Extending intravitreal retention of CNTF via fusion to the HA-binding protein VG1

Glaucoma is the leading cause of permanent blindness worldwide. Vision loss in glaucoma stems from the loss of retinal ganglion cells (RGC). Ciliary neurotrophic factor (CNTF) has been shown to protect RGCs in pre-clinical models of glaucoma. However, CNTF has a short ocular retention time following intraocular injections due to rapid diffusion. To address this, we developed a fusion protein between CNTF and the hyaluronic acid- (HA) binding G1 domain of endogenous extracellular matrix (ECM) protein, versican (VG1; CNTF-VG1). We hypothesize that a CNTF-VG1 protein will have an extended ocular retention time compared to CNTF due to HA-binding with endogenous vitreous-HA. We demonstrate that CNTF-VG1 non-covalently binds HA with high affinity and is bioactive. We show that the vitreous and thermal stability of CNTF-VG1 can be significantly increased through co-formulation with HA. In an in vivo pharmacodynamic study, we show that VG1 significantly increases the window of neuroprotective JAK/STAT3 pathway activation in un-injured mice (C57BL/6J, female) retina. Finally, we close our work by showing that CNTF is neuroprotective of primary mouse cortical neurons in an acute H2O2 cytotoxicity model. Taken together, our work demonstrates the potential of HA-binding domain VG1 to improve the ocular retention of neuroprotective CNTF following intraocular injection.