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

体内 SELEX 策略鉴定针对胶质母细胞瘤的有效适体-药物缀合物

• 批准号: 10721036
• 负责人: LOUIS JAMES MAHER
• 金额: $ 41.47万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-21 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10721036
• 关键词: 3-Dimensional; Address; Adult; Affinity; Antibodies; Antibody Diversity; Antibody Therapy; Antibody-drug conjugates; Binding; Biological; Blood - brain barrier anatomy; Brain; Brain Neoplasms; Cells; Central Nervous System; Characteristics; Chemicals; Coupled; Cultured Cells; Cytotoxic Chemotherapy; DNA; DNA Folding; DNA Library; Data; Development; Dose; Drug Kinetics; Epitopes; Event; Evolution; Excision; FDA approved; Genetic Recombination; Glioblastoma; Goals; Heterogeneity; In Vitro; Individual; Infiltration; Injections; Laboratories; Libraries; Ligands; Malignant Neoplasms; Methods; Modification; Morbidity - disease rate; National Institute of Neurological Disorders and Stroke; Neurologic; Normal Cell; Normal tissue morphology; Operative Surgical Procedures; Organ; Patients; Pharmaceutical Preparations; Phase; Population; Population Heterogeneity; Preparation; Primary Brain Neoplasms; Process; Proteins; Protocols documentation; RNA; Radiation Dose Unit; Reproducibility; Research; Resected; Rewards; Risk; Shapes; Single-Stranded DNA; Solid; Specificity; Targeted Toxins; Technology; Therapeutic; Therapeutic antibodies; Time; Tissue Banks; Tissues; Toxin; Toxin Conjugates; Training; Treatment Efficacy; Vertebrates; Vision; Work; aptamer; brain tissue; clinical efficacy; cytotoxicity; design; effective therapy; experience; in vivo; innovation; macromolecule; neoplastic cell; neurovascular unit; next generation sequencing; novel; novel strategies; patient derived xenograft model; precision medicine; process optimization; programs; public health relevance; screening; single cell sequencing; targeted agent; treatment strategy; tumor; tumor DNA; tumor heterogeneity

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.

项目描述/摘要 尽管几十年来，开发胶质母细胞瘤（GBM）的有效疗法仍然是一个重大挑战 深入研究。再加上瘤内的异质性和可塑性，正常脑组织的浸润 GBM 细胞带来的独特的治疗挑战。此外，形成特殊的神经血管单元 GBM 中的血脑屏障 (BBB) 部分完整，导致异质性、亚治疗性递送 大多数细胞毒性化疗针对每个 GBM 区域。我们之前已经证明了 否则，高效的抗体-药物偶联物在 GBM 中的递送效果受到了特别限制。 BBB。与抗体一样，单链 DNA 适体通过表位结合折叠成独特的 3 维形状 与抗体的亲和力相媲美，一些适体还可以有效地穿过血脑屏障。相比之下 基于抗体的疗法的艰苦开发、固相支持物合成、PCR 的整合 扩增和下一代测序技术实现大规模并行筛选策略， 称为“指数富集配体系统进化 (SELEX)”，用于识别个体 DNA 通过连续几轮的阴性和/或阳性，适配体具有所需的物理和生物学特征 选择。根据该策略的先前经验，我们假设肿瘤特异性 DNA 适体药物 使用体内 SELEX 可以有效地识别针对 BBB 分布进行优化的缀合物 (ApDC) 具有适体-药物缀合物和原位 GBM 患者来源的异种移植物库。针对肿瘤 异质性、体内选择将在多个 PDX 和单细胞测序技术中进行 将被用来识别与不同肿瘤细胞亚群结合的 ApDC，而不是与肿瘤细胞内的正常细胞结合的 ApDC。 脑。该应用程序的目标是开发一个强大的平台，用于有效筛选 GBM 特异性 ApDC。这将通过解决三个目标来实现。 目标 1 – R61：优化适体毒素库的设计和测序 我们将通过多轮 SELEX 优化文库处理过程中的毒素结合策略。 将我们证明 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/适体测序的毒素偶联 PCR 引物。 目标 2 – R61：确定最佳训练策略以识别脑肿瘤特异性 ApDC 我们将优化 DNA 文库注射和组织采集之间的时间。我们还将评估一本小说 SELEX 奖励策略基于仅在从 ApDC 裂解 MMAE 后选择性捕获适体。 目标 3 – R33：应用体内 SELEX 与原位 GBM PDX 来训练 ApDC 库 体内 SELEX 将使用 ApDC 文库在一组异质的原位 GBM PDX 上进行，以 了解靶向异质肿瘤并保护正常细胞群的潜力。