Light-induced adenovirus gene transfer, an efficient and specific gene delivery technology for cancer gene therapy.

A main issue for further clinical progress of cancer gene therapy is to develop technologies for efficient and specific delivery of therapeutic genes to tumor cells. In this work, we describe a photochemical treatment that substantially improves gene delivery by adenovirus, one of the most efficient gene delivery vectors known. Transduction of two different cell lines was studied by microscopy, flow cytometry, and an enzymatic assay, employing a beta-galactosidase-encoding adenovirus. The photochemical treatment induced a >20-fold increase in gene transduction, compared with conventional adenovirus infection, both when measured as the percentage of cells transduced, and when measured as the total beta-galactosidase activity in the cell population. The effect was most pronounced at lower virus doses, where in some cases the same transduction efficiency could be achieved with a 20 times lower virus dose than with conventional infection. Photochemical treatments are already in clinical use for cancer therapy, and generally are very specific and have few side effects. The photochemical internalization technology described thus has a clear potential for improving both the efficiency and the specificity of gene delivery in cancer gene therapy, making it possible to achieve efficient site-specific in vivo gene delivery by adenoviral vectors.

Photochemically enhanced adenoviral transduction in a multicellular environment.

Photochemical internalization (PCI) enhances adenovirus (Ad) transgene expression in a variety of cell lines in vitro. However, measurements of the photochemical effect on transduction in multicellular environments are lacking. In this study, spheroids of DU 145 prostate cancer cells were used as a model to evaluate Ad serotype 5 (Ad5) transduction in a multicellular environment in response to PCI treatment. Furthermore, the Ad5 was coated with poly(2-methyl-acrylic acid 2-[(2-(dimethylamino)-ethyl)-methyl-amino]-ethyl ester) (pDAMA) to evaluate whether physicochemical properties such as charge and size of viral vectors affect transduction of photochemically treated spheroids. Spheroids incubated with photosensitizer TPPS(2a) (1 microg ml(-1)) and infected with adenovirus contained 3-fold higher percentage of reporter gene expressing cells after exposure to blue light (0.42 J cm(-2)) compared to no light, as analysed by flow cytometry of dissociated spheroids two days after treatment. The cells within the infected spheroids were further divided into three sections corresponding to the interior, intermediate and peripheral layers of the spheroids. This was performed by staining the spheroids with a diffusion-limited dye prior to dissociation. Transduction of cells within photochemically treated and untreated spheroids was heterogeneous, with a radial reduction of transgene expression towards the inner section of the spheroid. The coating of Ad with pDAMA induced up to 2-fold decrease in transduction of cells in the interior section of spheroids compared to uncomplexed Ad, while transduction of the peripheral section remained unchanged. The decrease in transduction could be related to reduced diffusion due to the size of the Ad-pDAMA complexes.

Photochemically enhanced transduction of polymer-complexed adenovirus targeted to the epidermal growth factor receptor.

BACKGROUND: The development of methods for specific delivery of genes into target tissues is an important issue for the further progress of gene therapy. Biological and physical targeting techniques may be combined to redirect gene therapy vectors to specific cells and enhance gene transfer. METHODS: The polymer poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) was conjugated with avidin or poly(ethylene glycol) (PEG) and complexed with adenovirus serotype 5 (Ad5). Targeting of polymer-coated Ad5 to the epidermal growth factor receptor (EGFR) was accomplished by the binding of biotin-EGF to pDMAEMA-avidin. A photochemical treatment procedure using photosensitizer and light was applied to increase transduction with EGFR-targeted viral complexes. RESULTS: pDMAEMA-avidin efficiently enhanced transduction through unspecific viral uptake into cells, while pDMAEMA-PEG provided charge shielding of the complexes and increased the specificity to EGFR when biotin-EGF ligands were used. Transduction of PEG-containing, EGFR-targeted viral complexes was inhibited by 66% in coxsackie and adenovirus receptor (CAR)-deficient RD cells and by 47% in CAR-expressing DU 145 cells in receptor antibody experiments. The photochemical treatment had a substantial effect on transduction, enhancing the percentage of reporter gene positive cells from 20% to 75% of the total viable RD cell population and from 10% to 70% in DU 145 cells. CONCLUSION: Photochemical treatment of cells infected with targeted viral vectors exhibiting a neutral surface charge is a potent method for enhancing transgene expression.