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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，这些变体可显著增强在接受标准G-CSF（安进非格司亭）治疗的阿尔茨海默病（AD）患者中观察到的认知能力改善。除了在治疗AD方面显示出前景外，G-CSF还在肌萎缩性侧索硬化症（ALS）和中风患者中进行了试验，并减少了帕金森病（PD）灵长类动物模型中的运动缺陷。因此，穿越BBB的G-CSF变体可以在治疗一系列神经系统疾病，改善数千人的生命和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是基于以下假设：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脑后的寿命。我们在酵母表面展示蛋白工程平台上的独特应用使此类变体的开发成为可能。具体地，我们将进化具有高度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被路由到溶酶体进行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在中性pH下具有一系列结合亲和力并且在内体pH（5.5-6.0）下具有降低的亲和力的G-CSF变体。将纯化前导变体，并通过表面等离子体共振验证结合特性。细胞培养研究将验证变体穿过内皮细胞模型BBB、逃避成熟神经元中的溶酶体降解以及促进神经干细胞增殖的能力增强。结合和细胞培养试验数据将阐明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，指导我们选择一种或两种主要的G-CSF变体用于后续AD小鼠研究。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，他们将在两年的性能期后立即开始这些实验。我们乐观地认为，这些动物研究将是实现我们在未来五年内进行穿越BBB的G-CSF变体人体试验的目标的前奏。除了使G-CSF成为用于治疗AD和其他神经变性病症的临床上更相关的药剂之外，所采用的方法适用于开发另外的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），用于增加BBB穿越和神经营养活性持续时间。因此，这项工作将是开发多种新的神经营养剂的重要的第一步，用于广泛施用，可能组合，作为治疗AD和其他神经系统疾病的有效疗法。