Sustained Release of Sh3-RdCVF to Rescue Cone Photoreceptor Death in Retinitis Pigmentosa
Authors: Lia Huo1, Carter Teal1, Daniela Isaacs1, Margaret Ho1, Thierry Léveillard2, Shira Albeck3, Tamar Unger3, Robert Fluhr3, Anubhav Garg4, Molly Shoichet1. 1University of Toronto, 2Institut de la Vision, 3Weizmann Institute of Science, 4McMaster University.
Author Disclosures: L. Huo: None. C. Teal: None. D. Isaacs: None. M. Ho: None. T. Léveillard: None. S. Albeck: None. T. Unger: None. R. Fluhr: None. A. Garg: None. M. Shoichet: None.
Abstract Body:
Purpose: Retinitis Pigmentosa (RP) is a group of inherited retinal degenerative diseases that affects nearly 2 million people worldwide. In RP, an initial loss of night vision caused by the death of rod photoreceptors eventually progresses to the loss of high acuity vision due to the apoptosis of cone photoreceptors. Among other mechanisms, this two-phase degeneration is thought to be caused by the loss of rod-secreted protein factors that are necessary for cone survival. One recently discovered protein - rod-derived cone viability factor (RdCVF) - has been shown to increase cone viability both in vitro and in vivo. Though RdCVF is a promising therapeutic that could rescue dying cones, it cannot be translated to the clinic because of its rapid clearance, limited bioavailability, and the need for repeated invasive injections. Therefore, there is a need to develop a drug delivery system that can sustain the release of protein factors to the retina locally, while reducing the number of ocular injections. Affinity-based systems are a method for controlling release, whereby a drug-binding ligand is immobilized onto a polymer backbone and the non-covalent interactions between the ligand and the drug slow protein release. We previously developed a biocompatible hyaluronan (HA)-oxime hydrogel that is very stable due to its oxime crosslinks and displays properties similar to that of the vitreous. Here, we demonstrate protein release from this hydrogel for the first time, by modifying the HA-oxime gel with Sh3 binding peptides and expressing RdCVF with a Src homology 3 (Sh3) domain to slow protein release through Sh3 and Sh3-binding peptide interactions.
Study Design:
Methods: The delivery system was synthesized by incorporating Sh3-RdCVF in an injectable hyaluronan-based hydrogel crosslinked via oxime ligation, whereby Sh3-binding peptides were covalently bound to the polymer backbone through an inverse electron demand Diels-Alder ligation. To investigate tunability of release, the hydrogel was made with increasing molar excesses of Sh3-binding peptide to Sh3-RdCVF, and released protein was quantified using an ELISA at different time points for 14 days. Bioactivity was then tested on E6 chick retinal dissociates stained for the cone-specific marker visinin to assess if our hydrogel would affect the function of the released protein.
Results: Our results show sustained release of Sh3-RdCVF from our delivery system with tunability dependent on peptide to protein ratio: as the molar excess of Sh3 binding peptide increases with respect to the Sh3-RdCVF protein, the slower the protein release. Beyond prolonging protein release, our hydrogel formulation also extends linear diffusion and decreases diffusivity. Furthermore, the released protein is bioactive as shown by an increase in cone viability in chick retinal dissociates.
Conclusions: In conclusion, the release of RdCVF is not only controllable with our affinity-based release system, but also bioactive, thereby laying the foundation for in vivo studies in disease models of RP. Beyond RP, this versatile injectable hydrogel could be explored in many other retinal degenerative diseases and circumvent repeated invasive intravitreal or subretinal drug injections.
