Neurotrophin Drug Development for Parkinson's Disease

描述(申请人提供)：帕金森氏病(PD)在美国有100万人患病，是一种由黑质-纹状体束脑细胞丧失引起的退行性疾病。对大脑这一区域最有效的保护因素是神经营养因子，即胶质源性神经营养因子(GDNF)。然而，治疗帕金森病的GDNF药物开发失败了，因为神经营养素不能穿过血脑屏障(BBB)。因此，GDNF是通过经颅给药系统给帕金森病患者注射的，该系统不能有效地将药物输送到参与帕金森病的大脑部分。目前的研究将从基因上设计一种新的GDNF形式，使其能够通过受体介导的运输穿过血脑屏障。PI之前已经通过基因工程设计了一种针对小鼠转铁蛋白受体(TFR)的嵌合单抗，命名为cTfRMAb，它通过受体介导的小鼠TFR上的转运，在血到脑的方向上穿过BBB。此外，PI还进行了基因工程，并表达了GDNF与cTfRMAb的融合蛋白，命名为cTfRMAb-GDNF融合蛋白。瞬时表达实验验证了融合蛋白的双功能。本研究将利用中国仓鼠卵巢(CHO)细胞大量分泌cTfRMAb-GDNF融合蛋白，从而产生200 mg的融合蛋白，从而产生永久转基因的宿主细胞系。然后，该融合蛋白将用于小鼠的药代动力学研究，并在使用6-羟基多巴胺的C57BL/6小鼠的实验性帕金森病小鼠模型中进行时间反应和剂量反应疗效研究。此外，还将进行毒性研究，雄性和雌性C57BL/6小鼠长期使用cTfRMAb-GDNF融合蛋白治疗；然后将检查大脑和其他主要器官的组织学。抗融合蛋白抗体的形成将使用一种新的夹心ELISA法进行检测。实验性帕金森病的药物疗效将通过旋转行为测量和纹状体酪氨酸羟基酶测定来验证。这项研究将提供必要的临床前药理学，以支持IND申请使用基因工程形式的GDNF通过受体介导的转运穿过BBB来治疗人类帕金森病。 公共卫生相关性：帕金森氏症(PD)影响美国100万人。治疗帕金森病最有效的形式是一种名为GDNF的神经营养因子；然而，GDNF不能跨越血脑屏障(BBB)。目前的研究将在小鼠的实验性PD中测试一种新形式的GDNF的有效性，其中神经营养因子被重新设计为与单抗的融合蛋白，该融合蛋白通过受体介导的转运穿过BBB。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) affects 1 million people in the U.S., and is a degenerative disease caused by the loss of brain cells in the nigral-striatal tract. The most potent protective factor for this area of the brain is the neurotrophin, glial derived neurotrophic factor (GDNF). However, GDNF drug development in PD has failed, because the neurotrophin does not cross the blood-brain barrier (BBB). Consequently, the GDNF was administered to patients with PD via a trans-cranial drug delivery system that does not effectively deliver the drug to the part of the brain involved in PD. The present research will genetically engineer a new form of GDNF that is enabled to cross the BBB via receptor-mediated transport. The PI has previously genetically engineered a chimeric MAb against the mouse transferrin receptor (TfR), designated cTfRMAb, that crosses the BBB in the blood-to-brain direction via receptor-mediated transport on the mouse BBB TfR. In addition, the PI has genetically engineered, and expressed a fusion protein of GDNF and the cTfRMAb, which is designated the cTfRMAb-GDNF fusion protein. The bi-functionality of the fusion protein was verified in transient expression experiments. The present research will produce a permanently transfected host cell line using Chinese hamster ovary (CHO) cells that secrete the cTfRMAb-GDNF fusion protein in high amounts so that 200 mg of the fusion protein can be produced. This fusion protein will then be used for pharmacokinetics studies in the mouse, for time response and dose response efficacy studies in an experimental mouse model of PD using C57Bl/6 mice lesioned with intra-cranial 6- hydroxydopmaine. In addition a toxicity study will be performed where male and female C57Bl/6 mice are treated chronically with the cTfRMAb-GDNF fusion protein; the histology of brain and other major organs will then be examined. The formation of anti-fusion protein antibodies will be examined using a novel sandwich ELISA. The drug efficacy in experimental PD will be validated with rotation behavior measurements, and assays of striatal tyrosine hydroxylase. This research will provide the necessary pre-clinical pharmacology to support an IND filing for the treatment of humans with PD using genetically engineered forms of GDNF that cross the BBB via receptor-mediated transport. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) affects 1 million people in the U.S. The most potent form of treatment of PD is a neurotrophin called GDNF; however, GDNF does not cross the blood-brain barrier (BBB). The present research will test in experimental PD in mice the efficacy of a new form of GDNF, wherein the neurotrophin is re-engineered as a fusion protein with a monoclonal antibody that crosses the BBB via receptor-mediated transport.