Overcoming gene delivery barriers to the back of the eye

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ABSTRACT Gene therapy strategies including augmentation, editing or knockdown, can lead to restoration of vision for patients suffering from inherited retinal degenerations (IRDs). Viral and non-viral vectors efficiently deliver genes through the subretinal route of administration, but this requires a surgical procedure that detaches the fragile degenerating retina. Intravitreal injections offer a safer ocular route for gene delivery and is a widely used outpatient procedure, but significant biological barriers limit the transport of carriers from the vitreous chamber to the back of the eye. The vehicle, once delivered in the vitreous chamber, will have to move through the vitreous humor, which is composed of many proteins that restrict diffusion. If intact, the vehicle must then penetrate an inner limiting membrane functioning as a sieve and restricting permeability of substances into the retina. Finally, once the vehicle hits the target cell population, it must get internalized and then escape the endosome to allow for cytosolic delivery of nucleic acids. After subretinal injection, clinically approved lipid nanoparticles (LNPs) carrying mRNA can mediate protein expression in the retinal pigment epithelium (RPE) and photoreceptors, the cell types mainly afflicted in IRDs. However, our preliminary data show that these LNPs have limited expression post-intravitreal injection. There is a critical need to develop carriers that can traverse the retinal barriers after intravitreal injection. Our long-term goal is to develop novel, peptide targeted LNP systems that can deliver genes to the outer retina after an intravitreal injection with limited toxicity. We hypothesize that nanoparticle physicochemical properties like surface charge, size, stability, and ionizability will be the critical determinants that enhance permeation towards the retina. We posit that peptides can penetrate and target specific cell types within the outer retina. Our main objectives are to 1) evaluate the physicochemical parameters of nanoparticles that are a prerequisite for gene delivery to the outer retina and 2) identify peptides and their structural features that allow for cell-specific delivery. Individually evaluating each physicochemical characteristic of LNPs and all peptide moieties available is arduous. Thus, we will generate a diverse DNA barcoded LNP library and use a phage display peptide library to identify optimal nanocarriers and peptides, respectively. Most nanocarriers have had limited translation potential due to species-specific difference in ocular barriers. Therefore, we are evaluating our nanocarriers in an ex-vivo non-human primate (NHP) model for rapid screening of multiple vectors after intravitreal delivery. Overall, this application will 1) identify novel peptides and structural features of LNPs that enable gene delivery to the outer retina post- intravitreal administration, and 2) generate a translational, transfection efficiency platform that can be widely used for the evaluation of gene delivery systems.