Controlled premature termination of translation to generate designated truncated

描述（由申请人提供）：许多疾病，包括几种形式的癌症，取决于病理蛋白质的不适当表达，如癌基因。 通常，这些蛋白质的结构-功能掩盖了模块化结构，在蛋白质的C-末端部分具有必需的功能结构域（例如转录因子的反式激活结构域）。缺乏C-末端结构域的突变体可以具有改变的和拮抗的功能，并且在某些情况下，它们可以表现为显性阴性变体。这些变体有时通过选择性剪接/多聚腺苷酸化天然产生。然而，这些并不总是存在或不容易激活。 特异性诱导这些显性阴性变体的方法将是高度期望的，并且在广泛的疾病（包括癌症）中具有显著的治疗潜力。 为了产生一个预先设计的，理想的截短的变体，而不是病理性的，我们提出了一种新的通用方法的发展，基于一个全新的设计的反义分子，能够重定向终止翻译从正常翻译的全长ORF的上游指定的位置。 这些自缠绕反义翻译终止（SWATT）化合物通过引入无法通过进入的翻译核糖体的解旋酶活性解决的不可克服的空间位阻而起作用，所述核糖体通常可以解旋前体mRNA内的广泛二级结构。因此，该障碍将导致核糖体脱落和较短的C末端截短变体的表达。 我们将通过评估SWATT化合物在专门设计的报告荧光素酶构建体上的可行性来验证这种新策略的可行性，并最终通过使用Notch信号作为癌细胞中的原理验证底物来显示其生物相关性。缺乏胞内信号传导结构域的Notch的C末端显性负性形式的诱导应通过使Notch配体参与非生产性相互作用来抑制Notch信号传导，并因此防止其致癌功能。 SWATT化合物将提供一种额外的工具来剖析蛋白质在其生理背景下的结构-功能特性，并且它们将构成治疗癌症和其他疾病的新型治疗方法的基础。 公共卫生相关性：我们计划开发一种新技术，将有助于肿瘤进展的蛋白质的生产重新定向为具有相反抗肿瘤特性的相同蛋白质的变体（显性-阴性变体）。 这将通过在特定的、预先确定的点处将物理“路障”引入核糖体的翻译路径中来实现，从而迫使它们在那里中断蛋白质合成。 如果成功， 这项工作可能构成一种新的、替代性的癌症治疗方法的基础，并可能应用于广泛的肿瘤。

DESCRIPTION (provided by applicant): Many diseases, including several forms of cancer, depend on the inappropriate expression of pathological proteins, such as oncogenes. Frequently, the structure-function of these proteins belies a modular construction, with an essential functional domain in the C-terminal portion of the protein (for example the transactivation domain of a transcription factor). Mutants lacking the C-terminal domain can have altered and antagonistic functions, and in some cases they can behave as dominant- negative variants. Such variants are sometimes naturally generated by alternative splicing/polyadenylation. However, these are not always present or not easily activatable. A method to specifically induce these dominant negative variants would be highly desirable and have a significant therapeutic potential in a broad range of diseases, including cancer. In order to generate a pre-designed, desirable truncated variant instead of the pathological one we propose development of a novel universal methodology, based on a brand new design of antisense molecules that are able to redirect termination of translation from the normally translated full-length ORF to an upstream specified location. These Self-Wrapping Antisense Translation Termination (SWATT) compounds work by introducing an insurmountable steric block that cannot be resolved by the helicase activity of incoming translating ribosomes, which can normally unwind extensive secondary structures within the pre-mRNA. This roadblock would thus result in ribosome drop-off and expression of a shorter, C-terminally truncated variant. We will validate the viability of this novel strategy by assessing SWATT compounds on specifically designed reporter luciferase constructs and, eventually, by showing their biological relevance using Notch signaling as a proof-of-principle substrate in cancer cells. Induction of a C-Terminal dominant negative version of Notch, lacking the intracellular signaling domain, should inhibit Notch signaling by engaging Notch ligands in non-productive interactions, and thus prevent its oncogenic functions. SWATT compounds will provide an additional tool to dissect the structure-function properties of proteins in their physiological contexts, and they wil constitute the basis for a novel therapeutic approach in the treatment of cancer and other diseases. PUBLIC HEALTH RELEVANCE: We plan to develop a novel technology to re-direct the production of proteins that contribute to tumor progression to variants of the same proteins with opposite, anti- tumorigenic properties (Dominant-Negative variants). This will be obtained by introducing a physical 'road-block' into the path of ribosomes during translation at specific, pre-determined points, and therefore forcing them to interrupt protein synthesis there. If successful, this work might constitute the basis for a novel, alternative approach to cancer therapy, with potential application to a broad range of tumors.