Directed Evolution of Blood Brain Barrier-Traversing Granulocyte Colony-Stimulati

穿过血脑屏障的粒细胞集落刺激的定向进化

基本信息

批准号：
8688870
负责人：
Peter J Heinzelman
金额：
$ 11.16万
依托单位：
UNIVERSITY OF OKLAHOMA
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8688870
关键词：
Affinity Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Animal Model Animals Antibodies Binding Biological Assay Blood Blood - brain barrier anatomy Brain CSF3 gene Cell Culture Techniques Cell Proliferation Cell model Cells Cessation of life Chimeric Proteins Clinical Clinical Research Clinical Trials Cognition Cognitive Collaborations Complex Culture Media Data Development Dissociation Endosomes Endothelial Cells Engineering Environment Erythropoietin Escherichia coli Evolution Filgrastim Flow Cytometry Fluorescence-Activated Cell Sorting Goals Granulocyte Colony-Stimulating Factor Granulocyte Colony-Stimulating Factor Receptors Granulocyte-Macrophage Colony-Stimulating Factor Health Care Costs Human In Vitro Incubated Kinetics Laboratories Lysosomes Measures Mediating Methods Modeling Motor Mus Mutation Nerve Degeneration Nerve Growth Factor Receptors Neuroblastoma Neurodegenerative Disorders Neurology Neurons Outcome Parkinson Disease Patients Performance Positioning Attribute Predisposition Primates Property Protein Engineering Proteins Publishing Recycling Research Side Stroke Surface Surface Plasmon Resonance Therapeutic Time Treatment Efficacy Variant Work Yeasts base brain tissue clinically relevant directed evolution effective therapy granulocyte improved innovation monolayer motor deficit nerve stem cell nervous system disorder neurotrophic factor novel premature public health relevance receptor receptor binding research study transcytosis

项目摘要

DESCRIPTION (provided by applicant): We will employ in vitro laboratory directed evolution to engineer blood brain barrier (BBB)-traversing granulocyte colony-stimulating factor (G-CSF) variants that substantially augment cognitive performance improvements observed in Alzheimer's Disease (AD) patients treated with standard G-CSF (Amgen's Filgrastim). In addition to showing promise in treating AD, G-CSF has been trialed in Amyotrophic Lateral Sclerosis (ALS) and stroke patients and reduces motor deficits in Parkinson's Disease (PD) primate models. As such, BBB-traversing G-CSF variants can have a broad impact in treating a range of nervous system disorders, improving thousands of lives and saving millions of dollars in healthcare costs. The anticipated therapeutic efficacy of evolved G-CSF variants is based on the hypothesis that increasing G-CSF's ability to escape lysosomal degradation will improve its ability to cross the BBB and extend its lifetime after entering the brain. Our unique application o a yeast surface display protein engineering platform enables development of such variants. Specifically, we will evolve variants with highly pH- sensitive G-CSF receptor (G-CSFR) binding affinity. This sensitivity promotes G-CSF/G-CSFR complex dissociation within the acidic endosomal environment, sparing G-CSF from being routed to lysosomes for degradation and promoting recycling to the cell exterior. We will use yeast display to evolve G-CSF variants with a range of binding affinities at neutral pH and reduced affinities at endosomal pH (5.5-6.0). Leading variants will be purified and binding properties validated by surface plasmon resonance. Cell culture studies will validate variants' increased ability to cross an endothelial cell model BBB, escape lysosomal degradation in mature neurons, and promote neural stem cell proliferation. Binding and cell culture assay data will elucidate relationships among pH sensitivity, binding affinity, BBB model transcytosis and cell activation potency, guiding our choice of one or two leading G-CSF variants for follow-on AD mouse studies. Our collaborators in the lab of Dr. Juan Sanchez-Ramos, the first group to trial G-CSF in AD, will initiate these experiments immediately after this two-year performance period. We are optimistic that these animal studies will be a preface to realizing our goal of conducting BBB-traversing G-CSF variant human trials within the next five years. Beyond making G-CSF a more clinically-relevant agent for treating AD and other neurodegenerative conditions, the methods employed apply to evolving additional candidate therapeutic neurotrophic factors, particularly granulocyte macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), for increased BBB-traversal and neurotrophic activity duration. As such, this work will be an important initial step toward developing multiple new neurotrophic agents for widespread administration, possibly in combination, as effective therapeutics for treating AD and other nervous system disorders.

描述（由申请人提供）：我们将采用体外实验室定向进化来设计穿过血脑屏障（BBB）的粒细胞集落刺激因子（G-CSF）变体，这些变体可显着增强阿尔茨海默病（AD）患者中观察到的认知能力改善用标准 G-CSF（Amgen 的 Filgrastim）治疗。除了在治疗 AD 方面显示出前景外，G-CSF 还在肌萎缩侧索硬化症 (ALS) 和中风患者中进行了试验，并在帕金森病 (PD) 灵长类动物模型中减少了运动缺陷。因此，穿过 BBB 的 G-CSF 变体可以在治疗一系列神经系统疾病、改善数千人的生活并节省数百万美元的医疗费用方面产生广泛的影响。进化的 G-CSF 变体的预期治疗功效基于这样的假设：增加 G-CSF 逃避溶酶体降解的能力将提高其穿过 BBB 的能力并延长其进入大脑后的寿命。我们在酵母表面展示蛋白质工程平台上的独特应用使得能够开发此类变体。具体来说，我们将开发具有高度 pH 敏感性 G-CSF 受体 (G-CSFR) 结合亲和力的变体。这种敏感性促进 G-CSF/G-CSFR 复合物在酸性内体环境中解离，避免 G-CSF 被输送到溶酶体降解并促进再循环到细胞外部。我们将使用酵母展示来进化 G-CSF 变体，其在中性 pH 下具有一系列结合亲和力，在内体 pH (5.5-6.0) 下具有降低的亲和力。主要变体将被纯化并通过表面等离子共振验证结合特性。细胞培养研究将验证变体穿过内皮细胞模型 BBB、逃避成熟神经元溶酶体降解并促进神经干细胞增殖的能力增强。结合和细胞培养测定数据将阐明 pH 敏感性、结合亲和力、BBB 模型转胞吞作用和细胞活化效力之间的关系，指导我们为后续 AD 小鼠研究选择一种或两种主要的 G-CSF 变体。我们在 Juan Sanchez-Ramos 博士实验室的合作者是第一个在 AD 中试验 G-CSF 的小组，他们将在两年的实验期结束后立即启动这些实验。我们乐观地认为，这些动物研究将成为实现我们在未来五年内进行穿越 BBB 的 G-CSF 变异人体试验的目标的序言。除了使 G-CSF 成为治疗 AD 和其他神经退行性疾病的临床相关药物外，所采用的方法还适用于开发其他候选治疗性神经营养因子，特别是粒细胞巨噬细胞集落刺激因子 (GM-CSF) 和促红细胞生成素 (EPO)，以用于治疗 AD 和其他神经退行性疾病。增加 BBB 穿越和神经营养活动持续时间。因此，这项工作将是开发多种新型神经营养剂以广泛给药（可能联合用药）作为治疗 AD 和其他神经系统疾病的有效疗法的重要第一步。