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S. P. Read, S. M. Cashman, R. Kumar-Singh; POD Peptide Compacts DNA into Nanoparticles and Permits Gene and Recombinant Protein Delivery to Ocular Tissues in vivo. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1727.
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© ARVO (1962-2015); The Authors (2016-present)
In order to develop a non-viral gene and protein delivery vector, we designed the novel synthetic cationic peptide POD, or "protein for ocular delivery," shown to be a cell-penetrating peptide (CPP).We compared the efficacy of POD in delivering DNA with that of other peptides as well as its ability deliver a fusion protein to post-mitotic ocular tissues in vivo.
POD peptide was PEGylated (PEG-POD) and used to compact DNA into nanoparticles. Nanoparticles were analyzed using TEM, DLS, and plasmid labeling. Transfection efficiency and localization were determined 48 hours post-injection into the subretinal space of mice using luciferase and LacZ, respectively. Efficiency of ocular transfection was compared to two other CPPs, PEG-TAT and PEG-polylysine (PEG-CK30). In addition, particles were assayed for serum stability using DNaseI. A POD-GFP fusion protein was injected into the subretinal space, intravitreally, and applied topically to the cornea and uptake compared to GFP protein. Gross toxicity was assayed using electroretinograms (ERGs) performed 48 hours after injection.
PEG-POD was able to compact DNA and form nanoparticles of 136±27.2nm that could penetrate the RPE in vivo. PEG-POD significantly increased expression of plasmid DNA 215-fold (p<0.0001), PEG-TAT 56.52-fold (p<0.05) and PEG-CK30 24.73-fold (p<0.05) relative to DNA injected alone. In all cases β-galactosidase was observed primarily in the RPE. PEG-POD protects plasmid DNA from DNaseI digestion. Unlike GFP, POD-GFP efficiently transduced the retinal pigment epithelium (RPE) and outer nuclear layer (ONL) by subretinal delivery, ganglion cell layer (GCL) and ONL by intravitreal injection, and corneal epithelium when applied topically. In some cases, retinal transduction could be observed in up to 40% of the ONL. Analysis of the ERGs showed no significant difference between fusion protein, nanoparticle, or buffer injected eyes (p>0.05).
PEG-POD was found to significantly increase gene delivery relative to both DNA alone and other PEGylated peptides. Our results indicate that POD fusion proteins are able to deliver macromolecules to the retina and cornea and may have a significant therapeutic application in gene and protein delivery to these tissues. These findings highlight the use of PEGylated CPPs, and specifically POD, as novel gene and protein delivery vectors.
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