Optimizing NGF for Topical Treatment of Glaucoma

青光眼是世界上最主要的致盲性疾病之一。这种慢性和渐进的 视神经病变的特征在于视网膜神经节细胞（RGC）轴突的丧失， 构成视神经。降低升高的眼内压， 致病因素，减缓疾病的发作和进展，但没有治疗方法 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， 对中枢和周围神经的神经元发挥营养和分化活性 在退行性疾病中观察到具有保护和/或再生作用的系统，或 受伤后。局部应用于眼睛的神经生长因子已被证明可以显着防止RGC 在实验性大鼠青光眼模型中的变性。在3例晚期青光眼患者中， 局部用NGF（滴眼液）治疗可改善视敏度、对比敏感度和 电生理功能而没有不希望的副作用。NGF结合酪氨酸激酶 受体TrkA和受体P75 NTR（TNF受体超家族）。重要的是，NGF与 TrkA单独促进RGC的存活和增殖。相反，NGF与p75 NTR的结合导致 到凋亡。rNGF（重组NGF）目前在非人细胞系统中产生。由于 原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（胰蛋白酶） pro-NGF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 结合的单体和非共价同二聚体，来自当前生产工艺的表达产率是 NGF蛋白质含量低。 我们已经开发了一种具有成本效益和可扩展的表达系统，以产生治疗性人类 来自专有HEK 293细胞系的蛋白质。在初步研究中，我们建立了一个 NGF的细胞池，其表现出比目前的表达系统高>10倍的产量， 体内活性与鼠野生型NGF（wtNGF）相当。在这项研究中，我们将首先 优化NGF以选择性激活TrkA受体而不损害表达产率，或 蛋白质稳定性然后我们将选择最稳定的HEK 293细胞克隆用于TrkA选择性NGF突变体， 这将适用于未来的大规模cGMP生产。由于有大量 啮齿动物和人类眼睛之间的相似性，我们将验证有效性和安全性， 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，并选择最稳定的HEK 293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， 巩膜外静脉注射高渗盐水。

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.