Optimize RNA-aptamer for Biomarker Molecule CD30

优化生物标记分子 CD30 的 RNA 适体

• 批准号: 7745628
• 负责人: Xianbin Yang
• 金额: $ 12.36万
• 依托单位: AM BIOTECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-03-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7745628
• 关键词: Affect; Affinity; Anaplastic Cell; Animals; Antibodies; Aptamer Technology; B-Lymphocytes; Binding; Biological; Biological Assay; Biological Markers; Biotechnology; Cell Culture Techniques; Cell Line; Cell surface; Cells; Cellular Morphology; Chemistry; Child; Clinical; Clinical Research; Collaborations; Confocal Microscopy; Cultured Cells; DNA; Detection; Diagnosis; Diagnostic; Diffuse; Disease; Fingers; Flow Cytometry; Goals; Hospitals; Human; In Situ; In Vitro; Ki-1 Large-Cell Lymphoma; Ligands; Lymphoma; Malignant Neoplasms; Methodist Church; Methods; Microfluidics; Modification; Normal tissue morphology; Nucleic Acids; Oligonucleotides; Outcome; Oxygen; Phase; Play; Positioning Attribute; Process; Proteins; Protocols documentation; RNA; Reagent; Reporting; Research; Research Institute; Resistance; Role; Sensitivity and Specificity; Small Business Innovation Research Grant; Solutions; Specificity; Sulfur; Surface; T-Cell Lymphoma; T-Lymphocyte; TNFRSF8 gene; Technology; Testing; Time; Variant; Vertebral column; Work; aptamer; base; biological systems; cancer cell; clinical application; commercialization; cytotoxicity; density; disease diagnosis; experience; improved; innovation; inorganic phosphate; method development; monomer; neoplasm type; neoplastic cell; novel; nuclease; public health relevance; tool; tumor; vascular bed

DESCRIPTION (provided by applicant): Systemic Anaplastic Large Cell Lymphoma (ALCL) is the most common T-cell lymphoma in children. ALCL is characterized by its anaplastic cell morphology and high level expression of CD30 on the cell surface. This diffuse and homogeneous CD30 expression in ALCL cells and tumors has been considered as a diagnostic feature for the disease and anti-CD30 antibodies are widely used as a specific diagnostic probe in clinical settings. Developing an antibody is time-consuming and costly and batch-to-batch variations due to usage of different animal or cultured cells are common. To overcome these limitations a chemically synthesized specific probe for CD30 has been investigated. A recent study reported that a synthesized RNA aptamer could specifically bind to CD30 molecules in solution with high affinity. By using cultured lymphoma cells, our preliminary work indicates that this RNA aptamer also binds specifically to human cells via their surface CD30, suggesting that the aptamer may be used as a specific probe to detect ALCL cells for disease diagnosis. Since RNA is an easily degradable molecule when used in routine clinical lab testing conditions; developing a stable RNA aptamer probe is critical for clinical application. Additionally, the usefulness of an RNA aptamer as a diagnostic tool for ALCL would be further enhanced by improving the aptamer's sensitivity/affinity and specificity toward its target. As an initial proof of concept, which can be extended to a commercialized diagnostic product for clinical use; we propose to develop a new RNA reagent building block which can be used to improve the RNA aptamer. Our central hypothesis is that the phosphates of the RNA aptamer backbone play a critical role in its interaction with its target biomarker molecule CD30. Replacing the two non-bridging oxygens with sulfurs at several linkage positions will result in a phosphorodithioated (PS2) RNA aptamer (PS2- aptamer) that will increase the RNA aptamer's stability, cell-binding affinity and specificity. We plan to test our central hypothesis and accomplish the objective of this application by pursuing the following three specific aims: Aim 1. Develop a new chemistry for producing TOM-thiophosphoramidites and a new protocol for synthesis of PS2-RNAs; Aim 2. Develop parallel synthesis processes for PS2-aptamers on a high density chip for high throughput optimization of the anti-CD30 RNA aptamer; Aim 3. Validate specific cell-binding of the optimized CD30 PS2-aptamer and the original RNA aptamer to cultured lymphoma cell lines in vitro. Our ultimate goal is to advance into clinical studies an optimized PS2- aptamer that has excellent affinity and specificity for the CD30 ALCL tumor biomarker. PUBLIC HEALTH RELEVANCE: Systemic Anaplastic Large Cell Lymphoma (ALCL) is a devastating cancer that affects children and is characterized by high levels of the CD30 protein on the cancer cell surface. AM Biotechnologies in collaboration with The Methodist Hospital Research Institute will develop a diagnostic tool for ALCL using a molecule that will bind specifically and tightly to the CD30 protein on the surface of the tumor cells. This molecule will be constructed from a short strand of nucleic acids called an aptamer. Aptamers serve as excellent detection tools because they are extremely sensitive and very specific to the target.

描述（由申请方提供）：系统性间变性大细胞淋巴瘤（ALCL）是儿童中最常见的T细胞淋巴瘤。ALCL的特征是其间变性细胞形态和细胞表面高水平表达的CD 30。ALCL细胞和肿瘤中的这种弥漫性和均匀性CD 30表达被认为是该疾病的诊断特征，抗CD 30抗体被广泛用作临床环境中的特异性诊断探针。开发抗体是耗时且昂贵的，并且由于使用不同的动物或培养细胞而导致的批次间差异是常见的。为了克服这些限制，已经研究了化学合成的针对CD 30的特异性探针。最近的一项研究报道，合成的RNA适体可以在溶液中以高亲和力特异性结合CD 30分子。通过使用培养的淋巴瘤细胞，我们的初步工作表明，这种RNA适体也通过其表面CD 30特异性结合人类细胞，这表明该适体可用作检测ALCL细胞用于疾病诊断的特异性探针。由于RNA在常规临床实验室测试条件下使用时是一种易于降解的分子，因此开发稳定的RNA适体探针对于临床应用至关重要。此外，RNA适体作为ALCL诊断工具的有用性将通过改善适体对其靶标的灵敏度/亲和力和特异性而进一步增强。作为概念的初步证明，其可以扩展到用于临床使用的商业化诊断产品;我们建议开发一种新的RNA试剂构建块，其可以用于改进RNA适体。我们的中心假设是RNA适体骨架的磷酸盐在其与其靶生物标志物分子CD 30的相互作用中起关键作用。在几个连接位置用硫取代两个非桥接氧将产生二硫代磷酸化（PS2）RNA适体（PS2-适体），其将增加RNA适体的稳定性、细胞结合亲和力和特异性。我们计划通过追求以下三个具体目标来测试我们的中心假设并实现本申请的目标：目标1。开发生产TOM-硫代亚磷酰胺的新化学方法和合成PS2-RNA的新方案;目标2。在高密度芯片上开发PS2-适体的平行合成方法，用于抗CD 30 RNA适体的高通量优化;目标3。优化的CD 30 PS2-适体和原始RNA适体与体外培养的淋巴瘤细胞系的细胞特异性结合。我们的最终目标是将优化的PS2适体推进临床研究，该适体对CD 30 ALCL肿瘤生物标志物具有优异的亲和力和特异性。公共卫生关系：系统性间变性大细胞淋巴瘤（ALCL）是一种影响儿童的毁灭性癌症，其特征是癌细胞表面高水平的CD 30蛋白。AM生物技术公司与卫理公会医院研究所合作，将开发一种ALCL的诊断工具，使用一种分子，该分子将特异性地与肿瘤细胞表面的CD 30蛋白紧密结合。该分子将由称为适体的短链核酸构建。适体作为优秀的检测工具，因为它们是非常敏感和非常具体的目标。