Despite continuous improvement in gene delivery vector technology, development of methods for efficient and specific delivery of therapeutic genes to target cells still remains an important issue in the development of clinical gene therapy. Gene transfer is a complex process with many possible extracellular and intracellular barriers limiting the efficiency. Among the extracellular barriers can be mentioned enzymatic degradation of the DNA, unspecific interaction with blood components or nontarget cells, recognition by the immune system, slow transport through the target tissue and inefficient uptake into the target cells.The importance of the intracellular barriers varies considerably between different gene vector systems. Viral vectors, which are based on naturally occurring viruses having their own mechanisms to overcome most of the intracellular barriers, are much more efficient in gene transfer than synthetic non-viral vectors. Thus, just a few viral particles taken up by a cell may be sufficient to obtain transgene expression/transduction. In contrast, with non-viral vectors cells may not be transfected even though they have taken up ample amounts of exogenous DNA, indicating that overcoming intracellular barriers other than cellular uptake are of primary importance for improving the efficiency of these vectors. Intracellularly there are many obstacles for the functional delivery of transgenes. Nuclear transport and uptake of the transfecting DNA represent one important such barrier. Other intracellular obstacles can be e.g. dissociation of DNA from the non-viral complexing agent, degradation of the DNA, and sequestration of the DNA in endocytic vesicles. Finally transcription and translation of the transgene also represent possible barriers, especially in cases where it is desirable with long-term expression of the transgene.Another factor often limiting the success of gene therapy is lack of specificity of gene delivery and expression. For diseases like cancer, obtaining gene-mediated toxic effects selectively in tumors is of great importance for the success of the treatment. To improve the specificity of gene therapy both targeted delivery of a therapeutic gene and targeted gene expression are being employed. Targeted delivery can be achieved by physical targeting i.e. direct action of physical force to get the DNA into the target cell (e.g. electroporation, gene gun), or by biological targeting based on biological characteristics of the target cells. In the latter case gene vectors are usually coupled to targeting ligands that ensure attachment of the gene vector to specific cells via ligand-receptor interactions. For targeted gene expression different inducible or tissue specific promoters/enhancers have been exploited to obtain the expression of a therapeutic gene specifically e.g. in tumors. Photochemical internalization (PCI) is a recently developed light-dependent technology, where photochemical treatment, i.e. photosensitising compounds (photosensitizers) and light, are employed to improve both the efficiency and the specificity of different gene vectors. The technology aims at eliminating the endosomal barrier to increase the delivery of genes into the cytosol of the cell. Since the major mechanism of uptake of genes carried either by viral or non-viral vectors is through the endocytic pathway and to the endocytic vesicles, endosomal escape is a necessary step for further gene processing. In the PCI technology light activates photosensitizers localised in the endocytic vesicles, inducing rupture of the vesicular membranes and release of the trapped gene. The liberated genes can then be further delivered to the cell nucleus, transcribed and translated. Being a light dependent treatment PCI provides a possibility to liberate the genes or other endocytosed macromolecules in response to illumination, thereby triggering their activity only at specific (illuminated) sites.This study focuses on the employment of PCI as a gene delivery method in combination with different gene vectors in vitro. Previous studies have shown that PCI improves gene delivery and expression of the reporter gene encoding enhanced green fluorescent protein complexed with the polycation polylysine. In the present study we examine whether PCI could also have positive effects on gene transfer when the DNA is delivered by receptor-mediated endocytosis, employing transferrin for targeted delivery via the transferrin receptor. To further test the utility of the PCI method in gene therapy, we have also employed the technology with a therapeutically relevant gene, the gene encoding herpes simplex virus thymidine kinase (HSV-tk) followed by treatment with the HSV-tk activated prodrug ganciclovir (GCV).