Oral tolerance in enzyme replacement therapy of Morquio A disease

描述(申请人提供)：Morquio A病(粘多糖病IVA；MPS IVA)是一种常染色体隐性遗传性溶酶体储存障碍(LSD)，由N-乙酰氨基半乳糖-6-硫酸酯硫酸酯酶(GALNS)缺乏引起。未降解的角蛋白硫酸盐(KS)会积聚在骨骼中，导致全身性骨骼发育不良。自20多年前PI及其合作者开始Morquio A研究以来，GALNS的纯化、人GALNS基因的克隆、人类基因组基因的克隆、常见突变的鉴定、小鼠基因的克隆、GALNS的三级结构、Morquio A小鼠模型的建立、EL ISA法的KS分析、Morquio的教育CD、CHO生产的重组人GALN、串联质谱仪KS分析、国际Morquio注册表的建立、Morquio A基因敲除小鼠的首次ERT尝试以及Morquio A患者的生长曲线都取得了进展。我们已经发表了50多篇关于Morquio A的同行评议文章，申请和/或发布了7项与治疗和诊断相关的专利。我们正在开发新的创新的酶替代疗法(ERT)来靶向Morquio A的骨骼。同时，重复使用这种酶可能会导致不良的免疫反应。50-90%的LSD患者静脉注射抗输液酶抗体。LSD患者对ERT的免疫反应已被广泛报道，并被认为是治疗的主要并发症之一。目标：直接的携带者目标是对LSD，特别是Morquio A病的ERT免疫反应产生“口服耐受性”。运营商的长期目标是优化现有的ERT方案，以更具可行性的方式改变ERT对LSD的疗效。我们的目标是：1)建立Morquio A小鼠对人重组GALN的口服耐受方案；2)比较耐受和不耐受Morquio A小鼠ERT的疗效。研究计划：为了抑制ERT的免疫反应，将设计一种新的口服耐受方案，通过口服合成的GALNS多肽。在Morquio A基因敲除小鼠中，将通过多次低剂量喂养方案来研究合成GALNS多肽对口服耐受性的诱导。在对GALNS诱导口服耐受后，我们将在Morquio A小鼠身上进行ERT，以评估病理损害的改善。实验计划是1)合成GALNS多肽，2)通过多次低剂量喂养合成GALNS多肽诱导Morquio A小鼠耐受，3)炎症反应的抑制将通过流式细胞仪调节T细胞数量的增加来衡量，4)GALNS抗体的存在或不存在，5)Morquio A小鼠将接受ERT治疗，6)我们通过测量抗体和评估病理改善来评估免疫耐受.实现所提出的目标将解决当前ERT中尚未满足的挑战之一，并可能显著改变治疗效果. 与公共卫生相关：Morquio A病是一种罕见的疾病，由溶酶体酶缺乏引起，导致糖链主要积聚在骨骼中，表现为身材矮小、胸部突出、脊髓受压、膝部撞击和关节松动的全身性骨骼疾病。最近的先进技术导致了所谓的“酶替代疗法”的发展，以弥补酶的缺陷。为了避免对输注酶的免疫反应和由此产生的不良反应，我们将通过口服合成肽的方法来开发对输注酶的免疫耐受新方法。

DESCRIPTION (provided by applicant): Morquio A disease (mucopolysaccharidosis IVA; MPS IVA) is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of N-acetyl-galactosamine-6-sulfate sulfatase (GALNS). Undegraded keratin sulfate (KS) will be accumulated especially in bone, leading to systemic skeletal dysplasia. Since the PI and co-investigators started Morquio A research over 20 years ago, purification of GALNS, cloning of human GALNS cDNA, cloning of human genomic gene, identification of common mutations, cloning of mouse gene, tertiary structure of GALNS, establishment of Morquio A murine models, KS assay by ELISA, Educational CD for Morquio, recombinant human GALNS produced in CHO, KS assay by tandem mass spectrometry, development of International Morquio Registry, the first attempt of ERT on Morquio A knock-out mouse, and growth chart for Morquio A patients were achieved. We have published over 50 peer-review articles on Morquio A. Seven patents related to therapy and diagnosis were applied and/or issued. We are developing new innovative enzyme replacement therapy (ERT) to target bone for Morquio A. Meanwhile, repeated administration of the enzyme could induce an undesirable immune response. 50-90% patients with LSDs administered intravenously raised antibody against infused enzyme. Immune response to ERT in LSDs has been widely reported and presented as one of the major complications of treatment. Goals: The immediate carrier goal is to develop "oral tolerance" for immune response to ERT of LSDs, in particular, Morquio A disease. The long-term carrier goal is to optimize current ERT regimen and to change efficacy of ERT for LSDs with more feasibility. We will aim: 1) to establish an oral tolerance protocol to the human recombinant GALNS in Morquio A mice; 2) to compare the efficacy of ERT between the tolerized and non-tolerized Morquio A mice. Research Plan: For suppressing the immune response to ERT, a novel oral tolerance protocol will be designed by administering synthetic GALNS peptides orally. Induction of oral tolerance with synthetic GALNS peptides will be investigated by a multiple low-dose feeding regimen in a Morquio A knock-out mouse. After induction of oral tolerance against GALNS, we will perform ERT on Morquio A mouse to assess improvement of pathological lesions. The experimental plan is 1) synthetic GALNS peptides will be made, 2) oral tolerance is induced by a multiple low-dose feeding regimen of synthetic GALNS peptides in Morquio A mouse, 3) the inhibition of an inflammatory response will be measured by the increment of regulatory T cell populations by flow cytometry, 4) the presence or absence of antibodies against GALNS will be measured, 5) Morquio A mice will be treated by ERT, and 6) we assess the immunological tolerance by measuring antibodies and assessing pathological improvement..To accomplish the proposed aims will resolve one of unmet challenges in current ERT and could change efficacy of the therapy dramatically. PUBLIC HEALTH RELEVANCE: Morquio A disease is a rare disorder caused by a deficiency of one of lysosomal enzymes, leading to accumulation of a sugar of chain mainly in bone, and showing systemic skeletal disease with short stature, prominent chest, spinal cord compression, knock-knee, and loose joints. Recent advanced technology has led to development of so-called "enzyme replacement therapy" to make up for the deficient enzyme. To avoid immunological reaction against infused enzyme and resultant unwanted side effect is urgently demanded, therefore, we will develop novel immunological tolerance method to the infused enzyme by oral administration of synthetic peptide for the enzyme.