IgA肾病是常见的原发性肾小球肾炎，异常糖基化IgA1及其免疫复合物在肾系膜区的沉积是IgAN关键病理机制，目前尚缺乏针对该发病机制环节的干预方法。新近报道IgA蛋白酶可特异降解人正常IgA1免疫复合物，但未证明对异常糖基化IgA1的作用，且缺乏靶向性。我们前期研究发现，病原菌来源IgA蛋白酶可高效降解异常糖基化IgA1及其免疫复合物。本项目提出应用脂质体包装IgA蛋白酶，靶向降解系膜区异常糖基化IgA1免疫复合物的干预方法。拟对流感嗜血杆菌来源IgA蛋白酶进行PEG化阳离子脂质体包装，利用脂质体的粒径、电荷和肾小球滤过膜孔径、电荷性质，调控IgA蛋白酶脂质体的肾系膜靶向传递，特异清除IgAN小鼠肾小球免疫复合物。本研究将为IgA蛋白酶应用于IgAN临床治疗提供科学依据。

IgA nephropathy (IgAN) is the most common primary glomerulonephritis. The mesangial deposition of aberrantly glycosylated IgA1 and its immune complex is a key pathogenic mechanism for IgAN. However, there is yet no effective treatment for IgAN by targeting this process. A current study suggested that IgA protease can specifically degrade normal IgA1 and its immune complex. However, no evidence has been shown that IgA protease can also degrade aberrantly glycosylated IgA1. Moreover, there is also lack of the kidney-specific IgA protease delivery system, which may cause systemic immune response and physiological toxicity, therefore preventing the use of IgA protease clinically. Our preliminary studies found that the bacteria-derived IgA protease is also able to efficiently degrade aberrantly glycosylated IgA1 immunoglobin and its immune complex, providing the experiment proofs for future clinic treatment of IgAN by IgA protease. In this proposal, we aims to develop a targeted therapy for IgAN by specifically degrading mesangial IgA1-immune complexes with cationic liposome-encapsulated IgA protease. We plan to first encapsulate the IgA protease that is engineered from the H. influenzae, with PEGlylated cationic liposome and test the hypothesis that PEGlyated cationic liposome-formulated IgA protease can size- and charge-selectively localize to the mesangial area and specifically degrade the deposited IgA1 and its immune complexes locally in passive mouse model of IgAN. We expect that PEG-cationic liposome-IgA protease can size- and charge-selectively localize to the mesangial area to specifically degrade the deposited IgA1 and its immune complexes locally. Thus, the outcomes from this study are highly significant both scientifically and clinically.

IgA肾病是常见的原发性肾小球肾炎疾病，异常糖基化IgA1及其免疫复合物在肾系膜区的沉积是IgAN关键病理机制，目前尚缺乏针对该发病机制环节的干预方法。IgA蛋白酶是特定病原菌分泌的可以特异降解人IgA1免疫球蛋白的内肽酶，我们及其他研究团队的前期研究业已证明IgA蛋白酶可以特异性清除沉积于肾系膜的IgA1免疫复合物，从而用于IgAN治疗。在本研究中，我们克隆表达了高活性IgA蛋白酶，并采用新型药物传递系统（脂质体和纳米粒）对IgA蛋白酶进行包装，以实现IgA蛋白酶的靶向性药物传递和高的药效活性。我们首先采用兼并引物PCR扩增法成功克隆了具有高酶活性的H. influenzae 49247菌株IgA蛋白酶基因，经过基因工程改造融合组氨酸标签，实现了可溶IgA蛋白酶在E. coli菌株中组成型表达、自主分泌、简易快速纯化，且所得酶产量高、纯度高、活性高。我们对纯化的IgA蛋白酶同时进行阳离子脂质体和纳米粒包装，经过条件优化，成功制备了粒径约为60、90和120nm的IgA蛋白酶阳离子脂质体以及粒径约为120nm的阳离子纳米粒，且粒径电荷分布均一。然而，体内靶向性分布分析发现所有规格的IgA蛋白酶脂质体均未表现出明显的肾脏靶向性，而IgA蛋白酶纳米粒却表现出较好的肾靶向性。Passive IgAN小鼠体内肾系膜IgA1-IgG免疫复合物降解能力分析发现，注射10 ug IgA蛋白酶纳米粒可以有效清除肾系膜沉积的免疫复合物，且清除效率显著高于同等剂量单纯IgA蛋白酶注射。本项目的研究结果将为IgA蛋白酶纳米粒应用于未来IgAN临床治疗提供理论基础。