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

描述（由申请人提供）：遗传性溶酶体贮积性粘多糖病IIIB （MPS IIIB；也称为Sanfilippo B综合征）导致儿童期早期开始的进行性智力损伤和行为问题，最终导致神经系统破坏和死亡，通常在生命的第三个十年。酶替代疗法（ERT）已经成功开发并应用于其他粘多糖疾病，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，理论上应该可以用ERT治疗。然而，开发ERTF用于MPS IIIB的障碍是MPS IIIB中缺乏的酶(？- n -乙酰氨基葡萄糖苷酶（NAGLU）天然具有足够的甘露糖6-磷酸片段，可通过甘露糖6-磷酸受体途径使细胞有效摄取，但重组产生的NAGLU进入细胞效率较低，因为它含有很少或没有甘露糖6-磷酸化。为了克服这一障碍，我们开发了胰岛素样生长因子2 （IGF2）和NAGLU的融合蛋白（rhNAGLU-IGF2）。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提供了一种方法。我们提出IGF2肽融合到NAGLU将增加NAGLU的治疗潜力，使酶能够有效地进入细胞并运输到溶酶体。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-IGF2是否安全有效地减少MPS IIIB （Naglu-/-）小鼠的溶酶体储存（目的1），并确定脑室内rhNAGLU-IGF2的脑分布（目的2）。为了实现这些目标，我们将rhNAGLU、rhNAGLU- igf2融合物或载体对照注入MPS IIIB小鼠脑室内。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，以及分布和渗透研究，作为给药后剂量和时间的函数。我们的初步数据显示rhNAGLU- IGF2进入MPS IIIB成纤维细胞的效率远远高于rhNAGLU。这项工作的临床相关性得到以下方面的支持：a)重组ERT已被批准并上市用于治疗其他几种MPS疾病和溶酶体贮积病；b)鞘内输送酶替代疗法的临床试验正在进行中，用于其他三种MPS类型，但不包括MPS IIIB；c)最近开始了一项igf2标记的溶酶体酶（酸性α -葡萄糖苷酶）的临床试验。所有这些不仅建立了翻译相关性的优先级，而且将指导我们提出的rhNAGLU-IGF2应用程序的开发。因此，我们乐观地认为，rhNAGLU-IGF2可能会成功地开发用于治疗一种目前没有治疗方法的毁灭性致命疾病，而且，这种方法最终也可能适用于其他基于遗传的溶酶体储存疾病。

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.