Functional assay development for tafazzin enzyme replacement candidate selection

用于 tafazzin 酶替代候选选择的功能测定开发

基本信息

批准号：
10722856
负责人：
MICHAEL T CHIN
金额：
$ 62.3万
依托单位：
TUFTS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-15 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10722856
关键词：
3-Methylglutaconic aciduria type 2 Acyltransferase Affect Algorithms Antibody Formation Autopsy Biochemical Biological Biological Assay Cardiolipins Cardiomyopathies Cell membrane Cells Cessation of life Child Childhood Congestive Heart Failure Cultured Cells Cyclic Neutropenia Defect Development Disease Dose Drug Kinetics Drug or chemical Tissue Distribution Endosomes Endotoxins Enzyme Tests Enzyme-Linked Immunosorbent Assay Enzymes Evaluation Freedom Functional disorder Genes Genetic Diseases Heart failure Hereditary Disease Human In Vitro Intravenous Knockout Mice Label Left Ventricular Function Life Link Lipids Live Birth Longitudinal Studies Measurement Measures Mitochondria Modeling Monitor Morphology Mus Muscle Muscle hypotonia Mutation Organ Oxygen Consumption Patients Penetration Peptides Pharmacology Phase Physiological Plasma Population Primary idiopathic dilated cardiomyopathy Production Proteins Protocols documentation Quality Control Reactive Oxygen Species Reagent Recombinants Research Project Grants Respiration Serum Standardization Therapeutic Tissues Toxic effect Toxicology assay development candidate identification candidate selection clinical development cohort commercialization detection assay effective therapy effectiveness measure efficacious treatment efficacy evaluation efficacy study enzyme activity enzyme deficiency enzyme replacement therapy immunogenicity in vitro activity in vivo in vivo evaluation infancy intraperitoneal lead candidate manufacture manufacturing scale-up middle age mitochondrial dysfunction mitochondrial membrane mouse model peptide drug premature process evaluation process optimization therapeutic enzyme uptake

项目摘要

Abstract: Functional assay development for tafazzin enzyme replacement candidate selection In the proposed research project, we will perform key biochemical, cellular and physiological studies to evaluate the potential of various modified recombinant tafazzin enzyme therapeutics to become the first effective treatment for Barth Syndrome, a rare, life-threatening disorder with no efficacious therapy. Barth Syndrome is a rare X-linked disorder affecting 1:300,000 live births, resulting from defects in the gene encoding Tafazzin, an acyltransferase that modifies cardiolipin to the tetralinoleoyl form and is essential for mitochondrial respiration. Patients with Barth Syndrome develop cardiomyopathy, muscular hypotonia and cyclic neutropenia during childhood, rarely surviving to middle age. At present, no effective therapy exists for these patients. We are developing potential enzyme replacement therapeutics in which recombinant tafazzin is modified to contain a cellular penetrating peptide that promotes uptake into tafazzin-deficient cells, sometimes in conjunction with an endosomal escape peptide to facilitate escape from lysosomal degradation. We have found that these recombinant tafazzin enzyme replacement therapeutics (rTERTs) can enter tafazzin-deficient cells, localize to mitochondria and correct both cardiolipin remodeling and mitochondrial respiration defects, in vitro and in vivo to varying degrees. To facilitate the commercialization of these reagents, we will develop standardized biochemical, cellular and physiological assays to directly compare enzymatic function, cellular uptake, rescue of cardiolipin remodeling defects, and rescue of defective mitochondrial respiration in cultured cells. Based on these studies, we have developed a selection algorithm to identify a cohort of potential lead candidates that demonstrate the greatest biochemical and biological activity in vitro. The top candidates identified by this algorithm will undergo production process optimization and evaluation for activity in vivo. The in vivo evaluation will consist of pharmacokinetics, tissue distribution, toxicology and efficacy studies in tafazzin knockout mice using an in house tafazzin LC-MS peptide detection assay, an in house ELISA assay to measure antibody formation, in conjunction with measurements of tafazzin function and left ventricular function. We believe that our studies will establish standardized assays to characterize potential enzyme replacement therapies for Barth Syndrome in the R61 phase, thereby facilitating evaluation of potential lead candidates in a mouse model of Barth Syndrome in the R33 phase, ultimately leading to the identification of a lead candidate for further clinical development.

摘要：用于 tafazzin 酶替代候选选择的功能测定开发在拟议的研究项目中，我们将进行关键的生化、细胞和生理学研究评估各种改良重组tafazzin酶疗法成为第一个的潜力巴斯综合症是一种罕见的危及生命的疾病，目前尚无有效的治疗方法。巴特综合症是一种罕见的 X 连锁疾病，影响 1:300,000 活产，由基因缺陷引起编码 Tafazzin，一种酰基转移酶，可将心磷脂修饰为四亚油酰基形式，对于线粒体呼吸。巴特综合征患者会出现心肌病、肌张力低下和儿童时期出现周期性中性粒细胞减少症，很少能存活到中年。目前尚无有效疗法这些病人。我们正在开发潜在的酶替代疗法，其中重组 tafazzin 是修饰后含有细胞穿透肽，有时可促进缺乏 tafazzin 的细胞的摄取与内体逃逸肽结合以促进逃逸溶酶体降解。我们有发现这些重组tafazzin酶替代疗法（rTERT）可以进入tafazzin缺陷的患者细胞，定位于线粒体并纠正心磷脂重塑和线粒体呼吸缺陷，体外和体内都有不同程度的影响。为了促进这些试剂的商业化，我们将开发标准化的生化、细胞和生理测定，可直接比较酶功能、细胞培养物中心磷脂重塑缺陷的摄取、挽救以及线粒体呼吸缺陷的挽救细胞。基于这些研究，我们开发了一种选择算法来识别一组潜在的领导者在体外表现出最大生化和生物活性的候选者。最佳候选人通过该算法鉴定的产品将进行生产工艺优化和体内活性评估。这体内评价将包括tafazzin的药代动力学、组织分布、毒理学和功效研究使用内部 tafazzin LC-MS 肽检测分析（一种内部 ELISA 分析）对敲除小鼠进行结合 tafazzin 功能和左心室的测量来测量抗体形成功能。我们相信，我们的研究将建立标准化测定法来表征潜在的酶 R61 期 Barth 综合征的替代疗法，从而促进潜在先导药物的评估在 R33 期的巴斯综合征小鼠模型中进行候选研究，最终鉴定出进一步临床开发的主要候选者。