A Combinatorial Library of Biodegradable Polyesters Enables Non-viral Gene Delivery to Post-Mitotic Human Stem Cell-Derived Polarized RPE Monolayers

Abstract: Safe and effective delivery of DNA to post-mitotic cells, especially highly differentiated cells, remains a challenge despite significant progress in the development of gene delivery tools. Biodegradable polymeric nanoparticles (NPs) offer an array of advantages for gene delivery over viral vectors due to improved safety, carrying capacity, ease of manufacture, and cell-type specificity. Here we demonstrate the use of a high-throughput screening (HTS) platform to synthesize and screen a library of 148 biodegradable polymeric nanoparticles, successfully identifying structures that enable efficient transfection of human pluripotent stem cell differentiated human retinal pigment epithelial (RPE) cells with minimal toxicity. These NPs can deliver plasmid DNA (pDNA) to RPE monolayers more efficiently than leading commercially available transfection reagents. Novel synthetic polymers are described that enable high efficacy non-viral gene delivery to hard-to-transfect polarized human RPE monolayers, enabling gene loss- and gain-of-function studies of cell signaling, developmental, and disease-related pathways. One new synthetic polymer in particular, 3,3′-iminobis(N,N-dimethylpropylamine)-end terminated poly(1,5-pentanediol diacrylate-co-3 amino-1-propanol) (5–3-J12), was found to form self-assembled nanoparticles when mixed with plasmid DNA that transfect a majority of these human post-mitotic cells with minimal cytotoxicity. The platform described here can be utilized as an enabling technology for gene transfer to human primary and stem cell-derived cells, which are often fragile and resistant to conventional gene transfer approaches. Lay Summary: Many retinal diseases are attributable to dysregulation in gene expression or lack of expression of specific genes, allowing for the possibility of prevention or cure of these diseases by effective delivery of nucleic acids coding for the necessary gene to the retina. Delivery of nucleic acids to cells of the retina is challenging due to the non-dividing nature of most retinal cells, preventing DNA from reaching the nucleus. To overcome this barrier, we engineered and tested a library of nanoparticle formulations to identify polymers that enabled safe and effective delivery of nucleic acid cargoes to retinal pigment epithelial cells. The nanoparticle technology explored here has the potential to be utilized for therapeutic delivery of nucleic acids to retinal cells, possibly enabling treatment for otherwise untreatable retinal diseases for which a specific genetic deficit is known but no drugs are available.