Abstract
A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer∶DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (p<0.001 for both). Ten formulations exceeded 30% transfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×103-fold and 1.5±0.7×103-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p = 0.003 for RPE/choroid, p<0.001 for neural retina). The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases.
Highlights
Many of the most debilitating ocular diseases are caused by gene deletion mutations
The ability of an ocular disease to be treated with a single gene replacement therapy was shown to be successful in principle in a canine model of Leber’s Congenital Amaurosis in which retinal pigment epithelium (RPE) 65 was replaced using a recombinant adeno associated viral (AAV) delivery system that resulted in visual restoration in these animals [1]
The AAV vector is limited in what ocular diseases it could potentially treat because it is capable of optimally carrying 4.7–4.9 kilobases with a maximum carrying capacity of 5.2 kb [3] while many ocular diseases are caused by mutations to genes that are larger than 5 kb
Summary
Many of the most debilitating ocular diseases are caused by gene deletion mutations. The ability of an ocular disease to be treated with a single gene replacement therapy was shown to be successful in principle in a canine model of Leber’s Congenital Amaurosis in which RPE 65 was replaced using a recombinant adeno associated viral (AAV) delivery system that resulted in visual restoration in these animals [1]. The AAV vector is limited in what ocular diseases it could potentially treat because it is capable of optimally carrying 4.7–4.9 kilobases (kb) with a maximum carrying capacity of 5.2 kb [3] while many ocular diseases are caused by mutations to genes that are larger than 5 kb. Notable examples include Best’s disease, caused by a mutation in Bestrophin-1 (14.6 kb) [4], or Stargardt’s disease, caused by a mutation in the ABCA4 gene (6.8 kb) [5] To address this problem, we recently reported that our non-viral delivery system using poly(beta-amino) esters (PBAEs) can accommodate large inserts to deliver up to 100 plasmids and ,500 kbp of nucleic acid per nanoparticle [6]
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