COBRE P3: HUMANIZING ALGINATE DEPOLYMERASE

COBRE P3：人性化海藻酸盐解聚酶

• 批准号: 8167472
• 负责人: Karl E Griswold
• 金额: $ 26.53万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167472
• 关键词: Alginates; Biological Response Modifier Therapy; Biopolymers; Carbohydrates; Chronic; Clinical; Computer Retrieval of Information on Scientific Projects Database; Cystic Fibrosis; Engineering; Enzymes; Exhibits; Funding; Grant; Human; Human Genome; Hydrolase; Immune response; Infection; Institution; Muramidase; Patients; Polymers; Premature Mortality; Production; Pseudomonas aeruginosa; Relative (related person); Research; Research Personnel; Resources; Source; Therapeutic; United States National Institutes of Health; base; directed evolution; enzyme activity; immunogenicity; microbial; mucoid; poly(beta-D-mannuronate) lyase; therapeutic target; Alginates; Biological Response Modifier Therapy; Biopolymers; Carbohydrates; Chronic; Clinical; Computer Retrieval of Information on Scientific Projects Database; Cystic Fibrosis; Engineering; Enzymes; Exhibits; Funding; Grant; Human; Human Genome; Hydrolase; Immune response; Infection; Institution; Muramidase; Patients; Polymers; Premature Mortality; Production; Pseudomonas aeruginosa; Relative (related person); Research; Research Personnel; Resources; Source; Therapeutic; United States National Institutes of Health; base; directed evolution; enzyme activity; immunogenicity; microbial; mucoid; poly(beta-D-mannuronate) lyase; therapeutic target

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Premature mortality in cystic fibrosis typically results from chronic P. aeruginosa infection of the patient's airways. The mucoid strains of P. aeruginosa associated with cystic fibrosis are characterized by their production of the exopolysaccharide alginate, a known pathogenic factor. Alginate lyases are enzymes that degrade the alginate polymer, and have shown great promise for symptomatic treatment of cystic fibrosis. However, the microbial origin of these biocatalysts predisposes them towards high level immunogenicity, and brings into question their utility in a clinical setting. Unfortunately, as is the case with numerous microbial biotherapeutic candidates, alginate lyases have no exact human counterpart. However, the human genome encodes numerous enzymes with similar catalytic functions, i.e. degradation of carbohydrate-based biopolymers. The existence of human enzymes exhibiting similar catalytic functions but different substrate selectivities suggests that the alginate lyase therapeutic activity could be deimmunized by engineering a human carbohydrate hydrolase to act on the alginate substrate. Specifically, directed evolution of a human enzyme with altered substrate selectivity could yield a biocatalyst that efficiently degrades the alginate therapeutic target, but because of its human origin would be less likely to illicit a deleterious immune response in human patients. Directed evolution will be employed to alter the substrate selectivity of human lysozyme to generate a humanized alginate depolymerase. The activity of these enzymes will be compared to an authentic bacterial alginate lyase, and the immunogenicity of the engineered enzymes will be assessed relative to the wild type human lysozyme template.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 囊性纤维化的过早死亡率通常是由于患者气道的慢性铜绿假单胞菌感染引起的。与囊性纤维化相关的铜绿假单胞菌的粘液菌株的特征是它们的产生外多糖藻酸盐（一种已知的致病因子）。藻酸盐裂解酶是降解藻酸盐聚合物的酶，并显示出对囊性纤维化的症状治疗的巨大希望。但是，这些生物催化剂的微生物起源使它们倾向于高水平的免疫原性，并在临床环境中质疑它们的效用。不幸的是，就像许多微生物生物治疗候选者一样，藻酸盐裂解酶没有确切的人类对应物。但是，人类基因组编码具有相似催化功能的众多酶，即基于碳水化合物的生物聚合物的降解。人类酶的存在表现出相似的催化功能，但底物选择性不同，表明藻酸盐裂解酶治疗活性可以通过工程化的人类碳水化合物水解酶对藻酸盐底物作用来脱离。具体而言，人类酶具有改变底物选择性的定向演化可能会产生有效降解藻酸盐治疗靶标的生物催化剂，但由于其人类起源的可能性较小，在人类患者中不太可能具有有害的免疫反应。定向进化将用于改变人溶菌酶的底物选择性，以产生人源化的藻酸盐去聚合酶。这些酶的活性将与真实的细菌藻酸盐裂解酶进行比较，并将评估工程酶的免疫原性相对于野生型人类溶菌酶模板。