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眼部给予携带小鼠 Tph2 基因的 DNA 质粒：脑结构中重组 Tph2-FLAG 的证据。

Ophthalmic Administration of a DNA Plasmid Harboring the Murine Tph2 Gene: Evidence of Recombinant Tph2-FLAG in Brain Structures.

机构信息

Unidad de Investigación Biomédica en Infectología e Inmunología, Hospital de Infectología, Centro Médico Nacional "La Raza", IMSS, Paseo de las Jacarandas s/n esquina Calzada Vallejo, Col. La Raza, C.P. 02990, Mexico City, Mexico.

Unidad de Investigación Médica en Enfermedades Endocrinas, UMAE, Hospital de Especialidades, Centro Médico Nacional "Siglo XXI", IMSS, Mexico City, Mexico.

出版信息

Mol Biotechnol. 2020 Mar;62(3):200-209. doi: 10.1007/s12033-020-00239-x.

DOI:10.1007/s12033-020-00239-x
PMID:32030628
Abstract

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.

摘要

色氨酸羟化酶 2 型（Tph2）是大脑中 5-羟色胺（5-HT）生物合成的第一步限速酶。眼部给药（Op-Ad）是一种非侵入性方法，可通过眼睛将基因载体递送到大脑。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，外加 FLAG 标签。重组 Tph2-FLAG 在体外和体内进行了检测和测试，其中 25μg 的 pIRES-hrGFP-1a-Tph2-FLAG 通过 Op-Ad 递送到小鼠。该构建体能够在体外和体内表达和产生重组 Tph2-FLAG。体内实验表明，该构建体有效地穿过血眼屏障和血脑屏障，到达脑细胞，穿过视神经，并在不同脑区转录 mRNA-Tph2-FLAG。重组 Tph2-FLAG 观察到在杏仁核和脑干中，主要在中缝背侧和中缝内侧。Op-Ad 三天后，记录到 Tph2 的相对表达水平是基础水平的三倍。这些结果表明，通过眼睛给药的 pIRES-hrGFP-Tph2-FLAG 能够到达大脑，转录和翻译 Tph2。总之，这项研究表明了递送治疗基因的可行性，如 Tph2，它是 5-HT 生物合成的第一步限速酶。

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本文引用的文献

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Arch Med Res. 2017 Oct;48(7):616-622. doi: 10.1016/j.arcmed.2018.03.003. Epub 2018 Mar 16.
2
An intranasally delivered peptide drug ameliorates cognitive decline in Alzheimer transgenic mice.经鼻给药的肽类药物可改善阿尔茨海默病转基因小鼠的认知衰退。
EMBO Mol Med. 2017 May;9(5):703-715. doi: 10.15252/emmm.201606666.
3
Intranasal administration of plasmid DNA nanoparticles yields successful transfection and expression of a reporter protein in rat brain.
鼻腔内给予质粒 DNA 纳米粒可成功转染并在大鼠脑内表达报告蛋白。
Gene Ther. 2014 May;21(5):514-21. doi: 10.1038/gt.2014.28. Epub 2014 Mar 27.
4
Lack of brain serotonin affects postnatal development and serotonergic neuronal circuitry formation.缺乏脑内血清素会影响产后发育和血清素能神经元回路的形成。
Mol Psychiatry. 2013 Oct;18(10):1106-18. doi: 10.1038/mp.2012.128. Epub 2012 Sep 25.
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Gene transfer for ocular neovascularization and macular edema.基因转移治疗眼部新生血管和黄斑水肿。
Gene Ther. 2012 Feb;19(2):121-6. doi: 10.1038/gt.2011.164. Epub 2011 Nov 10.
6
Deficient serotonin neurotransmission and depression-like serotonin biomarker alterations in tryptophan hydroxylase 2 (Tph2) loss-of-function mice.色氨酸羟化酶 2（Tph2）功能丧失型小鼠中血清素神经递质传递不足和类似抑郁的血清素生物标志物改变。
Mol Psychiatry. 2012 Jul;17(7):694-704. doi: 10.1038/mp.2011.50. Epub 2011 May 3.
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