In vivo SELEX strategies to identify potent aptamer-drug conjugates for glioblastoma

项目说明/摘要 尽管几十年来，胶质母细胞瘤(GBM)的有效治疗方法的开发仍然是一个重大挑战 密集的研究。再加上肿瘤内的异质性和可塑性，正常脑组织的浸润 由基底膜细胞引起的治疗挑战独一无二。此外，形成脑血管的专门神经血管单位 血脑屏障(BBB)在GBM中部分完整，并导致异质性、亚治疗性的 大多数细胞毒性化疗针对每个基底膜的区域。我们之前已经证明， 否则，高度有效的抗体-药物结合物在GBM中受到限制，因为跨 BBB。像抗体一样，单链DNA适配子通过表位结合折叠成独特的三维形状 与抗体和一些适配子的亲和力相媲美的亲和力也能有效地穿过血脑屏障。与之相对的是 基于抗体的治疗、固体支持合成的集成、聚合酶链式反应的艰苦发展 扩增和下一代测序技术使大规模并行筛选策略成为可能， 被称为指数富集法的配体的系统进化(SELEX)，以识别个体DNA 通过连续几轮阴性和/或阳性的具有所需物理和生物特征的适配子 选择。根据这一策略的先前经验，我们假设肿瘤特异性dna适配子药物 利用体内SELEX可以有效地识别为跨血脑屏障分布而优化的偶联物(ApDC) 通过适体-药物结合物和原位GBM患者来源的异种移植物的文库。解决肿瘤问题 异质性、体内选择将跨多个PDX执行，以及单细胞测序技术 将被用来识别与不同的肿瘤细胞亚群结合的ApDC，而不是 大脑。此应用程序的目标是开发一个强大的平台，用于高效筛选特定于GBM的 APDC。这将通过解决三个目标来实现。 目标1-R61：优化适体毒素文库的设计和测序 我们将通过多轮SELEX优化文库加工过程中毒素结合的策略。 将我们展示MMAE毒素稳定性的初步数据扩展到PCR热循环，我们将优化 用于文库制备和单细胞RNA/适配子序列的毒素连接的聚合酶链式反应 目标2-R61：确定识别脑肿瘤特异性ApDC的最佳训练轮策略 我们将优化DNA文库注射和组织收集之间的时间。我们还将评估一部小说 SELEX奖励策略，基于仅在从APDC切割MMAE后选择性地捕获适配子。 目的3-R33：应用在体SELEX和原位GBM PDX来训练APDC文库 体内的SELEX将与APDC文库一起跨越一组不同的原位GBM PDX来执行 了解靶向异质肿瘤和保留正常细胞群体的潜力。

PROJECT DESCRIPTION/ABSTRACT Development of effective therapies for glioblastoma (GBM) remains a major challenge despite decades of intensive research. Coupled with intra-tumoral heterogeneity and plasticity, the infiltration of normal brain tissue by GBM cells poses unique therapeutic challenges. Further, the specialized neurovascular unit that forms the blood-brain barrier (BBB) is partially intact in GBM and results in heterogeneous, sub-therapeutic delivery of most cytotoxic chemotherapies to regions of every GBM. We have previously shown that the efficacy of otherwise highly potent antibody-drug conjugates is specifically limited in GBM by poor delivery across the BBB. Like antibodies, single-strand DNA aptamers fold into unique 3-dimensional shapes with epitope binding affinities that rival those of antibodies, and some aptamers also efficiently traverse the BBB. In contrast to the laborious development of antibody-based therapeutics, the integration of solid-support synthesis, PCR amplification, and next-generation sequencing technologies enable massively parallel screening strategies, known as ‘systematic evolution of ligands by exponential enrichment (SELEX)’, to identify individual DNA aptamers with desired physical and biological features through successive rounds of negative and/or positive selection. Based on prior experience with this strategy, we hypothesize that tumor-specific DNA aptamer-drug conjugates (ApDCs) optimized for distribution across the BBB can be efficiently identified using in vivo SELEX with libraries of aptamer-drug conjugates and orthotopic GBM patient-derived xenografts. To address tumor heterogeneity, in vivo selection will be performed across multiple PDXs, and single cell sequencing technology will be leveraged to identify ApDCs that bind to diverse subsets of tumor cells and not normal cells within the brain. The goal of this application is to develop a robust platform for efficient screening of GBM-specific ApDCs. This will be accomplished by addressing three Aims. Aim 1 – R61: Optimize design and sequencing of aptamer-toxin libraries We will optimize strategies for toxin conjugation during library processing through multiple SELEX rounds. Extending our preliminary data demonstrating MMAE toxin stability to PCR thermal cycling, we will optimize toxin-conjugated PCR primers for library preparation and single-cell RNA/aptamer-seq. Aim 2 – R61: Determine optimum training round strategy to identify brain tumor-specific ApDCs We will optimize the time between DNA library injection and tissue collection. We also will evaluate a novel SELEX reward strategy based on selectively capturing aptamers only after cleavage of MMAE from an ApDC. Aim 3 – R33: Apply in vivo SELEX with orthotopic GBM PDXs to train ApDC libraries In vivo SELEX will be performed with an ApDC library across a heterogenous set of orthotopic GBM PDXs to understand the potential for targeting heterogeneous tumor and sparing normal cell populations.