Optimize RNA-aptamer for Biomarker Molecule CD30

描述（由申请人提供）：全身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的特点是细胞形态间变性和细胞表面CD30的高表达。CD30在ALCL细胞和肿瘤中的弥漫性和均匀性表达被认为是该疾病的诊断特征，抗CD30抗体被广泛用作临床环境中的特异性诊断探针。开发一种抗体既耗时又昂贵，而且由于使用不同的动物或培养细胞而导致批次之间的差异很常见。为了克服这些限制，研究了一种化学合成的CD30特异性探针。最近的一项研究报道，合成的RNA适体可以高亲和力地特异性结合溶液中的CD30分子。通过培养的淋巴瘤细胞，我们的初步工作表明，该RNA适体也可以通过人类细胞表面CD30特异性结合，这表明该适体可以作为检测ALCL细胞的特异性探针用于疾病诊断。由于RNA在常规临床实验室检测条件下是一种容易降解的分子；开发稳定的RNA适体探针对临床应用至关重要。此外，RNA适体作为ALCL诊断工具的用途将通过提高适体对其靶标的敏感性/亲和力和特异性进一步增强。作为概念的初步证明，可扩展为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，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目标生物标志物分子CD30的相互作用中起着关键作用。用硫取代两个非桥接氧在几个连接位置将导致磷酸化二硫化（PS2） RNA适体（PS2-适体），这将增加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检验我们的中心假设并实现本应用程序的目标：开发一种新的合成tom -硫代磷酰胺的化学方法和一种新的合成ps2 - rna的方法；目标2。开发高密度芯片上ps2适配体的平行合成工艺，以实现抗cd30 RNA适配体的高通量优化；目标3。验证优化后的CD30 ps2适体和原始RNA适体在体外培养淋巴瘤细胞系上的特异性细胞结合。我们的最终目标是将一种优化的PS2-适体推进临床研究，该适体对CD30 ALCL肿瘤生物标志物具有出色的亲和力和特异性。公共卫生相关性：全身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CD30蛋白水平高。AM生物技术公司将与卫理公会医院研究所合作开发一种ALCL的诊断工具，该工具使用一种分子，该分子将特异性地与肿瘤细胞表面的CD30蛋白紧密结合。这种分子将由一种叫做适体的核酸短链构成。适体是一种很好的检测工具，因为它们对目标非常敏感和特异性。

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.