Genetically encoded tools to control any mammalian cell function with any desired stimulus

通过任何所需刺激控制任何哺乳动物细胞功能的基因编码工具

• 批准号: RGPIN-2019-04183
• 负责人: Truong, Kevin
• 金额: $ 3.06万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=737468
• 关键词: Genetically; encoded; tools; control; any

The development of genetically encoded tools like channelrhodopsin-2 has allowed the stimuli of light to control membrane depolarization of neurons, sparking a steady stream of breakthroughs in our understanding of brain function. While light as a stimuli has strengths (e.g. precise spatial and temporal activation), it also has weaknesses (e.g. low tissue penetration and invasive animal surgery). Furthermore, other than membrane depolarization as a cell function, biologists have sought control over cell death to determine the biological role of the particular cell types and control over gene expression to determine the function of particular genes under regulation. My ultimate research objective is to engineer genetically encoded tools that will allow the control of any mammalian cell function with any desired stimuli. Our group believes this is possible through Ca2+ rewiring - combining proteins that generate Ca2+ signals upon binding desired stimuli with proteins that control cell function when activated by Ca2+ signals. First in the proposal, we will develop genetically-encoded tools to allow Ca2+ influx and membrane depolarization to be controlled by magnetic fields. Magnetic fields are permeable through tissues that will allow neurons to be stimulated deep inside the brain with no surgery in contrast with light-based or electrode-based approaches. Second, we will develop Ca2+-activated caspases. Caspases are central cysteine proteases in the apoptotic cell death that function to dismantle the cell machinery by cleaving after an aspartate residue in their substrate. By co-expressing the chimeric caspase with a chimeric receptor that generates a Ca2+ signal in response to binding its target ligand, we aim to induce cell death in response to any stimuli via Ca2+ rewiring. Lastly, we will develop inducible gene expression systems. Through the ubiquitous NFAT pathway, Ca2+ oscillations activate gene expression. By co-expressing a chimeric receptor with natural or synthetic genes that enhance Ca2+ oscillations, we aim to induce gene expression in response to any stimuli via Ca2+ rewiring. The ability to control any mammalian cell function (e.g. Ca2+ influx, cell death and gene expression) with any desired stimuli (e.g. magnetic fields) would allow us to ask biological questions that are currently not possible. For example, magnetic field stimuli would allow activation of the areas deep inside the brain such as the hippocampus to probe questions about the formation of memories. Cell ablation rewired to any stimuli would allow cell death to be customized to the microenvironment (e.g. patterns of morphogens) to probe questions about their importance in development of tissues. Gene expression rewired to any stimuli would expand the current paucity of reliable inducible promoters for biomedical research. If rewired to magnetic fields, we could perform targeted deep tissue ablation and gene expression in the brain and spinal cord.

视紫红质通道蛋白 2 等基因编码工具的发展使得光刺激能够控制神经元膜去极化，从而在我们对大脑功能的理解中不断取得突破。虽然光作为刺激具有优点（例如精确的空间和时间激活），但它也有缺点（例如组织渗透性低和侵入性动物手术）。此外，除了作为细胞功能的膜去极化之外，生物学家还寻求控制细胞死亡以确定特定细胞类型的生物学作用，并控制基因表达以确定受调节的特定基因的功能。 我的最终研究目标是设计基因编码工具，允许通过任何所需的刺激来控制任何哺乳动物细胞的功能。我们的小组认为，这可以通过 Ca2+ 重新布线实现——将在结合所需刺激时产生 Ca2+ 信号的蛋白质与在被 Ca2+ 信号激活时控制细胞功能的蛋白质相结合。首先，我们将开发基因编码工具，通过磁场控制 Ca2+ 流入和膜去极化。磁场可透过组织，与基于光或基于电极的方法相比，无需手术即可刺激大脑深处的神经元。其次，我们将开发Ca2+​​激活的半胱天冬酶。半胱天冬酶是细胞凋亡中的核心半胱氨酸蛋白酶，其功能是通过切割底物中的天冬氨酸残基来拆除细胞机器。通过共表达嵌合 caspase 和嵌合受体，嵌合受体响应结合其靶配体而产生 Ca2+ 信号，我们的目标是通过 Ca2+ 重新布线诱导细胞死亡，以响应任何刺激。最后，我们将开发诱导型基因表达系统。通过无处不在的 NFAT 途径，Ca2+ 振荡激活基因表达。通过共表达嵌合受体与增强 Ca2+ 振荡的天然或合成基因，我们的目标是通过 Ca2+ 重新布线诱导基因表达以响应任何刺激。通过任何所需的刺激（例如磁场）控制任何哺乳动物细胞功能（例如 Ca2+ 流入、细胞死亡和基因表达）的能力将使我们能够提出目前不可能的生物学问题。例如，磁场刺激可以激活大脑深处的区域，例如海马体，以探究有关记忆形成的问题。细胞消融重新连接到任何刺激将使细胞死亡能够根据微环境（例如形态发生素的模式）进行定制，以探究它们在组织发育中的重要性的问题。重新连接任何刺激的基因表达将扩大目前生物医学研究中可靠的诱导型启动子的匮乏。如果重新连接到磁场，我们就可以在大脑和脊髓中进行有针对性的深层组织消融和基因表达。