Tryptophan hydroxylase-type 2 (Tph2) is the first rate-limiting step in the biosynthesis of serotonin (5-HT) in the brain. The ophthalmic administration (Op-Ad) is a non-invasive method that allows delivering genetic vehicles through the eye and reaches the brain. Here, the murine Tph2 gene was cloned in a non-viral vector (pIRES-hrGFP-1a), generating pIRES-hrGFP-1a-Tph2, plus the FLAG-tag. Recombinant Tph2-FLAG was detected and tested in vitro and in vivo, where 25 μg of pIRES-hrGFP-1a-Tph2-FLAG was Op-Ad to mice. The construct was capable of expressing and producing the recombinant Tph2-FLAG in vitro and in vivo. The in vivo assays showed that the construct efficiently crossed the Hemato-Ocular Barrier and the Blood-Brain Barrier, reached brain cells, passed the optical nerves, and transcribed mRNA-Tph2-FLAG in different brain areas. The recombinant Tph2-FLAG was observed in amygdala and brainstem, mainly in raphe dorsal and medial. Relative Tph2 expression of threefold over basal level was recorded three days after Op-Ad. These results demonstrated that pIRES-hrGFP-Tph2-FLAG, administrated through the eyes was capable of reaching the brain, transcribing, and translating Tph2. In conclusion, this study showed the feasibility of delivering therapeutic genes, such as the Tph2, the first enzyme, rate-limiting step in the 5-HT biosynthesis.

中文翻译：

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含有鼠Tph2基因的DNA质粒的眼科给药：脑结构中重组Tph2-FLAG的证据。

色氨酸羟化酶2型（Tph2）是大脑中5-羟色胺（5-HT）生物合成中的第一个限速步骤。眼科给药（Op-Ad）是一种非侵入性方法，可通过眼睛传递遗传载体并到达大脑。在这里，将鼠Tph2基因克隆到非病毒载体（pIRES-hrGFP-1a）中，生成pIRES-hrGFP-1a-Tph2，加上FLAG标签。在体内和体外检测并测试了重组Tph2-FLAG，其中25μgpIRES-hrGFP-1a-Tph2-FLAG对小鼠是Op-Ad。该构建体能够在体外和体内表达和产生重组Tph2-FLAG。体内试验表明，该构建体有效地跨越了血脑屏障和血脑屏障，到达了脑细胞，通过了视神经，并在不同的大脑区域转录了mRNA-Tph2-FLAG。重组Tph2-FLAG在杏仁核和脑干中观察到，主要在背侧和内侧。在Op-Ad后三天记录了相对于基础水平三倍的Tph2表达。这些结果表明，通过眼睛施用的pIRES-hrGFP-Tph2-FLAG能够到达大脑，转录和翻译Tph2。总而言之，这项研究表明了在5-HT生物合成中传递治疗性基因（例如第一个酶Tph2）的治疗基因的可行性。