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

描述（由申请方提供）：遗传性溶酶体贮积病粘多糖贮积症IIIB（MPS IIIB;也称为Sanfilippo B综合征）导致进行性智力障碍和行为问题，始于儿童早期，通常在30岁时达到神经系统破坏和死亡的顶峰。酶替代疗法（ERT）已成功开发并在临床上用于治疗其他粘多糖，特别是MPS I、II和VI。MPS IIIB也是由于可溶性溶酶体水解酶缺乏所致，理论上可以用ERT治疗。然而，开发MPS IIIB的ERTF的障碍是MPS IIIB中缺乏的酶（？N-乙酰葡糖胺糖苷酶，或NAGLU）天然具有足够的甘露糖6-磷酸部分以使得能够通过甘露糖6-磷酸受体途径有效地细胞摄取，但是重组产生的NAGLU由于其含有很少或不含有甘露糖6-磷酸化而低效地进入细胞。为了克服这一障碍，我们开发了胰岛素样生长因子2（IGF 2）和NAGLU的融合蛋白（rhNAGLU-IGF 2）。IGF 2是甘露糖6-磷酸受体（一种清道夫受体，也称为IGF 2受体）的天然配体，因此为NAGLU提供了一种在不存在甘露糖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。我们提出，IGF 2肽融合NAGLU将增加NAGLU的治疗潜力，使酶有效地进入细胞和运输到溶酶体。为了进一步发展这一点，我们提出了临床前研究，以确定脑室内rhNAGLU-IGF 2是否安全有效地减少MPS IIIB（Naglu-/-）小鼠中的溶酶体蓄积（目的1），并确定脑室内rhNAGLU-IGF 2的脑分布（目的2）。为了实现这些目标，我们将向MPS IIIB小鼠脑室内递送rhNAGLU、rhNAGLU-IGF 2融合物或溶媒对照。然后，我们将进行详细的病理学，免疫组织化学，共聚焦显微镜和生化功能分析，以及分布和渗透研究作为剂量和时间的函数后交付。我们的初步数据显示rhNAGLU-IGF 2进入MPS IIIB成纤维细胞的效率远高于rhNAGLU。这项工作的临床相关性得到以下方面的支持：a）重组ERT已获批并上市用于治疗其他几种MPS疾病和溶酶体贮积病; B）鞘内给药酶替代疗法的临床试验正在进行中，用于治疗其他三种MPS类型，但不包括MPS IIIB; c）IGF 2标记的溶酶体酶（酸性α-葡萄糖苷酶）的临床试验最近开始。所有这些不仅为翻译相关性建立了优先权，而且将指导我们提出的rhNAGLU-IGF 2应用的开发。因此，我们乐观地认为，rhNAGLU-IGF 2可能被成功开发用于治疗一种目前尚无治疗方法的毁灭性致命疾病，此外，这种方法最终也可能适用于其他基于遗传的溶酶体贮积病。

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.