A poly(ethylene) glycolylated peptide for ocular delivery compacts DNA into nanoparticles for gene delivery to post‐mitotic tissues in vivo

Abstract

Background We have previously shown that a novel synthetic peptide for ocular delivery (POD) can efficiently compact DNA and deliver it to cells in vitro. This observation prompted us to develop use of POD as a nonviral vector in vivo. Methods POD peptide was modified using poly(ethylene) glycol (PEG-POD) and used to compact DNA into nanoparticles that were then analysed using electron microscopy, dynamic light scattering, and fluorescent labeling. Transfection efficiency and localization were determined 48 h post-injection into the subretinal space of the mouse eye using luciferase and LacZ, respectively. Efficiency of ocular transfection was compared to two other PEGylated peptides: PEG-TAT and PEG-CK30. Results PEG-POD can compact DNA and form discrete nanoparticles of approximately 136 nm that can penetrate and transduce the retinal pigment epithelium (RPE) in vivo. PEG-POD significantly increased expression of plasmid DNA by 215-fold, PEG-TAT by 56.52-fold, and PEG-CK30 by 24.73-fold relative to DNA injected alone. In all cases β-galactosidase was observed primarily in the RPE layer after subretinal injection. Electrophysiological analyses of PEG-POD transduced retina indicates an absence of PEG-POD-mediated toxicity. PEG-POD can protect plasmid DNA from DNaseI digestion, resulting in significant transfection of the lung after intravenous injection in mice. Conclusions PEG-POD was found to significantly increase gene delivery relative to both DNA alone and other pegylated peptides. These findings highlight the use of pegylated peptides, and specifically PEG-POD, as novel gene delivery vectors.

Reducible PEG-POD/DNA Nanoparticles for Gene Transfer In Vitro and In Vivo: Application in a Mouse Model of Age-Related Macular Degeneration

“…Previously we attached a 10 kDa PEG to the free sulfhydryl group on the N-terminal cysteine residue of POD using the maleimide group of methoxy-PEG-maleimide (mPEG-MALM) 18 . This PEG-POD conjugation, involving a thioether bond, is hereafter referred to as PEG-S-POD.…”

“…Previously, we have described a novel peptide known as peptide for ocular delivery (POD) that can efficiently compact DNA and enable gene transfer to cells in culture 17 . These POD/DNA nanoparticles, however, failed to deliver DNA to post-mitotic cells in vivo 18 . We determined that the addition of polyethylene glycol (PEG) to POD prior to complexing with DNA led to the formation of homogeneous nanoparticles of approximately 120–150 nm in size, not substantially larger than an adenovirus (∼100 nm) 18 .…”

“…These POD/DNA nanoparticles, however, failed to deliver DNA to post-mitotic cells in vivo 18 . We determined that the addition of polyethylene glycol (PEG) to POD prior to complexing with DNA led to the formation of homogeneous nanoparticles of approximately 120–150 nm in size, not substantially larger than an adenovirus (∼100 nm) 18 . We found that these PEGylated POD/DNA nanoparticles could now enable delivery of DNA to retinal cells of mice and attenuate the progression of light-induced apoptosis and ensuing retinal degeneration in a murine model of retinitis pigmentosa 18, 19.…”

Microspheres and Microcapsules for Protein Drug Delivery

“…Furthermore, PEGylation of polymers or particles helped to prevent the particles from aggregating in vitreous (212). A cell-penetrating peptide for ocular delivery (POD)[CGGG(ARKKAAKA)4], which could bind the plasma membrane of human embryonic retinoblasts and enter the cytoplasm, was PEGylated and used to compact DNA into nanospheres for ocular gene delivery (213). The PEG-POD significantly increased the expression of plasmid DNA by 215-fold compared to the DNA injected alone.…”

Vector platforms for gene therapy of inherited retinopathies

“…The most relevant NP tested so far are peptides for ocular delivery (POD) and CK30PEG-NP. POD is a cell-penetrating peptide that was demonstrated to enter retinal cells in vivo (Read et al, 2010b), and POD nanoparticles localize to RPE and PR following subretinal injection (Johnson et al, 2008b, 2010). POD nanoparticles conjugated with polyethylene glycol (PEG–POD) containing an expression cassette for GDNF and injected subretinally result in reduction of ONL apoptosis induced by blue-light (Read et al, 2010a).…”