title: Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide.
abstract: Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (x=81.51%) was significantly better (P=0.0036) than those that possessed 4 positive charges (x=56.8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P<0.0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases.
authors.affiliation: Centre for Ophthalmology and Visual Science, Lions Eye Institute, University of Western Australia, 2 Verdun Street, Nedlands, WA 6009, Australia.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Marano
authors.forename: Norbert
authors.initials: N
authors.lastname: Wimmer
authors.forename: Philip S
authors.initials: PS
authors.lastname: Kearns
authors.forename: Bradley G
authors.initials: BG
authors.lastname: Thomas
authors.forename: Istvan
authors.initials: I
authors.lastname: Toth
authors.forename: Meliha
authors.initials: M
authors.lastname: Brankov
authors.forename: P Elizabeth
authors.initials: PE
authors.lastname: Rakoczy
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D020256:Choroidal Neovascularization / Q000378:metabolism* / Q000517:prevention & control*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D013329:Structure-Activity Relationship; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
chemical_list: D009841:Oligonucleotides; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A