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

描述(由申请人提供)：我们将利用体外实验室指导的进化来改造血脑屏障(BBB)-穿越粒细胞集落刺激因子(G-CSF)变体，显著增强接受标准G-CSF(Amgen‘s FilgrTim)治疗的阿尔茨海默病(AD)患者的认知能力改善。除了在治疗AD方面显示出希望之外，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，BBB-穿越G-CSF变种可以在治疗一系列神经系统疾病方面产生广泛影响，改善数千人的生活，并节省数百万美元的医疗成本。进化的G-CSF变异体的预期治疗效果是基于这样的假设，即增强G-CSF逃避溶酶体降解的能力将改善其穿越血脑屏障的能力，并延长其进入大脑后的寿命。我们对酵母表面展示蛋白工程平台的独特应用使此类变体的开发成为可能。具体地说，我们将进化出具有高度pH敏感的G-CSF受体(G-CSFR)结合亲和力的变体。这种敏感性促进了G-CSF/G-CSFR复合体在酸性内体环境中的解离，避免了G-CSF被路由到溶酶体进行降解，并促进了循环到细胞外部。我们将使用酵母展示来进化G-CSF变异体，在中性pH下具有一系列结合亲和力，在内质体pH(5.5-6.0)时亲和力降低。主要的变异体将被提纯并通过表面等离子激元共振验证结合特性。细胞培养研究将验证变异体跨越内皮细胞模型血脑屏障的能力增强，逃避成熟神经元中溶酶体的降解，并促进神经干细胞的增殖。结合和细胞培养分析数据将阐明pH敏感性、结合亲和力、血脑屏障模型细胞转运和细胞激活能力之间的关系，指导我们选择一个或两个领先的G-CSF变体用于后续的AD小鼠研究。我们在胡安·桑切斯-拉莫斯博士实验室的合作者是第一个在AD中试验G-CSF的小组，他们将在这两年的实施期结束后立即启动这些实验。我们乐观地认为，这些动物研究将是实现我们在未来五年内进行血脑屏障穿越G-CSF变异体人类试验的目标的前奏。除了使G-CSF成为治疗AD和其他神经退行性疾病的更具临床相关性的药物外，所采用的方法还适用于进化出更多的候选治疗神经营养因子，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)，以延长BBB穿越和神经营养活动持续时间。因此，这项工作将是朝着开发多种新的神经营养剂的重要的第一步，以便广泛应用，可能联合使用，作为治疗AD和其他神经系统疾病的有效疗法。

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.