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 胶质母细胞瘤（GBM）的有效疗法的开发仍然是一个重大挑战，尽管几十年来， 深入研究。再加上肿瘤内的异质性和可塑性，正常脑组织的浸润 对治疗提出了独特的挑战。此外，形成血管的专门神经血管单位 血脑屏障（BBB）在GBM中是部分完整的，并导致异质的亚治疗递送。 大多数细胞毒性化疗都是针对每一个GBM的区域。我们之前已经证明， 否则，高效的抗体-药物缀合物在GBM中由于穿过GBM的差递送而受到特别限制。 BBB.与抗体一样，单链DNA适体折叠成独特的三维形状， 适体具有与抗体相匹敌的亲和力，并且一些适体还有效地穿过BBB。相对于 基于抗体的治疗方法的艰苦开发，固相支持合成的整合，PCR 扩增，和下一代测序技术使大规模平行筛选策略， 被称为“指数富集配体系统进化（SELEX）”，以识别单个DNA 通过连续轮的阴性和/或阳性检测， 选择.基于这种策略的先前经验，我们假设肿瘤特异性DNA适体-药物 可以使用体内SELEX有效地鉴定针对跨越BBB分布而优化的缀合物（ApDC 用适体-药物缀合物和原位GBM患者来源的异种移植物的文库。治疗肿瘤 异质性，将在多个PDX和单细胞测序技术中进行体内选择 将被用来鉴定与肿瘤细胞的不同亚群而不是正常细胞结合的ApDC。 个脑袋本申请的目标是开发一个强大的平台，用于有效筛选GBM特异性 ApDC。这将通过实现三个目标来实现。 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优化文库处理过程中的毒素缀合策略。 将我们的初步数据扩展到证明MMAE毒素对PCR热循环的稳定性，我们将优化 用于文库制备的毒素缀合的PCR引物和单细胞RNA/适体-seq. 目的2 - R61：确定最佳训练轮策略以鉴定脑肿瘤特异性ApDC 我们将优化DNA文库注射和组织收集之间的时间。我们也会评估一本小说 基于仅在从ApDC切割MMAE后选择性捕获适体的SELEX奖励策略。 目的3 - R33：应用具有原位GBM PDX的体内SELEX来训练ApDC文库 体内SELEX将用ApDC文库跨一组异源的原位GBM PDX进行，以 了解靶向异质性肿瘤和保留正常细胞群体的潜力。