Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.

BACKGROUND: A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. METHODS: We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. RESULTS: The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. CONCLUSIONS: Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases.

Efficient gene therapy based targeting system for the treatment of inoperable tumors.

BACKGROUND: A considerable percentage of tumors are not amenable to surgery. We have designed a simple and powerful targeting system that offers an alternative option for the multi-component pre-targeting strategies used clinically. This targeting system can be used for any type of solid tumors independent of the tumor type, thereby omitting the need to engineer unique antibodies for each specific application or tumour type. In the present study, we show the expression of a chimeric fusion protein, which contains the low-density lipoprotein receptor transmembrane domains and avidin, after local gene transfer and its ability to bind biotinylated compounds in vivo. METHODS: Semliki Forest virus and lentivirus vectors were used to express the fusion protein with a high affinity binding site for biotinylated compounds in the tumor. Three different animal models and imaging modalities were used for the demonstration of the functionality and efficacy of the targeting system in vitro and in vivo. RESULTS: We demonstrate targeting of biotinylated compounds after local gene transfer in vivo using two different gene transfer vectors. The findings were confirmed by immunohistochemistry, single-photon emission computed tomography and magnetic resonance imaging. The therapeutic efficacy was tested in a syngeneic rat glioma model by injecting biotinylated-(90) Yttrium into the tail vein of glioma bearing rats. The study demonstrates that animals, which were treated by using the gene therapy based targeting system, lived significantly longer than control animals. CONCLUSIONS: Our gene therapy based targeting system is a promising tool for the treatment of inoperable tumors and other disease conditions, as well as diagnostic imaging.

Non-invasive Imaging in Gene Therapy.

Several methods are available for non-invasive imaging of gene delivery and transgene expression, including magnetic resonance imaging (MRI), single photon emission tomography (SPECT)/positron emission tomography (PET), and fluorescence and bioluminescence imaging. However, these imaging modalities differ greatly in terms of their sensitivity, cost, and ability to measure the signal. Whereas MRI can produce a resolution of approximately 50 mum, optical imaging achieves only 3-5 mm but outperforms MRI in terms of the cost of the imaging device. Similarly, SPECT and PET give a resolution of only 1-2 mm but provide for relatively easy quantitation of the signal and need only nanograms of probe, compared with the microgram or milligram levels required for MRI and optical imaging. To develop safer and more efficient gene delivery vectors, it is essential to perform rigorous in vivo experiments, to image particle biodistribution and transduction patterns, and to quantify the transgene expression profile. Differences between modalities have a significant effect on the resultant imaging resolution for gene therapy. This review describes the methodologies in use and highlights recent key approaches using the latest imaging modalities in gene therapy. Future trends in gene therapy imaging are also discussed.