Graduate Thesis Or Dissertation
 

Non-viral Vectors for Ocular Gene Delivery

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  • Nucleic acid therapeutics has cemented itself as one of the most efficacious and robust avenues for treatment of a wide range of diseases from vaccine development to treatment of liver, retina, cancer applications. Non-viral mediated gene delivery using lipid-based nanoparticle (LNP) carriers has proven its compatibility with addressing unexpected, fast paced health problems afflicting humans around the world as is the case with the COVID19 pandemic. Although LNPs continue to be ferociously developed for treatments of disease, challenges remain for the wide application of these carriers to diseases without druggable strategies. Among these we find that cell-specific targeting, endosomal escape efficacy as well as delivery of wide range of nucleic acid cargos is far from optimal. A more comprehensive knowledge of the mechanisms and diverse strategies to elucidate previous unknowns of these challenge areas would aid in the further development of LNP-based nucleic acid therapeutics and possibly result in the application to orphan diseases as of today. In this dissertation, we set out to find ways of improving mRNA gene delivery, LNP tissue penetration and specificity to the retina. We employed peptide phage display molecular tools in order to find promising candidates that can achieve reliable delivery of mRNA to the back of the eye. We examined peptide ligands’ abilities to selectively target, bind, and internalize into target cell populations and we then validated said candidates in animal models using fluorescent tags. We proceeded to select the best performing peptide ligands from our screen and conjugated them on the surface of LNPs and observed promising gene delivery patterns in mouse and non-human primate (NHP) retina. Of note, we have identified a peptide ligand, MH42, which shows substantial gene expression in crucial NHP retina cell layers such as photoreceptor nuclei, retinal pigment epithelium and Müller glia. These results merit the further exploration in high-relevance animal models such as NHPs, both mechanistically and physiologically to advance gene therapy for the retina. Encouraged by the dynamics of mRNA delivery in vivo, we proceeded to expand our understanding of the endosomal escape dynamics once LNPs have internalized in cells. We employed an endogenous lectin sensor, Galectin-8 (Gal8), and fused it with a green fluorescent protein reporter in order to probe for endosomal escape rate differences across different LNP formulations. We stably transfected cells and established a Gal8-GFP reporter cell in which Gal8 redistribution following endosomal escape events provided an easily detectable readout compatible with downstream applications. We were able to confirm that cholesterol analog substitution inside of our multi-component LNPs in fact led to increase endosomal escape using live-cell confocal imaging. We also confirmed that our enhanced LNP (eLNP) formulation boasted a poly-faceted profile with defined edges which we postulated destabilized the particle ever-slightly and contributed to the increased endosomal escape and consequent gene expression observed. We also confirmed the temporal characteristics of the endosomal escape events we visualized in our reporter cell system by co-localizing said events with late endosomal markers. This further bolstered the rigor of the Gal8-GFP sensor to detect endosomal escape events reliably with a high background to noise ratio making it an ideal cell reporter to test any novel formulations of interest. Additionally, we hypothesized that with our mechanistic knowledge of the delivery kinetics and gene expression capabilities of eLNPs, we could accomplish delivery of larger mRNA cargos such as prime editors, which has not been reported previously. We tested the encapsulation parameters and ratios of RNA species to be co-encapsulated in an effort to prime the non-viral delivery of these late-generation precise genome editors. We reported successful co-entrapment of large prime editor (PE) mRNA as well as corresponding prime editing guideRNA (pegRNA) and a nicking small guideRNA (nsgRNA) and followed up with efficient prime editing as evidenced using fluorescence-activated cell sorting analysis. We achieved upwards of 53% prime editing rates with our best performing eLNP-prime editor system which contained two guides, PE mRNA as cargos while the eLNP had fully substituted ß-sitosterol as the structural lipid for enhanced endosomal escape. We showed that our eLNPs are robust at encapsulating heterogeneous cargoes at different ratios but also capable of successfully delivering these past the endosomal barriers for genome manipulation. This works contributes to the continued development of non-viral delivery avenues for prime editing of target cells and one day animal models. Our findings systematically address the three main limitations of LNP-mediated delivery of mRNA: 1. Cell-specificity is improved via peptide ligands on surface of LNPs, 2. Improved endosomal escape is corroborated when comparing standard, cholesterol-based LNPs with our ß-sitosterol eLNPs in our Gal8-GFP cell reporter system, and 3. Proof of principle studies provide the first report of delivery of large, heterogeneous cargoes with highly efficacious prime editing rates in reporter cells. Altogether we have attempted to inch closer to solving the biggest impediments for LNP-mediated nucleic acid delivery for therapeutic applications.
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  • This project was supported through funding from the National Eye Institute 1R21EY031066 (G.S.), ONPRC Pilot Grant (R.C.R.), ONPRC Core Grant P51 OD011092 and S10RR024585, CEI Core Grant P30 EY010572 from the National Institutes of Health
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  • Pending Publication
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  • 2022-06-11 to 2023-07-12

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