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

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血脑屏障（BBB）穿越粒细胞集落刺激因子（G-CSF）变异，这些变异在使用标准G-CSF（安进的Filgrastim）治疗的阿尔茨海默病（AD）患者中观察到的认知能力改善。除了在治疗阿尔茨海默氏症（AD）方面表现出希望外，G-CSF还在肌萎缩侧索硬化症（ALS）和中风患者身上进行了试验，并减少了帕金森病（PD）灵长类动物模型的运动缺陷。因此，穿越血脑屏障的G-CSF变异可以在治疗一系列神经系统疾病方面产生广泛的影响，改善数千人的生命并节省数百万美元的医疗费用。进化的G-CSF变体的预期治疗效果是基于这样的假设，即增加G-CSF逃避溶酶体降解的能力将提高其穿过血脑屏障的能力，并延长其进入大脑后的寿命。我们对酵母表面显示蛋白工程平台的独特应用使此类变体的开发成为可能。具体来说，我们将进化出具有高度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，并促进细胞外部的再循环。我们将使用酵母展示来进化G-CSF变体，这些变体在中性pH下具有一定的结合亲和力，在内体pH（5.5-6.0）下具有较低的亲和力。主要变体将被纯化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通过内皮细胞模型血脑屏障的能力增加，逃避成熟神经元的溶酶体降解，并促进神经干细胞增殖。结合和细胞培养实验数据将阐明pH敏感性、结合亲和力、血脑屏障模型胞饮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变体用于后续的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，将在这两年的表现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血脑屏障的G-CSF变异人体试验目标的序曲。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），以增加血脑屏障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药物的重要的第一步，这些药物可以广泛使用，可能是联合使用，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.