Controlled Targeting of Proteins to Cell Compartments

描述(由申请人提供)：信号序列可以被利用和控制，以用于基因治疗的下一阶段：受控的靶向和基因产品的输送。为了实现这一点，蛋白质中存在的固有信号将被移除，取而代之的是可切换的机制。最理想的是，基因产物可以被定向到特定的亚细胞室，在那里它们是唯一活跃的，并以受控的方式从亚细胞室中移除。定位可控蛋白质可以用来纠正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“开/关开关”，以控制蛋白质的亚细胞靶向。这个系统提供的蛋白质在到达活跃的隔室时应该保持其治疗效果，在这种情况下，就是细胞核。当需要在特定的亚细胞室中进行受控反应时，或者在特定的亚细胞室中长时间、高水平表达有毒的治疗性蛋白时，这种系统将是有用的。我们项目的第一个目标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，而当激素被移除或不存在时触发的输出开关将蛋白质引导到细胞质。第二个目标是确定定向到亚细胞隔间的蛋白质的量是否可以调节和调节。可以通过改变信号强度(S)和/或通过改变添加的外源激素的剂量来调节针对特定亚细胞隔室的蛋白质的量。第三个目标是确定目标蛋白在被定向到其工作的亚细胞室时是否仍然有效(在其不活跃的室中是否无毒)。被选择运送到细胞核的蛋白质是核辅阻遏子NCoR的抑制域，它具有下调孕激素受体活性(与生殖癌有关)的治疗用途。第四个目标是通过使用其他核受体(包括雌激素受体、糖皮质激素受体和蜕皮激素受体)的替代配体结合域，扩大向细胞核提供诱导性的药物的范围。总而言之，最终目标是根据基因产物或治疗性蛋白质的位置和数量来调节其向其亚细胞部位的输送，在那里它保持其生物活性。

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.