Optimizing NGF for Topical Treatment of Glaucoma

优化 NGF 局部治疗青光眼

基本信息

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
项目类别：
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&apos 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

项目摘要

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，是一种慢性且进行性的疾病。视神经病变的特征是视网膜神经节细胞 (RGC) 轴突丧失，构成视神经降低升高的眼压的唯一可改变的。致病因素，减缓疾病的发生和进展，但尚无治疗方法恢复视神经损伤 (8, 9) 神经生长因子 (NGF) 是一种内源性神经营养因子。对中枢和周围神经系统的神经元发挥营养和分化活性在退行性疾病或中观察到的具有保护和/或再生作用的系统研究表明，在眼睛受伤后，局部使用 NGF 可以显着预防 RGC。青光眼实验大鼠模型中的变性在 3 名晚期青光眼患者中，局部 NGF（滴眼液）治疗可改善视力、对比敏感度和 NGF 与两种酪氨酸激酶结合，具有电生理功能，且无不良副作用。重要的是，NGF 与受体 TrkA 和受体 P75NTR（TNF 受体超家族）结合。 TrkA 单独促进 RGC 的存活和增殖，相反，NGF 与 p75NTR 的结合会导致 RGC 的存活和增殖。 rNGF（重组NGF）目前在非人类细胞系统中产生。前序列对于有效折叠或重折叠的重要性，体外（胰蛋白酶）或体内 NGF 前体的体内（弗林蛋白酶）蛋白水解后修饰，以及形成二硫键的要求键合单体和非共价同二聚体，当前制造工艺的表达产量为低，NGF 蛋白质量低。我们开发了一种经济有效且可扩展的表达系统来生产治疗性人类在初步研究中，我们建立了一种来自专有 HEK293 细胞系的蛋白质。 NGF 细胞池的产量比现有表达系统高 10 倍以上体内活性与小鼠野生型 NGF (wtNGF) 相当。优化 NGF 以选择性激活 TrkA 受体而不影响表达产量或然后我们将为 TrkA 选择性 NGF 突变体选择最稳定的 HEK293 细胞克隆，这将适合未来大规模的 cGMP 生产，因为有大量的。啮齿动物和人眼之间的相似性，我们将验证其有效性和安全性建立青光眼大鼠模型的具体目标包括： 1：优化 rNGF 作为 TrkA 特异性激动剂并选择合适的最佳稳定 HEK293 细胞克隆用于未来大规模 cGMP 生产。 2：确定用TrkA选择性NGF突变体局部治疗是否更有效在大鼠青光眼模型中，与 wtNGF 相比，保留了视网膜完整性和功能巩膜外静脉注射高渗盐水。