The use of synthetic RNA for research purposes as well as RNA-based therapy and vaccination has gained increasing importance. Given the anatomical seclusion of the eye, small interfering RNA (siRNA)-induced gene silencing bears great potential for targeted reduction of pathological gene expression that may allow rational treatment of chronic eye diseases in the future. However, there is yet an unmet need for techniques providing safe and efficient siRNA delivery to the retina. We used magnetic nanoparticles (MNPs) and magnetic force (Reverse Magnetofection) to deliver siRNA/MNP complexes into retinal explant tissue, targeting valosin-containing protein (VCP) previously established as a potential therapeutic target for autosomal dominant retinitis pigmentosa (adRP). Safe and efficient delivery of VCP siRNA was achieved into all retinal cell layers of retinal explants from the RHO P23H rat, a rodent model for adRP. No toxicity or microglial activation was observed. VCP silencing led to a significant decrease of retinal degeneration. Reverse Magnetofection thus offers an effective method to deliver siRNA into retinal tissue. Used in combination with retinal organotypic explants, it can provide an efficient and reliable preclinical test platform of RNA-based therapy approaches for ocular diseases.

The treatment of retinal diseases by intravitreal injections requires frequent administration unless drug delivery systems with long retention and controlled release are used. In this work, we focused on pullulan (≈67 kDa) conjugates of dexamethasone as therapeutic systems for intravitreal administration. The pullulan–dexamethasone conjugates self-assemble into negatively charged nanoparticles (average size 326 ± 29 nm). Intravitreal injections of pullulan and pullulan–dexamethasone were safe in mouse, rat and rabbit eyes. Fluorescently labeled pullulan particles showed prolonged retention in the vitreous and they were almost completely eliminated via aqueous humor outflow. Pullulan conjugates also distributed to the retina via Müller glial cells when tested in ex vivo retina explants and in vivo. Pharmacokinetic simulations showed that pullulan–dexamethasone conjugates may release free and active dexamethasone in the vitreous humor for over 16 days, even though a large fraction of dexamethasone may be eliminated from the eye as bound pullulan–dexamethasone. We conclude that pullulan based drug conjugates are promising intravitreal drug delivery systems as they may reduce injection frequency and deliver drugs into the retinal cells.

Rhodopsin (RHO) misfolding mutations are a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). The most prevalent mutation, RHO^P23H, results in its misfolding and retention in the endoplasmic reticulum (ER). Under homeostatic conditions, misfolded proteins are selectively identified, retained at the ER, and cleared via ER-associated degradation (ERAD). Overload of these degradation processes for a prolonged period leads to imbalanced proteostasis and may eventually result in cell death. ERAD of misfolded proteins, such as RHO^P23H, includes the subsequent steps of protein recognition, targeting for ERAD, retrotranslocation, and proteasomal degradation. In the present study, we investigated and compared pharmacological modulation of ERAD at these four different major steps. We show that inhibition of the VCP/proteasome activity favors cell survival and suppresses P23H-mediated retinal degeneration in RHO^P23H rat retinal explants. We suggest targeting this activity as a therapeutic approach for patients with currently untreatable adRP.

Introduction: Small interfering RNA (siRNA) is an important RNA interference tool that has found significant application in ocular therapy. For this reason, there is a considerable demand of siRNA delivery platforms able to effectively transfect cells within the retina. Up to now several strategies have been investigated for this application such as lipids, nanoparticles and polymers but in all the cases invasive injections are the only administration routes explored. Moreover, a major limitation shared by all these delivery systems is the lack of active targeting toward the posterior segment of the eye that hampers high transfection efficiencies.Purpose: We aim to synthesize and formulate a new magnetic nanoparticles‐mediated delivery technology that shall allow safe and efficient siRNA delivery to the retina assisted by magnetic targeting.Methods: (1) Cationic MNPs have been synthesized, characterized, and their ability to form complexes with siRNA has been demonstrated by Zeta Potential. (2) The in vitro MNPs mediated gene silencing have been tested on green fluorescent protein (GFP)‐stably transfected cells (661W & RPE). Also, gene silencing of the endogenous glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) gene has been monitored. (3) Finally, the in vitro toxicity of the formulations has been determined in term of apoptosis, cell proliferation and protein production.Results: MNPs were able to efficiently complex siRNA while maintain their size and charge. MNPs complexed with siRNA achieved high transfection efficiency and gene silencing of GFP as shown by fluorescence microscopy and flow cytometry. The knock‐down efficiency has been confirmed by targeting GAPDH by immunohistochemistry. Finally, proliferation, apoptosis and total protein content assays demonstrate the full biocompatibility of these gene delivery platforms.Conclusions: Magnetic nanoparticles are a promising platform for gene therapy in the context of ocular therapy and allow to achieve high transfection efficiencies in retinal cell lines.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No 722717.