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Baculoviruses as gene delivery vectors for mammalian cells

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   Baculoviruses are a family of rod-shaped dsDNA viruses whose host range is restricted to intervertebrates, mainly insects. Among the baculoviruses, the most characterized species belonging to the nuclear polyhedrosis viruses (NPVs) have been widely used for production of recombinant proteins in insect cell cultures and insect larvae. More recently, since the discovery that these viruses can transduce efficiently many different mammalian cell types, the applications of the baculovirus technology have expanded to include their use as vectors for (i) production of foreign proteins in mammalian cells; (ii) gene therapy applications; and (iii) applications related to vaccine antigen delivery (Kost et al., 2005).  
   The use of baculoviruses for mammalian transduction offers a number of advantages as compared to mammalian virus-derived vectors. These include the ease of production in insect cell lines; their ability to package large fragments of foreign genetic material; their inability to function productively and produce progeny virus in cells other than those of their normal hosts; the lack of induced cytotoxicity even at the high multiplicity values used for efficient mammalian cell transduction; and the absence of pre-existing immunity against them in mammalian organisms.   
   We have previously reported on the development of baculovirus vectors derived from Bombyx mori nuclear polyhedrosis virus (BmNPV), which are capable of delivering efficiently mammalian expression cassettes into established and primary cultures of mammalian cells without affecting their molecular phenotype or their capacity to differentiate in vitro toward their normal phenotype (Figure 1; Kenoutis and al., 2006).

Figure 1. Baculovirus-transduced Schwann cells retain a normal phenotype and express their phenotypic markers. (A to L), double immunofluorescence labeling of transduced Schwann cells for GFP (green) and the characteristic marker proteins (red) GFAP (A to C), S-100 (D to F), p75 NGF receptor (G to I) and P) myelin (J to L) followed by confocal microscopy (Kenoutis and al., 2006).

   Moreover, we have used recombinant BmNPV-based vectors to transduce HEK293 and primary Schwann cells (SCs) and express the therapeutic protein L1 in its native or a chimeric form, L1-Fc. This study has shown that this protein can be successfully expressed in the transduced mammalian cells and is functional in various in vitro assays (Figure 2; Lavdas et al., 2010).

Figure 2. Baculovirus-transduced SCs seeded on cerebellar slices are able to myelinate CNS axons. Both L1-Fc-transduced SCs (shown here) and GFP-transduced SCs (not shown) formed peripheral myelin internodes (red) around CNS axons (green) in the cerebellum (Lavdas et al., 2010).
   However, there are several issues that need to be addressed before engineered baculoviruses can be employed safely as gene therapy vectors in humans. One of them concerns the residual expression of baculovirus genes in infected mammalian cells. To address this issue, we produced infectious ie1-deficient viral particles lacking the gene encoding a major transcriptional regulator of the virus, IE1, which regulates multiple aspects of the baculovirus infection cycle, using insect host cells stably transformed with various IE1 expression constructs (Efrose et al., 2010). Our data demonstrated not only that such ie1 gene knockout viruses are unable to replicate and direct viral gene transcription in normal insect host cells to any appreciable degree but, also, that they are essentially devoid of the residual gene expression, which normally occurs in mammalian cells when the latter are transduced with wild type baculoviruses (Figure 3; Efrose et al., 2010).

Figure 3: Accumulation patterns of viral gene transcripts in HEK293 cells transduced with AcBacWT (red bars) or AcBacIE1KO virus (blue bars) at 3 days post-infection. Viral transcripts were detected by real-time PCR using gene-specific primers, and relative gene expression levels were calculated with respect to the level of cellular gapdh transcripts (Efrose et al., 2010).


   The ie1-deficient baculoviruses are thus considered to be safer transduction vehicles for gene therapy applications due to reduced risks for physiological changes in infected cells and induction of cellular immune responses in individuals transplanted with ex vivo transduced cells.
   Finally, to address a second drawback of the baculoviruses related to gene therapy applications, its presence as an episomal entity in the transduced cells, and achieve genomic integration and stable transformation of mammalian cells, a second generation of BmNPV-based recombinant baculoviruses employing the piggyBac transposition system was developed (unpublished data). The newly developed baculoviruses incorporate (i) a target piggyBac transposition cassette allowing the cloning of open reading frames for reporter or therapeutic protein expression under CMV promoter control and (ii) a mammalian piggyBac transposase expression cassette. Upon infection of mammalian cells, the latter causes transgene excision from the viral genome and integration into the genomes of recipient cells (Figure 4).


Figure 4: GFP expression in HEK293 cells transduced with recombinant baculoviruses containing a GFP expression cassette flanked by piggyBac recognition sequences alone (upper panels) or the same cassette and a piggyBac expression cassette (lower panels), both under CMV promoter control. Note the prolonged GFP expression in the cells of the lower panels, which is due to transposition and stable integration of the GFP transgene from the viral genome into the genome of the transduced host cells. Expression in these cells has been stable for 6 months, the time when the experiment was terminated.



   The piggyBac transposition cassette-containing recombinant baculoviruses therefore allows permanent transgene integration into the genomes of the target cells. This represents a major advantage over the first generation of baculovirus vectors, which can only achieve transient transgene expression. However, given that piggyBac directs essentially random integration of the transgene sequences into the host genome, which may give rise to harmful mutations including the induction of transforming phenotypes, further research is in progress that aims at the restriction of the number of integration sites in the human genome to only a few of known functional properties.



1. Kost et al., (2005) Kost T-A, Condreay J-P, Jarvis D-L (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23: 567-75.
2.  Kenoutis C et al. (2006) Baculovirus-mediated gene delivery into Mammalian cells does not alter their transcriptional and differentiating potential but is accompanied by early viral gene expression. J Virol 80: 4135-46.
3. Efrose et al. (2010) Baculoviruses deficient in ie1 gene function abrogate viral gene expression in transduced mammalian cells. Virology 406:293-301.
4. Lavdas et al. (2010) Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility. J Neurochem 115:1137-1149.