Affinity-Based Hydrogels for the Sustained Delivery of Cone-Protective Factors to Promote Cone Survival in Retinitis Pigmentosa

Retinitis pigmentosa (RP) is the leading cause of inherited blindness, affecting nearly 2 million people worldwide. As the rod photoreceptors degenerate, peripheral and night vision is lost, followed by the death of cone photoreceptors that are responsible for daylight and high acuity central vision. RP is heterogenous and is caused by 3000 mutations in more than 70 genes, making a gene-agnostic treatment compelling. Rods secrete rod-derived cone viability factor (RdCVF), which protects cones. Supplementing exogenous RdCVF, as intrinsic levels decrease, holds great therapeutic potential; however, the rapid clearance, limited bioavailability, and required repeated subretinal injections hinder the translation of RdCVF to the clinic. To address this, we synthesized a hyaluronan-oxime crosslinked hydrogel functionalized with Src homology 3 (Sh3) binding peptides, enabling sustained release of an engineered Sh3-RdCVF fusion protein through reversible affinity interactions. This slow-release RdCVF (SR-RdCVF) was evaluated in three distinct RP models: an autosomal recessive rd1 mouse harboring a nonsense mutation in the rod phosphodiesterase -subunit (Pde6b), leading to cyclic guanosine monophosphate cGMP build-up; an autosomal dominant P23H rat characterized by a missense mutation in rhodopsin,leading to its misfolding; and human retinal organoids derived from patients with non-syndromic autosomal recessive RP with a mutation in usherin, leading to truncated usherin and structural
deficits in photoreceptor connecting cilia. We demonstrated that a single intravitreal injection of
our slow-release system sustained the benefit of RdCVF for at least 3 weeks in rd1 mice whereas
a single RdCVF bolus injection was cleared within a week. Our SR-RdCVF led to increased
cone survival in rd1 mice at 3 and 4 weeks post-injection, and functional improvement at 4
weeks post-injection. SR-RdCVF also increased cone survival and outer segment length in P23H
rats; however, no cone functional rescue was observed. Moreover, we demonstrated, for the first
time, that human cone photoreceptors respond to RdCVF and increase gene expression of cone
and outer segment markers in USH2A patient-derived retinal organoids when treated with SRRdCVF for 2 weeks. Thus, the release of RdCVF from an affinity-based hydrogel improved cone
survival in three models of RP with a single administration and outperformed bolus RdCVF that
failed to confer cone protection.

100 µL of HAO or HOPE (0.4 cm²) were allowed to gel as a cylindrical shape overnight at 37 °C in a 16-well chamber slide and carefully removed by detaching the glass slide bottom. The compressive moduli of HAO and HOPE were then measured using a Mach-1 micromechanical system (Biomomentum) connected to a Universal Motion Controller (Newport). The sample height was measured with an initial force of 0.01 N. The compressive modulus was determined by applying an additional 10% strain in five sequential 2% strain steps. The slope of the resulting stress-strain curves for each sample was averaged to calculate the compressive modulus.