**4. Use of parvovirus vectors in cancer gene therapy**

Gene therapy is one of the most promising approaches for cancer treatment because it has the potential to provide tumor cell selectivity and/or protection of untransformed cells of the body. In order to transduce the gene of interest, either nonviral vectors or viral vectors are used. Nonviral vector strategies include naked plasmid DNA, liposome-DNA complexes, peptide-bound DNA and electroporation [49]. The most widely used viral vectors are retroviruses, adenoviruses and herpesviruses. The preliminary data looks very promising, but most vectors suffer from downsides that limit their utility for gene therapy. While nonviral vector systems have low transfection efficiencies, most of the viral systems have the problems of poor tumor targeting, immunogenicity and low transduction efficiencies. Certain parvoviruses are characterized by their oncotropism, oncosuppression and ability to mediate long-term gene expression. Together with their human apathogenicity, these characteristics make them very interesting vector systems for cancer gene therapy. Viruses of the Parvovirinae subfamily, of the Parvoviridae family, have the ability to infect a variety of different vertebrates. Although the natural hosts of parvovirus H1, MVM and LuIII are rodents, these parvoviruses can also infect human cells [50]. Similarly, many AAV serotypes are endemic in humans and non-human primates. Despite this, neither of these viruses are pathogenic in humans.

Gene therapy strategies for tumor cells have to be highly specific, particularly when the vector is to be used systemically, in order to prevent damage to healthy tissues. The therapeutic index of most existing vectors is low [51]. They non-specifically transduce normal cells as well along with targeting the tumor cells resulting in undesired damage and cell death. In general, there are two different ways to achieve specificity of a gene therapy vector: transductional targeting and transcriptional targeting [51, 52].

Transductional targeting describes the selective uptake of the vector into the cells of interest, where the transgene is transcribed. Selective uptake can be achieved by various strategies, such as modification of the viral capsid or pseudotyping of viruses. AAVs have serotype-specific tissue tropism; thus, one approach to achieving tissue-specific transduction with a therapeutic gene is the use of different AAV serotypes [53, 54]. For example, AAV-2 preferentially transduces the liver, AAV-1 transduces the muscle and AAV-5 transduces airway epithelium [53, 55]. However, serotype-specific tissue tropism enhances transgene expression in certain tissues but does not provide absolute specificity of transgene expression in other tissues. Various re-targetting strategies have been tried to enhance the specificity, efficiency and safety of AAV vectors, in particular: (1) direct re-targeting by modification of the viral capsid using the optimal insertion site that ensures the presentation of a targeting peptide on the viral surface but does not interfere with packaging [56–58] and (2) indirect re-targeting using a molecule bound to the viral surface that binds specifically and stably to the target cell (e.g. glycoside molecules and bispecific antibodies) [59, 60]. In another study, two unique features of AAV and B19 virus were exploited to create a chimeric recombinant vector system to specifically target the primitive erythroid progenitors in human bone marrow cells [61]. Recombinant B19 virus vectors are much more efficient than the recombinant AAV vectors in transducing primary human erythroid progenitor cells. Further refinement of this vector system can be useful in cancer gene therapy applications for erythroid cell lineage in the human hematopoietic system.

In transcriptional targeting, even though the transgene might be taken up by many different cells, it is transcribed only in the target cells. Using this approach, transgenes are expressed selectively by replacing the natural promoter or by modifying the transcription-factor-binding sites within a promoter. Transcriptional targeting of AAV is mostly used to enhance transgene expression in a tissue-specific manner rather than to restrict its expression to certain tissues. There are various promoters that have been used successfully like an albumin gene promoter and a retroviral long terminal repeat promoter to express human α1 -antitrypsin in hepatocytes [62], a myelin basic protein (MBP) gene promoter to direct MBP expression specifically to oligodendrocytes [63, 64] and regulatory elements of the F4/80-gene promoter for specific expression in primary microglia [65]. Transcriptional targeting of ARPs has the benefit that the vectors are already selective for cancer cells. Transcriptional targeting of ARPs has been used to achieve cell-type specific transgene expression of the parvovirus LuIII. rLuIII vectors expressing the luciferase marker gene under the control of a chimeric promoter containing a liver-specific enhancer. It directed the preferential expression of the luciferase marker in transduced human hepatoma cells [66, 67]. Another approach targeted colon carcinoma by using hybrid H-1-MVM parvovirus vectors carrying binding sites for the heterodimeric β-catenin/Tcf transcription factor in the P4 promoter; this transcription factor functions in the wnt signalling pathway, which is constitutively activated in colon carcinoma.

Gene therapy strategies for cancer can be grouped as follows: (1) Immunogene therapy with the aim of achieving either an antitumor vaccine effect or enhancing T-cell antitumor effector capability; (2) anti-angiogenic gene therapy to reduce the supply of oxygen and nutrients to the tumor; (3) cytoreductive gene therapy by gene transfer to a large number of tumor cells in situ to achieve nonimmune tumor reduction by direct cytotoxicity or by an indirect bystander effect; and (4) transduction of HSCs with drug-resistance genes to enhance their resistance to cytotoxic drugs. Depending on the desired gene therapy approach, there are different requirements the vector must fulfil with regard to safety and efficiency of the vector, specific targeting of gene transduction, expression level of the transduced gene and ease of manufacture [68].
