB (pAAV8-CMV-mTFEB), or AAV8 vectors expressing green fluorescent proteins (GFPs) in the barrel cortex. The brain tissue samples were collected at 2 months after injection. Western blotting and immunofluorescence staining showed that mTFEB protein levels were significantly increased in the brain tissue samples of mice injected with mTFEB-overexpressing vectors compared with those injected with PBS or GFP-overexpressing vectors. pAAV8-CMV-mTFEB injection resulted in significant elevations in the mRNA and protein levels of lysosomal biogenesis indicators in the brain tissue samples. No significant changes were observed in the expressions of GFAP, Iba1, and proinflammation mediators in the pAAV8-CMV-mTFEB-injected brain compared with those in the control groups. Collectively, our results suggest that AAV8 successfully mediates mTFEB overexpression in the mouse brain without inducing apparent local inflammation, supporting the safety of TFEB-based gene therapy in treating neurodegenerative diseases.

中文翻译：

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小鼠颅内注射基于转录因子EB（TFEB）的基因治疗的安全性

基于转录因子EB（TFEB）的基因疗法是一种通过促进自噬/溶酶体介导的降解和错误折叠蛋白的清除来治疗神经退行性疾病的有前途的治疗策略，而这些错误折叠的蛋白会导致这些疾病的发病机理。但是，最近的发现表明TFEB具有促炎特性，引起了对其临床应用的安全性担忧。为了研究TFEB是否会在大脑中诱导明显的炎症反应，向雄性C57BL / 6小鼠注射了磷酸盐缓冲液（PBS），过表达小鼠TFEB的腺相关病毒血清型8（AAV8）载体（pAAV8-CMV-mTFEB），或者AAV8载体在桶状皮质中表达绿色荧光蛋白（GFP）。注射后2个月收集脑组织样品。Western印迹和免疫荧光染色显示，与注射过PBS或GFP的载体相比，注射mTFEB的载体的小鼠的脑组织样品中的mTFEB蛋白水平显着增加。pAAV8-CMV-mTFEB注射导致脑组织样品中溶酶体生物发生指示物的mRNA和蛋白质水平显着升高。与对照组相比，在注射了pAAV8-CMV-mTFEB的大脑中，GFAP，Iba1和促炎介质的表达未见明显变化。总的来说，我们的结果表明AAV8成功介导了小鼠大脑中mTFEB的过度表达而没有引起明显的局部炎症，从而支持了基于TFEB的基因疗法在治疗神经退行性疾病中的安全性。