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 (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.
