Optimizing NGF for Topical Treatment of Glaucoma

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 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 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 P NTR TNF receptor superfamily Importantly the binding of NGF to TrkA alone promotes RGC s survival and proliferation In contrast binding of NGF to p NTR 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 HEK cell line In the preliminary studies we have established a cell pool of NGF which exhibited andgt 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 HEK 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 To optimize rNGF as a TrkA specific agonist and select top stable HEK cell clones suitable for future large scale cGMP manufacturing 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 We will improve nerve growth factor and develop a cost effective stable eye drop for chronic glaucoma treatment