Novel nanoparticle-based enzyme replacement therapy for Hunter Syndrome

摘要 意义：在活产儿总患病率为1：5-7000的情况下，溶酶体储存疾病(LSD)作为一类 是最常见的儿童疾病之一。LSD是由溶酶体功能障碍引起的，主要原因是 溶酶体酶突变，通常在生命的头20年是致命的。中枢神经系统病理存在 然而，在大约75%的LSD患者中，目前市场上的酶替代疗法(ERT)对LSD无效 具有显著的中枢神经系统病理，因为在治疗性血清中，大脑中的酶水平可以忽略不计 酶的浓度。在身体组织中低效的酶摄取，中和抗体的形成， 成本效益差，以及每周静脉注射是其他不太理想的属性。亨特综合征，引起 在1：100,000名男婴中，通过X连锁艾杜糖酸-2-硫酸酯酶(I2S)突变，与严重的 神经病理学和广义躯体组织病理学。ELAPRASE®，人类重组I2S，不能解决 然而，CNS病理服务面向所有患者，每年的销售额接近6亿美元。鞘内I2S是 目前正在开发中。创新：Neuronano Pharma建议开发一种创新的聚合物- 基于纳米颗粒的I2S制剂，将被皮下注射以实现脑治疗 I2S水平。该产品将对亨特综合征患者产生革命性影响，进而， 这项技术有望应用于其他由酶突变引起的LSD。方法： 来自亚历山大·卡巴诺夫博士实验室的初步数据和含有酶的图片显示，大脑 使用这些制剂，可以实现递送、保护免受抗体反应和可忽略的毒性。 在北卡罗来纳大学结构生物学中心进行表达和纯化后，I2S将被整合到 纳米级核-壳多离子络合物(PIC)在水条件下通过 阴离子酶与含有阳离子嵌段的共聚物混合时的静电相互作用 和亲水性非离子嵌段。卡巴诺夫博士和朱迪·里弗尔博士是这一领域的专家，将担任 顾问。I2S PIC配方将根据物理化学性质、酶 活性、细胞毒性、溶酶体定位和脑水平。两个配方将被选为完整的 威廉·班克斯博士(VA Puget Sound Health Care System)的血脑药代动力学研究 障碍专家。除了自己的实验室设施外，NNP还可以通过以下途径获得所有必要的设备 北卡罗来纳大学纳米医学表征核心设施。预期结果：优化的I2S PIC将是 与脑实质和溶酶体相比，被确定将实现至少五倍的I2S增加 在SC注射后释放I2S和至少50%的血清生物利用度。延长的血清T1/2和宽体细胞 与游离I2S相比，组织摄取也是可取的。在第二阶段，I2S PIC将在I2S淘汰赛中进行测试 小鼠表现出脑内酶活性增强，对神经病理有效，并具有保护作用 来自免疫反应。优化的I2S PIC将被放大，并在Pre-Ind GLP毒理学中进行测试。

ABSTRACT Significance: With overall prevalence of ~1:5-7000 live births, lysosomal storage diseases (LSD) as a class are one of the most common childhood diseases. LSD are caused by lysosomal dysfunction, mainly due to lysosomal enzyme mutations, and are usually fatal in the first two decades of life. CNS pathology is present in ~75% of LSD, however, currently marketed enzyme replacement therapy (ERT), is not effective for LSD with significant CNS pathology because negligible enzyme levels are achieved in brain at therapeutic serum concentrations of enzyme. Inefficient enzyme uptake in somatic tissues, neutralizing antibody development, poor cost-effectiveness, and weekly IV infusions are other suboptimal attributes. Hunter Syndrome, caused by X-linked iduronate-2-sulfatase (I2S) mutations in 1:100,000 male births, is associated with severe neuropathology and broad somatic tissue pathology. Elaprase®, human recombinant I2S, does not address CNS pathology yet is offered to all patients and has sales approaching $600M yearly. Intrathecal I2S is currently in development. Innovation: NeuroNano Pharma proposes to develop an innovative polymer- based nanoparticle formulation of I2S which will be delivered subcutaneously to achieve therapeutic brain levels of I2S. This product will have a transformative impact on Hunter Syndrome patients and, by extension, holds promise for application of the technology to other LSD caused by enzyme mutations. Approach: Preliminary data from Dr. Alexander Kabanov’s laboratory with PICs containing enzymes shows that brain delivery, protection from antibody response, and negligible toxicity can be achieved with these formulations. After expression and purification at the UNC Center for Structural Biology, I2S will be incorporated into nanoscale core-shell polyion complexes (PICs) which form spontaneously in aqueous conditions via electrostatic interactions upon mixing of the anionic enzyme with a copolymer consisting of a cationic block and a hydrophilic nonionic block. Dr. Kabanov and Dr. Judy Riffle, experts in this field, will serve as consultants. I2S PIC formulations will be characterized with respect to physicochemical properties, enzyme activity, cytotoxicity, lysosome localization and brain levels. Two formulations will be selected for full pharmacokinetic characterization by Dr. William Banks (VA Puget Sound Health Care System), a blood brain barrier expert. In addition to its own laboratory facilities, NNP has access to all necessary equipment through the UNC Nanomedicines Characterization Core Facility. Expected Results: An optimized I2S PIC will be identified that will achieve at least a five-fold increase of I2S in brain parenchyma and lysosomes compared to free I2S and at least 50% serum bioavailability after SC injection. Extended serum t1/2 and broad somatic tissue uptake compared to free I2S is also desirable. In phase II, the I2S PIC will be tested in I2S knockout mice to demonstrate enhanced enzyme activity in the brain, efficacy against neuropathology, and protection from immune response. An optimized I2S PIC will be scaled up and tested in pre-IND GLP toxicology.