Controlled Targeting of Proteins to Cell Compartments

描述（由申请人提供）：可以利用和控制信号序列用于基因治疗的下一阶段：基因产物的受控靶向和递送。为了实现这一点，蛋白质中存在的固有信号将被去除，并被可转换机制所取代。最佳地，基因产物可以被引导至特定的亚细胞区室，在那里它们是唯一活性的，并且以受控的方式从区室中去除。定位可控蛋白可用于校正在某些癌症和其他疾病状态中发生的内源性蛋白质的错误定位。我们的项目的主要目标是使用激素诱导的核输入信号和输出信号作为双向的“开/关开关”，用于控制蛋白质靶向亚细胞区室。由该系统递送的蛋白质在到达活性区室（在这种情况下，细胞核）时应保持其治疗效果。当需要特定亚细胞区室中的受控反应时，或当在特定亚细胞区室中长时间高水平表达时用于去除有毒的治疗性蛋白质时，这样的系统将是有用的。我们项目的第一个目的是确定蛋白质是否可以通过激素诱导的输入/输出开关双向靶向正确的亚细胞区室，并确定输入和输出的机制。由激素调节的输入开关将蛋白质引导到细胞核，而当激素被去除或不存在时触发的输出开关将蛋白质引导到细胞质。第二个目的是确定是否可以调节和调谐针对亚细胞区室的蛋白质的量。靶向特定亚细胞区室的蛋白质的量可以通过改变信号的强度和/或通过改变所添加的外部激素的剂量来调节。第三个目的是确定靶向蛋白质在导向其工作亚细胞区室时是否仍然有活性（并且在其非活性区室中无毒）。选择用于递送至细胞核的蛋白质是核辅阻遏物NCoR的阻遏结构域，其具有下调孕酮受体活性（涉及生殖癌）的治疗用途。第四个目标是通过使用来自其他核受体（包括雌激素受体、糖皮质激素受体和蜕皮激素受体）的替代配体结合结构域来扩大赋予细胞核诱导性的试剂的范围。总之，最终目标是调节基因产物或治疗性蛋白质在其亚细胞位点的位置和量，在亚细胞位点基因产物或治疗性蛋白质维持其生物活性。

DESCRIPTION (provided by applicant): Signal sequences can be harnessed and controlled for use in the next phase in gene therapy: controlled targeting and delivery of gene products. To achieve this, the inherent signals present in a protein will be removed and replaced with a switchable mechanism. Optimally, gene products could be directed to a specific subcellular compartment where they are exclusively active, and removed from the compartment in a controlled manner. Localization controllable proteins could be used to correct for mislocalization of endogenous proteins that occurs in some cancers and other disease states. The main goal of our project is to use hormone inducible nuclear import signals and export signals as a bidirectional "on/off switch" for controlled targeting of proteins to subcellular compartments. The protein delivered by this system should retain its therapeutic effect when it reaches the active compartment, in this case, the nucleus. Such a system would be useful when a controlled response in a specific subcellular compartment is desired, or for removal of the therapeutic protein that is toxic when expressed for prolonged, high levels in a particular subcellular compartment. The first aim of our project is to determine if a protein can be bidirectionally targeted to the correct subcellular compartment via a hormone inducible import/export switch, and also to determine the mechanism of import and export. The import switch, regulated by hormone, directs the protein to the nucleus, while the export switch, triggered when hormone is removed or absent, directs the protein to the cytoplasm. The second aim is to determine if the amount of protein directed to a subcellular compartment can be regulated and tuned. The amount of protein targeted to a specific subcellular compartment can be regulated by altering the strength of the signal(s) and/or by changing the dose of the external hormone added. The third aim is to determine if the targeted protein is still active when directed to its working subcellular compartment (and non-toxic in its inactive compartment). The protein selected for delivery to the nucleus is the repression domain of the nuclear corepressor NCoR, which has a therapeutic use for down regulation of progesterone receptor activity (implicated in reproductive cancers). The fourth aim is to expand the range of agents that confer inducibility to the nucleus by using alternative ligand binding domains from other nuclear receptors including the estrogen receptor, glucocorticoid receptor, and ecdysone receptor. In summary, the ultimate goal is to regulate the delivery, in terms of the location and amount, of a gene product or therapeutic protein to its subcellular site where it maintains its biological activity.