Optimizing NGF for Topical Treatment of Glaucoma

优化 NGF 局部治疗青光眼

• 批准号: 9341566
• 负责人: Soon Seog Jeong
• 金额: $ 29.98万
• 依托单位: HUMAN CELL CO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9341566
• 关键词: Adverse effects; Agonist; Alpha Cell; Alzheimer' s Disease; Apoptosis; Attenuated; Binding; Bioreactors; Blindness; Cell Density; Cell Line; Cell Survival; Cells; Chemicals; Child; Chronic; Clinical Trials; Clone Cells; Contrast Sensitivity; Corneal Diseases; Cyclic GMP; Degenerative Disorder; Development; Disease Progression; Electrophysiology (science); Electroretinography; Endotoxins; Escherichia coli; Exhibits; Eye; Eyedrops; FDA approved; Fibroblasts; Future; Glaucoma; Growth; Growth Factor; Human; Hypoxic-Ischemic Brain Injury; In Vitro; Injection of therapeutic agent; Injury; Licensing; Modeling; Modification; Mus; NGFR Protein; Nerve Growth Factors; Neurons; Neurotrophic Tyrosine Kinase Receptor Type 1; Optic Nerve; Patients; Pattern; Peptide Signal Sequences; Peripheral Nervous System; Phase; Physiologic Intraocular Pressure; Process; Production; Proteins; Rattus; Receptor Protein-Tyrosine Kinases; Recombinants; Retinal; Retinal Ganglion Cells; Risk Factors; Rodent; Safety; Saline; Serum; Suspensions; System; Technology; Therapeutic; Topical application; Trypsin; Tumor Necrosis Factor Receptor; Veins; Visual Acuity; Visual Fields; blind; cost effective; disulfide bond; drotrecogin alfa; expression vector; improved; in vivo; manufacturing process; monomer; mutant; neuroprotection; neurotrophic factor; neurotropin; optic nerve disorder; prevent; promoter; regenerative; retinal axon; therapeutic protein

Glaucoma is one of the leading causes of blindness in the world. This chronic and progressive optic neuropathy is characterized by loss of axons of the retinal ganglion cells (RGC) that constitute the optic nerve. Reduction of elevated intraocular pressure, the only modifiable causative factor, slow the onset and progression of the disease, yet no treatment is available to restore optic nerve damage (8, 9). Nerve growth factor (NGF) is an endogenous neurotrophin that exerts trophic and differentiating activity on neurons of the central and peripheral nervous systems with protective and/or regenerative effects observed in degenerative diseases or following injury. NGF applied topically to the eye has been shown to significantly prevent RGC degeneration in experimental rat models of glaucoma. In 3 patients with advanced glaucoma, treatment with topical NGF (eye drop) improved visual acuity, contrast sensitivity, and electrophysiological functions without undesired side effects. NGF binds to both tyrosine kinase receptor TrkA and receptor P75NTR (TNF receptor superfamily). Importantly, the binding of NGF to TrkA alone promotes RGC’s survival and proliferation. In contrast, binding of NGF to p75NTR leads to apoptosis. rNGF (recombinant NGF) is currently produced in non- human cell systems. Due to the importance of pro-sequence for efficient folding or refolding, the in vitro (trypsin) or in vivo (furin) post-proteolytic modifications of pro-NGF, and the requirement of forming disulfide bonded monomer and non-covalent homodimer, expression yields from current manufacturing process are low and the NGF protein was in low quality. We have developed a cost-effective and scalable expression system to produce therapeutic human proteins from a proprietary HEK293 cell line. In the preliminary studies, we have established a cell pool of NGF which exhibited >10-fold higher yield than current expression systems with the ex vivo activity comparable to that of the murine wildtype NGF (wtNGF). In this study, we will first optimize the NGF to selectively activate TrkA receptor without compromising the expression yield or protein stability. We will then select top stable HEK293 cell clones for TrkA selective NGF mutant, which will be suitable for future large-scale cGMP manufacturing. Since there are substantial similarities between the rodent and human eyes, we will validate the efficacy and safety in an established rat model of glaucoma. Specific aims include: 1: To optimize rNGF as a TrkA-specific agonist and select top stable HEK293 cell clones suitable for future large-scale cGMP manufacturing. 2: To determine whether topical treatment with TrkA selective NGF mutant more effectively preserves retinal integrity and function compared to the wtNGF in a rat glaucoma model of episcleral vein by hypertonic saline injections.

青光眼是世界上导致失明的主要原因之一。这是一种慢性的、渐进的 视神经病变的特征是视网膜神经节细胞(RGC)轴突的丧失 构成视神经。降低升高的眼压，这是唯一可以改变的 致病因素，延缓疾病的发生和发展，但没有可用的治疗方法 修复视神经损伤(8、9)。神经生长因子(NGF)是一种内源性神经营养因子 对中枢和外周神经的神经元起营养和分化作用 在退行性疾病中观察到具有保护和/或再生作用的系统或 在受伤之后。将NGF局部应用于眼睛，已被证明显著防止RGC 实验性青光眼大鼠模型的变性。在3名晚期青光眼患者中， 局部使用NGF(眼药水)可提高视力、对比敏感度和 电生理功能，无不良副作用。NGF与两种酪氨酸激酶结合 受体TrkA和受体p75NTR(肿瘤坏死因子受体超家族)。重要的是，NGF与 TrkA单独促进RGC的存活和增殖。相比之下，NGF与p75NTR导联的结合 到细胞凋亡。重组神经生长因子(RNGF)目前在非人类细胞系统中产生。由于 前序列对高效折叠或重折叠的重要性，体外(胰酶)或在 前体NGF的活体(呋喃)蛋白水解物修饰及其形成二硫化物的要求 键合单体和非共价均二聚体，当前制造工艺的表达产率为 低，NGF蛋白质量较低。 我们已经开发了一种经济高效且可扩展的表达系统来生产治疗性人类 来自一种专有HEK293细胞系的蛋白质。在初步研究中，我们已确立了一套 NGF的细胞库，其产量是目前表达系统的10倍 体内活性与小鼠野生型神经生长因子(WtNGF)相当。在这项研究中，我们将首先 优化NGF以选择性激活TrkA受体，而不影响表达产量或 蛋白质稳定性。然后我们将为TrkA选择性NGF突变体选择最稳定的HEK293细胞克隆， 适合未来大规模生产cGMP。因为有大量的 由于啮齿类动物与人类眼睛的相似性，我们将在 建立大鼠青光眼模型。具体目标包括： 1：优化rNGF作为TrkA特异性激动剂，筛选最稳定的HEK293细胞克隆 为今后cGMP的规模化生产做准备。 2：确定TrkA选择性NGF突变体局部治疗是否更有效 与wtNGF相比，在大鼠青光眼模型中保持视网膜的完整性和功能 巩膜外静脉注射高渗盐水。