Light Directed Gene Transfer by Photochemical Internalisation

Abstract

Numerous gene therapy vectors, both viral and non-viral, are taken into the cell by endocytosis, and for efficient gene delivery the therapeutic genes carried by such vectors have to escape from endocytic vesicles so that the genes can further be translocated to the nucleus. Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer by many such vectors. To improve endosomal escape we have developed a new technology, named photochemical internalisation (PCI). In this technology photochemical reactions are initiated by photosensitising compounds localised in endocytic vesicles, inducing rupture of these vesicles upon light exposure. The technology constitutes an efficient light-inducible gene transfer method in vitro, where light-induced increases in transfection or viral transduction of more than 100 and 30 times can be observed, respectively. The method can potentially be developed into a site-specific method for gene delivery in vivo. This article will review the background for the PCI technology, and several aspects of PCI induced gene delivery with synthetic and viral vectors will be discussed. Among these are: (i) The efficiency of the technology with different gene therapy vectors; (ii) use of PCI with targeted vectors; (iii) the timing of DNA delivery relative to the photochemical treatment. The prospects of using the technology for site-specific gene delivery in vivo will be thoroughly discussed, with special emphasis on the possibilities for clinical use. In this context our in vivo experience with the PCI technology as well as the clinical experience with photodynamic therapy will be treated, as this is highly relevant for the clinical use of PCI-mediated gene delivery. The use of photochemical treatments as a tool for understanding the more general mechanisms of transfection will also be discussed.

Photochemically enhanced gene transfection increases the cytotoxicity of the herpes simplex virus thymidine kinase gene combined with ganciclovir

“…[1][2][3] Recently, we have developed a technology, generally named photochemical internalization (PCI), for the specific light-directed delivery of different macromolecules, including proteins and genes, into the cytosol of target cells. 4,5 The method is based on photochemical principles also used in photodynamic therapy of cancer (PDT)-a localized cancer treatment involving tumorlocalizing photosensitizers followed by focused light activation. 6 In the PCI technology, photosensitizers localizing in endocytic vesicles are used.…”

A gene delivery system based on the N-terminal domain of human topoisomerase I

“…In this scenario, PTD-mediated gene delivery is probably through the non-endosomal pathway [21,55e58]. More recent hypotheses include the artifactual uptake of peptides upon even mild cell fixation [59], improved endosomal escape as the result of photochemical reactions initiated by photosensitization of compounds localized in endocytic vesicles, which induces rupture of these vesicles upon light exposure [60], ATP-and temperature-dependent involvement of endocytosis [59,61], which was subsequently identified as macropinocytosis [24,25,62], clathrin-dependent endocytosis [63] and endosomal acidification [64]. Here, experiments conducted with cells that were pre-incubated for 1 h at 4 C [65] or with 10 mM sodium azide and 6 mM 2-deoxy-D-glucose to deplete cellular ATP [59] revealed that the cellular uptake of 6 Â His-PTD-TopoNs/DNA complexes was energy-and temperature-dependent (Fig.…”

Chemically Programmed Polymers for Targeted DNA and siRNA Transfection

“…They demonstrated the disruption of endosomal membranes by reactive oxygen species generated by light activated coincubated photosensitizers (PS) such as porphyrin and phthalocyanine derivatives. This smart strategy was used for controlled and targeted release of endosomally enclosed complexes into the cytosol, thus considerably enhancing nucleic acid delivery with polyplexes [149,150] and PEG-shielded, receptor-targeted systems [64,151,152]. In some applications, however, the PSs applied as separate molecules showed significant phototoxicity due to possible interactions of the PSs with the plasma membrane and cytoplasmic organelles after endosomal escape, inducing cell death.…”