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even more challenging. Further knowledge on molecular mechanisms behind cellular entry will create the basis for further engineering of the baculovirus envelope, allowing better possibilities for cell/tissue-specific entrance. Pseudotyping with known viral surface proteins may also be useful to achieve this goal (52,99,166).

Current baculovirus vectors allow only transient transgene expression, which is desirable in the treatment of cancer or cardiovascular disorders (167). However, more prolonged expression is needed for the treatment of genetic diseases (1). As an attempt to reach this goal, AAV baculovirus hybrid vector was described (107). The strategy to combine elements from known integrating viruses (3) and viruses capable of episomal replication (168) may well prove to be useful. In these kind of vectors, the addition of gene expression control by tetracylin-based system will be desirable (169,170). As time progresses, more baculovirus hybrid vectors will appear to ease the preparation of viruses that are difficult to produce at present (81,105,106,108,171).

A novel system was recently developed where the fusion protein (enhanced GFP) is displayed in large quantities on the surface of the baculovirus capsid without compromising the viral titer or functionality (117). The system is based on the production of the desired peptides or proteins as either C-terminally or N-terminally linked fusion proteins with the baculovirus major capsid protein, vp39 (Fig. 2). This system has many advantages as compared with the baculovirus surface display system, based on baculovirus major envelope glyco-protein, gp64 (Fig. 2) (121,172-174). In vp39, no structural motifs have been recognized either for association with molecules within the stromal matter or for capsid assembly (175). Neither is it responsible for infectivity of the virus. This is important because the major restriction of the (baculo)virus surface display is the fact that displayed proteins may easily destroy the infectivity of the virus in insect cells, leading to low or no titer of the produced virus (176). In addition, immu-noelectron microscopy has shown that vp39 is uniformly distributed on the surface of the capsid (175). The baculovirus envelope display system allows fusions only to the N-terminal end of the gp64, whereas the results suggest that vp39 allows the tagging of both termini. Because the length of the capsid can extend relatively freely (29), it is reasonable to expect that this system may be compatible with even larger proteins than GFP. If the penetration of the baculovirus into mammalian cells and the release of the viral capsid into the cytoplasm prove to be a general phenomenon, the concept of baculovirus-mediated therapy may be further extended, with the possibility of using baculovirus capsid as a shuttle to transport therapeutic proteins directly into the cells as an alternative to traditional protein transduction strategies (177).

In conclusion, baculoviruses with many advantages provide an alternative to current gene therapy vectors (Table 1). They are especially useful for large expression constructs allowing the use of multigene strategies. Inherent safety, together with the ease and speed of production of these vectors, creates a powerful concept to test different gene delivery constructs also in a high-throughput manner.

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