近红外光控稀土基纳米光遗传学基因表达系统的构建及应用

The development of optogenetics opens up the novel thinking for inducing precise expression and regulation of exogenous gene. Light is an ideal inducer because of a series of advantages such as low cytotoxicity, easy obtainment and accessibility, high spatiotemporal resolution and so on. However, the present widely used photo-induced gene expression systems still suffer from poor specificity, low inducible multiple and hardgenic import because of transcription factors compromising of proteins. Additionally, in situ induction indeep tissues in vivo can hardly be achieved due to low tissue penetration depth of blue light as excitation source. All these problems are particularly urgent to be solved. Therefore, to achieve light-controlled gene expression and disease treatment in deep tissues, this project will construct a general NIR-responsive nano-optogenetic system containing rare-earth ion doped upconversion nanoparticles as mediums of light conversion for precise expression and regulation of exogenous gene in a modality of “LightOn”. Accordingly, at first, photosensitive proteinwill be introduced to Gal4/UAS two hybrid system to construct blue light activated transcription factors. Then controllable rare-earth ion doped upconversion nanoparticles with good biocompatibility will be prepared to serve as both mediums of light conversion and transfection reagents. Next, the photosensitive activating transcription factor GAVPO will be further optimized to initiate reporter gene plasmids which could express different target genes. Finally, animal models of diseases will be constructed and light-controlled gene expression applying to disease therapy will be examined. For example, insulin expressed in report plasmids could be applied to light-controlled therapy of diabetes mellitus type I in deep tissues.

光遗传学的发展为诱导外源基因精准表达调控开辟了全新的思路。光作为理想的诱导剂具有低毒性、易获取、易操控、高时空分辨率等优点。而目前大多诱导体系存在特异性差、诱导倍数低、由蛋白构成的转录因子不利于基因导入、且多以蓝光为诱导光源，无法实现活体深层组织的原位诱导等亟需克服的问题。基于此，本项目拟利用稀土上转换纳米粒子作为光转换媒介，构筑一种普适性的近红外光响应的外源基因精准表达调控的“LightOn”纳米光遗传学系统，用于目的基因在深层组织原位光控表达及疾病治疗：将光敏蛋白引入Gal4/UAS双杂交模型，构建蓝光激活的光控转录因子；合成一类具有良好生物相容性的高质量稀土上转换发光纳米粒子，既作为为光转换媒介，也可用转染试剂；利用优化了的光敏转录激活因子GAVPO, 可启动表达不同目的基因的报告质粒；构建活体动物疾病模型，诱导表达可用于疾病治疗的报告质粒如胰岛素，探索用于小鼠I型糖尿病等疾病治疗。

本项目旨在合成高质量稀土上转换纳米粒子，构筑一种普适性的近红外光响应的纳米光遗传学系统，用于目的基因在深层组织原位光控表达及疾病治疗。研究工作围绕稀土上转换多功能纳米复合材料设计合成、有机-无机纳米复合体系的设计与构筑、药物控制释放及肿瘤治疗等展开。在本项目的原有研究内容基础上，延续了稀土上转换纳米材料的精准控制合成这一核心主题，并经过科学研判，对相关研究内容进行了一定的调整和扩展，进一步开展了系列无机纳米佐剂及肿瘤纳米疫苗等相关的拓展研究，并已基本完成了各项研究任务和目标。主要研究内容包括：(1) 精准控制合成了几种稀土上转换纳米复合体系，如发展了肿瘤微环境响应的锰基纳米体系、生物可降解稀土上转换纳米粒子等；(2) 开发几种新型高效纳米声敏剂用于声动力治疗，实现氧气的可控释放和特定部位的输送，缓解了深部肿瘤组织中的乏氧等；(3) 发展了几种无机纳米佐剂及肿瘤纳米疫苗，提出了焦亡佐剂（PTAVs）的新概念，首次证实氧化锰纳米材料具有本征的免疫原性，提出了一种通过放大免疫原性细胞死亡的免疫调节效应来构建原位癌症疫苗的策略；(4) 利用异质纳米复合材料，实现了协同光热疗法/化学动力疗法/免疫疗法以及近红外光同步触发的高效光热/光催化/化学治疗等。依托该项目，项目负责人在Adv. Mater.，Angew. Chem. Int. Ed., ACS Nano, Adv. Funct. Mater.等国内外核心期刊以通讯作者身份发表论文30篇(其中IF>10的论文18篇)，应邀在材料及化学核心杂志Adv. Mater.、Small、Adv. Drug Delivery Rev.、J. Mater. Chem. B等上面撰写了多篇有关无机纳米材料及其生物医学应用最新进展的综述文章。参加国内外相关领域学术会议并做邀请/特邀报告14次；依托该项目培养中青年学术带头人1名 (国家优青)，中国科学院院长特别奖获得者1人，博士后1人，博士/硕士研究生15人；获得 “吉林省自然科学一等奖”(2022，排名第1)等奖励。