Glycosylation-independent enzyme therapy of the brain in Sanfilippo B syndrome

描述(申请人提供)：遗传性溶酶体储存疾病粘多糖症IIIB(MPS IIIB；也称为Sanfilippo B综合征)从儿童早期开始导致进行性智力障碍和行为问题，最终导致神经破坏和死亡，通常在生命的第三个十年。酶替代疗法(ERT)已经被成功地开发并应用于其他粘多糖病，特别是MPS I、II和VI。MPS IIIB也是由于缺乏可溶性溶酶体水解酶，理论上应该可以用ERT治疗。然而，为MPS IIIB开发ERTF的障碍是这样一个事实，即MPS IIIB(β-N-乙酰氨基葡萄糖苷酶，NAGLU)缺乏的酶自然具有足够的甘露糖6-磷酸部分，使细胞能够通过甘露糖6-磷酸受体途径有效地摄取，但重组产生的NAGLU由于含有很少或根本不含甘露糖6-磷酸化，所以进入细胞的效率低下。为了克服这一障碍，我们开发了一种胰岛素样生长因子2(IGF2)和NAGLU(rhNAGLU-IGF2)的融合蛋白。IGF2是甘露糖6-磷酸受体(一种清道夫受体，也称为IGF2受体)的天然配体，因此为NAGLU提供了一种在没有甘露糖6-磷酸残基的情况下利用该受体进行细胞摄取和溶酶体靶向的方法。我们认为，融合到NAGLU的IGF2肽将通过使酶有效地进入细胞并运输到溶酶体来增加NAGLU的治疗潜力。为了进一步发展这一点，我们建议进行临床前研究，以确定脑室内rh NAGLU-IGF2是否安全有效地减少MPS IIIB(Naglu-/-)小鼠的溶酶体存储(Aim 1)，并确定脑室内rh NAGLU-IGF2的脑分布(Aim 2)。为了实现这些目标，我们将把重组人NAGLU、重组人NAGLU-IGF2融合基因或载体对照基因注入MPS IIIB小鼠的脑室。然后，我们将进行详细的病理、免疫组织化学、共聚焦显微镜和生化功能分析，以及随分娩剂量和时间的变化而进行的分布和渗透研究。我们的初步数据显示，rh NAGLU-IGF2进入MPS IIIB成纤维细胞的效率远远高于rh NAGLU。这项工作的临床意义得到以下支持：a)重组ERT被批准用于治疗其他几种MPS疾病和溶酶体储存疾病，并已上市；b)鞘内给药的酶替代疗法正在进行中，用于其他三种MPS类型，但不是MPS IIIB；以及c)一种标记IGF2的溶酶体酶(酸性α-葡萄糖苷酶)的临床试验最近已经开始。所有这些不仅建立了翻译相关性的优先地位，而且将指导我们提议的应用程序的开发。因此，我们乐观地认为，rh NAGLU-IGF2可能被成功开发来治疗一种毁灭性的、致命的疾病，而这种疾病目前还没有治疗方法，而且这种方法最终也可能适用于其他基于基因的溶酶体储存疾病。

DESCRIPTION (provided by applicant): The genetic lysosomal storage disease mucopolysaccharidosis IIIB (MPS IIIB; also called Sanfilippo B syndrome) causes progressive intellectual impairment and behavioral problems beginning in early childhood, culminating in neurological devastation and death, usually by the third decade of life. Enzyme replacement therapy (ERT) has been successfully developed and clinically deployed for other mucopolysaccharidoses, notably MPS I, II, and VI. MPS IIIB is due to deficiency of a soluble lysosomal hydrolase as well, and theoretically should be treatable with ERT. However, the barrier to developing ERTF for MPS IIIB is the fact that the enzyme that is deficient in MPS IIIB (?-N-acetylglucosaminidase, or NAGLU) naturally has sufficient mannose 6-phosphate moieties to enable efficient cellular uptake via the mannose 6-phosphate receptor pathway, but recombinantly-produced NAGLU enters cells inefficiently because it contains little or no mannose 6-phosphorylation. To overcome this hurdle, we developed a fusion protein of insulin-like growth factor 2 (IGF2) and NAGLU (rhNAGLU-IGF2). IGF2 is a natural ligand of the mannose 6-phosphate receptor (a scavenger receptor that is also called IGF2 receptor), and thus provides a way for NAGLU to exploit this receptor for cellular uptake and lysosomal targeting in the absence of mannose 6-phosphate residues. We propose that the IGF2 peptide fused to NAGLU will increase the therapeutic potential of NAGLU by enabling the enzyme to efficiently enter cells and traffic to the lysosomes. To develop this further, we propose preclinicl studies to determine whether intraventricular rhNAGLU-IGF2 safely and effectively reduces lysosomal storage in MPS IIIB (Naglu-/-) mice (Aim 1) and to determine the brain distribution of intraventricular rhNAGLU-IGF2 (Aim 2). To achieve these objectives, we will deliver rhNAGLU, the rhNAGLU-IGF2 fusion, or vehicle control intraventricularly to MPS IIIB mice. We will then perform detailed pathologic, immunohistochemical, confocal microscopic, and biochemical functional analyses, as well as distribution and penetration studies as a function of dose and time following delivery. Our preliminary data show that rhNAGLU- IGF2 enters MPS IIIB fibroblasts far more efficiency than rhNAGLU. The clinical relevance of this work is supported by the following: a) recombinant ERT is approved and marketed for several other MPS disorders and lysosomal storage diseases; b) clinical trials of intrathecally-delivered enzyme replacement therapy are ongoing for three other MPS types, but not MPS IIIB; and c) a clinical trial of an IGF2-tagged lysosomal enzyme (acid alpha-glucosidase) has recently begun. All these not only establish precedence for translational relevance, but will guide development of our proposed application with rhNAGLU-IGF2. Thus, we are optimistic that rhNAGLU-IGF2 may be successfully developed to treat a devastating, fatal disease for which no treatment exists, and furthermore, that this approach may ultimately be adapted to other genetically-based lysosomal storage diseases as well.