The role of cell cycle on polyplex-mediated gene transfer into a retinal pigment epithelial cell line

✍ Scribed by Marjo Männistö; Seppo Rönkkö; Mikko Mättö; Paavo Honkakoski; Mika Hyttinen; Jukka Pelkonen; Arto Urtti

• Publisher: John Wiley and Sons
• Year: 2004
• Tongue: English
• Weight: 225 KB
• Volume: 7
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.693

✦ Synopsis

Abstract

Background

Retinal pigment epithelium (RPE) maintains the function of photoreceptors and eyesight and is an important target for gene delivery. Since in some diseases RPE cells proliferate uncontrollably, we investigated the role of cell cycle in non‐viral gene delivery into a RPE cell line (D407).

Methods

D407 (human) cells were transfected with cationic DNA complexes. Cells were synchronized into different phases of the cell cycle and transfected using poly‐L‐lysine (PLL) or polyethyleneimine (PEI) carriers for 3 h. The effects of different reporters (β‐galactosidase, luciferase) or promoters (CMV, SV40, tk, PDE‐β) on gene expression were evaluated 43 h later. Cellular uptake of ethidium monoazide/DNA complexes with PLL or PEI was determined by flow cytometry. Fluorescent DNA and the complexes were localized with a confocal microscope. The role of cell cycle in transcription was evaluated by stable luciferase‐expressing cells.

Results

PLL showed lower transfection levels than PEI in synchronized cells and only slight dependence on cell cycle. PEI showed minimal efficiency at G1 phase and maximum level at S phase. All promoters and reporter genes showed dependence on cell cycle. Cellular uptake of polyplexes was highest at S phase (80–90%) and lowest at G1 phase (5–30%). Confocal microscopy showed minor differences of free DNA between groups in the nucleus, where it was largely carrier‐bound. Cell cycle effects on luciferase expression were clear in stable cell line

Conclusions

Transfection by polyplexes in the RPE cell line is influenced by cellular uptake and transcription, and both processes are cell‐cycle‐dependent. The results have implications in retinal gene therapy. Copyright © 2004 John Wiley & Sons, Ltd.