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Development and Utilization of Splice-specific Antibodies

剪接特异性抗体的开发和利用

基本信息

批准号：
9795724
负责人：
Rachel O'Neill
金额：
$ 21.07万
依托单位：
UNIVERSITY OF CONNECTICUT STORRS
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9795724
关键词：
Affinity Alternative Splicing Amino Acids Anti-Idiotypic Antibodies Antibodies Antigens Apoptosis Appearance Area Bacteriophages Biological Blood Vessels Brain Cell Cycle Progression Chimeric Proteins Code Detection Development Diagnostic Directed Molecular Evolution Enzyme Inhibitor Drugs Enzyme-Linked Immunosorbent Assay Epitopes Event Exons Flow Cytometry Generations Genes Human Human Genome Immune response Immunofluorescence Immunologic Immunoprecipitation Individual Legal patent Length Libraries Malignant Neoplasms Mass Spectrum Analysis Methods Modeling Neoplastic Processes Nucleotides Oncogenic Outcome Pathway interactions Peptides Phage Display Phenotype Physiological Processes Play Property Protein Isoforms Proteins Proteome RNA Splicing Reagent Regulation Research Risk Role Sampling Scientist Serine Side Site Specificity Structural Protein Structure Supporting Cell Survival Rate Technology Therapeutic Transcript Validation Variant Western Blotting angiogenesis anti-cancer anticancer research base cancer biomarkers cancer type centromere protein A cross reactivity design improved in vivo inhibitor/antagonist innovation inorganic phosphate neurogenesis new technology novel novel strategies off-patent oncology overexpression prognostic protein complex protein function protein protein interaction protein structure technique development technology development tumor tumorigenesis

项目摘要

ABSTRACT In the human genome, alternative splicing events facilitate the generation of a proteome with a greater diversity than is observed in the protein-coding gene repertoire. In other words, the vast majority of human genes can each produce numerous different transcripts and subsequently multiple protein isoforms. However, aberrant splicing can produce an increase in novel isoforms or normally low-level isoforms leading to potentially detrimental effects including altered protein function, protein- protein interactions as well as remodeling of protein complexes and pathways. In fact, the increase in these alternatively-spliced isoforms is now recognized as a major contributor to oncogenic phenotypes, such as the development of tumors and new blood vessels by supporting cell invasion and proliferation. A central challenge to realizing the biological impact, prognostic and therapeutic potential of alternatively-spliced isoforms across multiple cancer types has been a lack of technology available for unambiguously differentiating highly similar protein variants in their native forms. Current methods of detecting and studying alternatively-spliced isoforms at the protein level rely solely on indirect methods such as tag-based detection and nonspecific affinity reagents that are unable to discriminate among multiple protein isoforms. Herein, we propose a novel approach to develop highly specific antibodies against alternatively-spliced protein isoforms using a targeting method at the splice site junction with near amino acid specificity. With this technology, antibodies can be generated to specifically detect a desired, alternatively-spliced protein isoform without cross-reactivity to the native full-length form. This technological advance will accelerate oncology research and enable scientists to study both high-value and novel protein isoforms and their direct role in specific cancer phenotypes, metastatic potential, tumor grade specificity and survival rates, and provide a validation means for assessing the therapeutic potential of anticancer alternative-splicing inhibitors.

摘要在人类基因组中，选择性剪接事件促进了蛋白质组的产生，比在蛋白质编码基因库中观察到的更大的多样性。换句话说，大多数人类基因可以各自产生许多不同的转录本，蛋白质异构体。然而，异常剪接可以产生新的同种型的增加，或者正常情况下，低水平的异构体导致潜在的有害影响，包括改变蛋白质功能，蛋白质- 蛋白质相互作用以及蛋白质复合物和途径的重塑。事实上，在这些选择性剪接的亚型中，现在被认为是致癌的主要贡献者。表型，例如通过支持细胞侵袭而形成肿瘤和新血管和扩散。实现生物学影响、预后和治疗的核心挑战是由于缺乏技术，多种癌症类型的选择性拼接亚型的潜力一直存在可用于明确区分其天然形式的高度相似的蛋白质变体。电流在蛋白质水平上检测和研究选择性剪接异构体的方法仅依赖于间接方法，例如基于标签的检测和非特异性亲和试剂，区分多种蛋白质异构体。在这里，我们提出了一种新的方法来开发高度使用靶向方法，具有近氨基酸特异性剪接位点连接。通过这项技术，可以产生抗体为了特异性地检测所需的、选择性剪接的蛋白质同种型，而不与天然蛋白质同种型发生交叉反应，全长形式。这一技术进步将加速肿瘤学研究并使科学家能够研究高价值和新型蛋白质亚型及其在特定癌症中的直接作用表型、转移潜能、肿瘤分级特异性和存活率，并提供验证用于评估抗癌选择性剪接抑制剂的治疗潜力的方法。