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受体（receptor for advanced glycation end products，ERK）的细胞信号传导导致促炎反应。RAGE介导的炎症与包括糖尿病、动脉粥样硬化和阿尔茨海默病在内的炎性疾病有关。剪接或蛋白水解切割形式的β-淀粉样蛋白被称为可溶性β-淀粉样蛋白（sNAs），其作为天然诱饵反作用β-淀粉样蛋白信号传导。在动物模型中已经证明，施用甘油可阻断动脉粥样硬化形成，并稳定血管壁上现有的斑块。此外，血管内皮还可以防止血管损伤引起的新生内膜的形成，从而抑制再狭窄。 我们已经开发了稳定表达sHBVDNA的中国卵巢癌（CHO）细胞系，以及产生同源sHBVDNA的亲和纯化策略。系统地研究了支架在再狭窄动物模型中的应用，并对数据进行了分析。我们的研究结果表明，在我们的实验室生产的sodium表现出1000倍以上的效力比以前报道的。除了阻断再狭窄外，我们还在梗死动物模型上测试了血管阻断，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soblastin的分子基础，并发现soblastin中的N-聚糖结构有助于其生物活性。 为了进一步开发作为有效的治疗产品的S7-S7，我们使用来自Invitrogen的GeneOptimizer算法来优化T7-S7-该工具去除cDNA序列中的序列重复、杀伤基序、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scDNA是否具有更高的表达水平，并与天然序列进行比较。这一步骤应能促进今后的小规模生产。 为了克服表达和检测sHBVDNA的技术障碍，我们还开发了一套表达模块，便于亚克隆，细胞表面表达。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：临床前体外。