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.

通过晚期糖基化终末产物 (RAGE) 受体的细胞信号传导导致促炎症反应。 RAGE 介导的炎症与糖尿病、动脉粥样硬化和阿尔茨海默病等炎症性疾病有关。 RAGE 的剪接或蛋白水解形式被称为可溶性 RAGE (sRAGE)，其功能是对抗 RAGE 信号传导的天然诱饵。动物模型已证明，给予 sRAGE 可阻止动脉粥样硬化形成，并稳定血管壁上现有的斑块。此外，sRAGE 还可以防止血管损伤引起的新内膜形成，从而抑制再狭窄。 我们开发了稳定表达 sRAGE 的中国仓鼠卵巢 (CHO) 细胞系，以及产生同质 sRAGE 的亲和纯化策略。 sRAGE在再狭窄动物模型中应用的系统研究已经完成，并对数据进行了分析。我们的结果表明，我们实验室生产的 sRAGE 的效力比之前报道的高出 1000 倍。除了阻断再狭窄之外，我们还在梗塞动物模型上测试了 sRAGE 阻断作用，并获得了有希望的初步结果。我们还进行了研究来探索所观察到的 sRAGE 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。 为了进一步将 sRAGE 开发为有效的治疗产品，我们使用 Invitrogen 的 GeneOptimizer 算法来优化 T7-sRAGE——该工具去除 cDNA 序列中的序列重复、杀手基序、剪接位点和 RNA 二级结构，并优化密码子使用（对于 CHO 细胞）和 GC 含量，而不改变蛋白质序列。我们计划测试新的 sRAGE cDNA 是否具有更高的表达水平，并与天然序列进行比较。这一步骤将促进未来 sRAGE 的规模化生产。 为了克服 sRAGE 表达和检测的技术障碍，我们还开发了一套促进亚克隆、细胞表面表达的表达模块。和哺乳动物膜蛋白的表位标记。这项发明已获得美国临时专利（No. 61/142,531），NIH 目前正在对这项发明进行广告宣传。研发状态：体外临床前。