Functional assay development for tafazzin enzyme replacement candidate selection

文摘：他发津酶替代物候选筛选功能分析方法的研究进展 在拟议的研究项目中，我们将进行关键的生化、细胞和生理研究，以 评价各种改良型重组他法津酶疗法成为首个治疗药物的潜力 有效治疗Barth综合征，这是一种罕见的、危及生命的疾病，但没有有效的治疗方法。巴思 综合征是一种罕见的X连锁疾病，影响1：30万活产婴儿，由基因缺陷引起 编码Tafazzin的一种酰基转移酶，将心磷脂修饰为四氢呋喃油酰形式，是 线粒体呼吸作用。Barth综合征患者出现心肌病、肌肉低张症和 儿童时期的周期性中性粒细胞减少症，很少能存活到中年。目前，还没有有效的治疗方法。 这些病人。我们正在开发潜在的酶替代疗法，其中重组他法津是 经修饰后含有一种细胞穿透肽，可促进对他法津缺陷细胞的摄取，有时 与内体逃逸多肽相结合，以帮助逃脱溶酶体降解。我们有 发现这些重组他法津酶替代疗法(RTERT)可以进入他法津缺陷型 细胞，定位于线粒体，纠正心磷脂重塑和线粒体呼吸缺陷，在 体外和体内都有不同程度的变化。为了促进这些试剂的商业化，我们将开发 直接比较酶功能的标准化生化、细胞和生理分析 培养细胞对心磷脂重塑缺陷的摄取、修复和线粒体呼吸缺陷的修复 细胞。在这些研究的基础上，我们开发了一种选择算法来识别一组潜在的铅 在体外表现出最大的生化和生物活性的候选人。排名第一的候选人 经该算法鉴定后，将进行生产工艺优化和体内活性评价。这个 体内评价将包括他法津的药代动力学、组织分布、毒理学和疗效研究。 用室内Tafazzin LC-MS多肽检测方法敲除小鼠，室内ELISA法检测 测量抗体形成，同时测量他法津功能和左心功能 功能。我们相信，我们的研究将建立标准化的分析方法来表征潜在的酶 R61期Barth综合征的替代疗法，从而促进对潜在铅的评估 R33期Barth综合征小鼠模型的候选者，最终导致识别出 未来临床开发的主要候选者。

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.