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

• 批准号: 8575714
• 负责人: Peter J Heinzelman
• 金额: $ 11.17万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 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/8575714
• 关键词: Affinity; Alzheimer' 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

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指示进化来设计血液脑屏障（BBB） - 传播粒细胞菌落刺激因子（G-CSF）变体，这些变体大大增加了在阿尔茨海默氏病（AD）患者中使用标准G-CSF（AMGERMGRAST）（AMGER）治疗的认知性能改善（AD）。除了显示出AD的希望外，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，并减少了帕金森氏病（PD）灵长类动物模型中的运动缺陷。因此，BBB引发的G-CSF变体可能会对治疗一系列神经系统疾病，改善数千人的生命并节省数百万美元的医疗保健费用产生广泛的影响。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逃脱溶酶体降解的能力将提高其越过BBB并延长大脑后延长其寿命的能力。我们独特的应用o酵母表面显示蛋白工程平台可以开发此类变体。具体而言，我们将使用高pH-敏感的G-CSF受体（G-CSFR）结合亲和力发展变体。这种敏感性促进了酸性内体环境中的G-CSF/G-CSFR复合物分离，从而使G-CSF从被路由到溶酶体进行降解并促进回收到细胞外部。我们将使用酵母显示器在中性pH下具有一系列结合亲和力的G-CSF变体，并在内体pH值下降（5.5-6.0）。领先的变体将被纯化并通过表面等离子体共振验证的结合特性。细胞培养研究将验证变异的跨越内皮细胞模型BBB的能力，逃脱成熟神经元中的溶酶体降解并促进神经干细胞增殖。结合和细胞培养分析数据将阐明pH敏感性，结合亲和力，BBB模型转胞胞病症和细胞激活效力之间的关系，从而指导我们选择一个或两个领先的G-CSF变体，以进行后续AD小鼠研究。我们在第一个在AD中试用G-CSF的小组Juan Sanchez-Ramos博士的合作者将在这两年的性能期间立即启动这些实验。我们乐观的是，这些动物研究将成为实现我们在未来五年内进行BBB转化的G-CSF变体试验的目标。 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.因此，这项工作将是开发多种新的神经营养剂，可能是结合使用的多种新神经营养剂，作为治疗AD和其他神经系统疾病的有效疗法。