Imaging Survivin mRNA for Cancer Detection

生存素 mRNA 成像用于癌症检测

• 批准号: 6731349
• 负责人: DONALD J HNATOWICH
• 金额: $ 17.89万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-03 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6731349
• 关键词: antisense nucleic acid; athymic mouse; autoradiography; biotechnology; cell line; diagnosis design /evaluation; in situ hybridization; messenger RNA; neoplasm /cancer genetics; neoplasm /cancer radiodiagnosis; neoplastic cell; nuclear medicine; nucleic acid probes; oncogenes; oncoproteins; pharmacokinetics; polymerase chain reaction; protein quantitation /detection; radiopharmacology; radiotracer; scintillation cameras; survivin; technetium; tissue /cell culture

DESCRIPTION (provided by applicant): The dichotomous expression of survivin mRNA in normal versus cancer cells is among the most tumor-specific of all human gene products thus garnering great interest as a potential diagnostic marker and a therapeutic target. The goal of this investigation is to develop a novel radiolabeled antisense probe against survivin mRNA as a next-generation radiopharmaceutical for in vivo mapping of this transcript. The dichotomous expression of survivin mRNA may lead to a selective retention of radioactivity on tumors mediated by the in vivo hybridization. Capturing the radioactive signal with a gamma camera will produce a "clean-cut" tumor imaging-antisense imaging. Using technetium-99m (99mTc) radiolabeled antisense DNA probe to RIalpha mRNA, we have observed what is almost certainly an antisense effect. Our results, therefore, show that antisense imaging is feasible and could already be achievable if the tumor uptake and tumor/nontumor ratios can be improved. We, therefore, propose a series of novelties designed to accomplish this goal: 1). Select a superior antsiense sequence targeting a common stretch shared by all human survivin mRNA splice variants. 2). Use a just-emerging synthetic oligomer morpholino (MORF) bearing superior properties as antisense chemical form. 3). Design a multifunctional Tat vector with cell transduction potency to enhance cell uptake and accelerate clearance. MORF will be conjugated to the overhung cysteine on the Tat vector to form chimeric antisense construct (Tat-MORF), which can easily be radiolabeled with 99mTc via a built-in N2S2 chelator by a one-step procedure. The 99mTc-Tat-MORF will be evaluated first in cell culture with tumor cells overexpressing the survivin mRNA (Survivin+) and tumor cells without expression of human survivin mRNA (Survivin-). The kinetics of cellular accumulation and efflux will be measured with antisense vs. scrambled control in Survivin+ and Survivin- tumor cells. Inhibition assay and the correlation of antisense accumulation to the level of target mRNA expression will be performed to reinforce the antisense mechanism, ie. the selective antisense accumulation in target cells is mediated by hybridization of antisense probes. For in vivo study, a dual-xenograft tumor model in nude mice will be established bearing Survivin+ and Survivin- tumors. This animal model will allow us to address almost all concerns for antisense tumor targeting. Longitudinal scintigraphies will be complemented with periodical biodistribution of tissue dissection and whole body autoradiographies to supplement relative tissue radioactivity measurements with absolute measurements. We believe that using this novel chimeric antisense probe as a next-generation radiopharmaceutical to target the dichotomous expression of survivin mRNA would allow us to obtain a "clean-cut" tumor imaging by antisense mechanism. This "proof-of-concept" of antisense imaging will be helpful to provide a paradigm of the merger of modern biology and conventional nuclear medicine.

描述（由申请人提供）： 在正常癌细胞和癌细胞中，源性mRNA的二分法表达是所有人类基因产物中肿瘤特异性中最特异性的之一，因此引起了潜在的诊断标记和治疗靶点的极大兴趣。这项研究的目的是开发针对Survivin mRNA的新型放射标记的反义探针，作为该转录本的体内映射的下一代放射性药物。 Survivin mRNA的二分法表达可能导致在体内杂交介导的肿瘤上的放射性选择性保留。使用伽马摄像机捕获放射性信号将产生“清洁”肿瘤成像 - 抗异源成像。使用Technetium-99m（99MTC）放射性标记的反义DNA探针到Rialpha mRNA，我们已经观察到了几乎可以肯定的反义作用。因此，我们的结果表明，如果可以改善肿瘤摄取和肿瘤/非肿瘤比率，则反义成像是可行的，并且可以实现。因此，我们提出了一系列旨在实现这一目标的新颖性：1）。选择一个针对所有人类Survivin mRNA剪接变体共享的共同拉伸的上蚂蚁序列。 2）。使用具有优质特性作为反义化学形式的纯粹出现的合成寡聚物（MORF）。 3）。设计具有细胞转导效力的多功能TAT载体，以增强细胞摄取和加速清除率。 MORF将与TAT载体上的Overhung半胱氨酸共轭，形成嵌合反义构建体（TAT-MORF），可以通过一个步骤通过内置的N2S2螯合剂轻松将99mtc放射性标记。 99MTC-TAT-MORF将首先在细胞培养中进行评估，肿瘤细胞过表达遗物mRNA（survivin+）和肿瘤细胞，而没有人类遗传mRNA的表达（Survivin-）。细胞积累和外排的动力学将通过反义与crec症的控制和Survivin-肿瘤细胞中的炒作对照进行测量。将进行抑制测定和反义积累与靶mRNA表达水平的相关性，以增强反义机制，即。反义探针的杂交介导了靶细胞中的选择性反义积累。为了进行体内研究，将建立裸鼠中的双Xenogroght肿瘤模型，该模型将建立携带survivin+和survivin-lamors。该动物模型将使我们能够解决对反义肿瘤靶向的所有问题。纵向闪烁图将与组织解剖和整个身体放射自显影的定期生物分布相辅相成，以通过绝对测量来补充相对组织放射性测量。我们认为，将这种新型的嵌合反义探针作为下一代放射性药物来靶向Survivin mRNA的二分法表达，这将使我们能够通过反义机制获得“清洁”的肿瘤成像。反义成像的“概念概念”将有助于提供现代生物学和常规核医学合并的范式。