Soluble Receptor for Advanced Glycation End Products for Therapeutic Application

用于治疗应用的高级糖基化终产物的可溶性受体

• 批准号: 8552494
• 负责人: Edward Lakatta
• 金额: $ 12.3万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552494
• 关键词: Advanced Glycosylation End Products; Advertising; Affinity Chromatography; Algorithms; Alzheimer' s Disease; Amino Acid Sequence; Animal Model; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Atherosclerosis; Award; Basic Science; Biochemical; Blood Vessels; Cardiovascular Diseases; Cell Line; Cell surface; Chemistry; Chinese Hamster Ovary Cell; Cleaved cell; Clinic; Clinical Trials; Codon Nucleotides; Complementary DNA; Data; Detection; Development; Diabetes Mellitus; Disease; Dose; Drug Delivery Systems; Epitopes; Exhibits; Future; Genetic Engineering; Goals; Guanine + Cytosine Composition; In Vitro; Infarction; Inflammation; Inflammatory; Inflammatory Response; Injury; Laboratories; Legal patent; Manuscripts; Mediating; Membrane Proteins; Molecular; Molecular Biology; Peptide Sequence Determination; Pharmaceutical Preparations; Physiology; Polysaccharides; Post-Translational Protein Processing; Process; Production; Property; RNA Splice Sites; RNA Splicing; Reporting; Signal Transduction; Staging; Structure; System; Testing; Therapeutic; United States National Institutes of Health; Vascular Diseases; Vascular Endothelial Cell; atherogenesis; base; clinical application; combat; experience; in vivo; monocyte; nanoparticle; neointima formation; particle; pre-clinical; prevent; receptor; research and development; restenosis; scale up; tool

Cellular signaling via receptor for advanced glycation end products (RAGE) results in pro-inflammatory responses. RAGE-mediated inflammation has been implicated in inflammatory diseases including diabetes, atherosclerosis, and Alzheimers disease. The spliced or proteolytically cleaved form of RAGE is referred as soluble RAGE (sRAGE), which functions as a natural decoy counter-effecting RAGE signaling. It has been demonstrated in animal models that administration of sRAGE blocks atherogenesis, and stabilizes existing plaques on the vessel wall. In addition, sRAGE also prevents the formation of neointima prompted by vascular injuries and hence inhibits restenosis. We have developed Chinese Hamster Ovary (CHO) cell lines that stably express sRAGE, and the accompanied affinity purification strategies that produce homogenous sRAGE. Systemic studies of sRAGE application in restenosis animal models have been completed, and data have been analyzed. Our results showed that sRAGE produced in our laboratory exhibits 1000 x higher potency than that of previously reported. In addition to blocking restenosis, we also tested sRAGE blockage on infarct animal models and obtained promising preliminary results. We also performed studies to explore the molecular basis of the observed high potency of sRAGE and found that N-glycan structure in sRAGE contributes to its bioactivity. To further develop sRAGE as an effective therapeutic product, we used GeneOptimizer algorithm from Invitrogen to optimize T7-sRAGE----this tool removes sequence repeat, killer motifs, splice sites and RNA secondary structures in the cDNA sequence and optimize codon usage (for CHO cell) and GC content without changing protein sequence. We plan to test whether the new sRAGE cDNA has a higher level of expression and compare to native sequence. This step should enhance future sRAGE scale-up production. To overcome technical hurdles for expression and detection of sRAGE, we also developed a set of expression modules that facilitate subcloning, cell-surface expression. and epitope tagging of mammalian membrane proteins. U.S. Provisional Patent (No. 61/142,531) has been awarded to this invention, and NIH is currently advertising the invention. R&D Status: Pre-clinical in vitro.

通过受体的细胞信号传导晚期糖基化最终产物（RAGE）导致促炎反应。愤怒介导的炎症与包括糖尿病，动脉粥样硬化和阿尔茨海默氏病在内的炎症性疾病有关。愤怒的拼接或蛋白水解裂片形式称为可溶性愤怒（SRAGE），该愤怒（Srage）是自然诱饵反应的愤怒信号传导。在动物模型中已经证明了SRAGE的给药可阻止动脉粥样硬化，并稳定了容器壁上现有的斑块。此外，Srage还防止了血管损伤引起的新内膜的形成，因此抑制再狭窄。 我们已经开发了稳定表达SRAGE的中国仓鼠卵巢（CHO）细胞系，并产生同质性的亲和力纯化策略。在再狭窄动物模型中应用SRAGE应用的系统性研究已经完成，并且已经分析了数据。我们的结果表明，在我们的实验室中产生的SRAGE表现出比先前报道的效力高1000 x。除了阻塞再狭窄外，我们还测试了梗死动物模型上的SRAGE阻塞，并获得了有希望的初步结果。我们还进行了研究，以探索观察到的高效力的分子基础，并发现Srage中的N-聚糖结构有助于其生物活性。 To further develop sRAGE as an effective therapeutic product, we used GeneOptimizer algorithm from Invitrogen to optimize T7-sRAGE----this tool removes sequence repeat, killer motifs, splice sites and RNA secondary structures in the cDNA sequence and optimize codon usage (for CHO cell) and GC content without changing protein sequence.我们计划测试新的Srage cDNA是否具有较高的表达水平，并与天然序列进行比较。此步骤应提高未来的SRAGE扩展生产。 为了克服用于表达和检测SRAGE的技术障碍，我们还开发了一组表达模块，可促进亚克隆，细胞表达。和哺乳动物膜蛋白的表位标记。美国临时专利（第61/142,531号）已授予本发明，NIH目前正在宣传本发明。研发状态：体外临床前。