Cell penetrating peptide POD mediates delivery of recombinant proteins to retina, cornea and skin

Abstract

Recently we described a novel cell penetrating peptide, POD (peptide for ocular delivery) that could deliver small molecules including fluorescent dyes into retinal cells. The objective of the current study was to examine whether biologically relevant macromolecules such as proteins, genetically fused with POD could also be delivered into retinal tissues in vivo. We generated a POD-GFP fusion protein and examined its cell and tissue penetrating properties. We found that endogenously expressed POD-GFP fusion protein localized to the nucleus, suggesting that POD acts as a nuclear localization signal. Adenovirus (Ad) vectors expressing POD-GFP fusion protein were constructed and the recombinant protein was purified from Ad-infected human embryonic retinoblasts (HER). Exogenously supplied POD-GFP fusion protein rapidly transduced A549 and HER cells and colocalized in part with markers of late endosomes, from which it could escape. Following subretinal delivery, POD-GFP localized to the retinal pigment epithelium and the photoreceptor cell bodies. When injected into the vitreous, POD-GFP localized to the ganglion cells and the inner nuclear layer of the retina as well as the lens capsule. Topical application of POD-GFP to ocular surfaces resulted in uptake by the corneal epithelium. POD-GFP also transduced non-ocular tissues, including the epidermis of the skin following topical application.

Corneal Penetrating Elastin-Like Polypeptide Carriers

“…POD was used to deliver green fluorescent protein (GFP) to the mouse cornea after topical application, where it entered the corneal epithelium, but was not detected in the corneal stroma. 31 Furthermore, a POD-fused polyethylene glycol nanoparticle was able to bind plasmid DNA and achieve transgene expression in the optic nerve head following subretinal injection. 32 These data, along with the studies on conventional CPPs, suggest a potential role for the use of CPPs to enhance uptake or corneal penetration of drugs following topical ocular application.…”

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).…”

Dendrimers for gene delivery – a potential approach for ocular therapy?

“…The dendrimer consistently yielded the highest gene transfer efficiency into primary human RPE cells, with the DNA concentration and DNA : dendrimer concentration playing a major role in the transfection efficiency. In the same year, Hudde et al [99] investigated activated PAMAM dendrimers for the delivery of plasmids [83] PLGA nanoparticles pSEC.shRNA (VEGF-A) Corneal neovascularization [84] Human serum albumin nanoparticles pDNA (SOD1) Age-related macular degeneration [85] CS-g-(PEI-b-mPEG) nanoparticles siRNA (IKKb) Bleb survival after trabeculectomy [86] PLA and PLGA nanoparticles pDNA (GFP, RNFP) Bovine primary RPE cells, human RPE cells (ARPE-19) [87] PLGA microspheres RNA aptamer (VEGF) Human umbilical vein endothelial cells (HUVEC) [88] Hyaluronic acid chitosan nanoparticles pDNA (EGFP or b-gal) Human corneal epithelial cells (HCEC) and normal human conjunctival (IOBA-NHC) cells [89] Calcium phosphate nanoparticles pDNA (EGFP) (with PEI) Immortalized human corneal endothelial cells (HCEC) [90] (B) Liposomes DOTAP/DOPE/PS/PEI pDNA (SEAP) Human RPE cells (ARPE-19) [91] DOTAP/DOPE/ DSPE-PEG or ceramide-PEG pDNA (Luciferase) Human RPE cells (407) [92] DOTMA/DOPE or DOTMA/cholesterol pDNA (Luciferase) Rabbit eye [93] Phospholipid/PEG-DSPE/cholesterol ODN Rabbit eye [94] (C) Dendrimers PLL dendrimers ODN (VEGF) Choroidal neovascularization [95][96][97] PAMAM dendrimers pDNA (GFP) Human primary RPE cells [98] PAMAM dendrimers pDNA (TNFR-Ig) Human or rabbit corneas [99] PLL and PEG-PLL dendrimers pDNA (Luciferase) Human RPE cells (D407) [100] (D) Others PEI PS-AsODN (anti-TGFb2) Rat retinal Müller glial cells [101] Cell penetrating peptide pDNA (GPFHis) Human embryonic retinoblast (HER) cells [102] Dihydrazide derivatized hyaluronic acid Nucleic acid (HAS) Dry eye syndrome [103] Cationic nanoemulsion AsODN (VEGF-R2) Corneal neovascularization [104] Cationic core-shell liponanoparticles pDNA (EGFP) Human conjunctival epithelial cells and rabbit eye [105] Electrotransfer to ciliary muscle pDNA (hTNFR-Is) Inflammatory, degenerative or angiogenic diseases [106] Ultrasound with DOTAP/DOPE/ DSPE-PEG liposomes pDNA (Luciferse) Bovine neural retina [107]…”