Ther. transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BVCAR to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BVCAR-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BVCAR in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BVCAR-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BVCAR-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between Gastrofensin AN 5 free base the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no Gastrofensin AN 5 free base effect on the efficiency of transduction by the BVCAR-Ad5GFP complex. Various situations in vitro or ex vivo in which our BVCAR-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed. Adenoviruses (Ads) are extensively used today as gene transfer vectors for in vitro, ex vivo, and in vivo gene transfer protocols (reviewed in reference 65). Cell entry of human Ad type 5 (Ad5), the serotype most widely used as a gene vector, occurs most efficiently by the receptor-mediated endocytosis pathway (reviewed in references 64 and 65), via Gastrofensin AN 5 free base the coxsackievirus B-adenovirus receptor (CAR) (3, 77) and v3/v5 integrins (84, 85), although alternative receptors have been described (11, 12, 14, 27). Cell surface expression of CAR differs with different cell types, and this represents one of the major determinants of the efficiency of Ad5-mediated transduction (43). The ubiquitous nature of CAR is Rabbit Polyclonal to NUMA1 responsible for transduction of nontarget tissues by Ad vectors. Paradoxically, many target cells such Gastrofensin AN 5 free base as dermal fibroblasts, synoviocytes, mesenchymal stem cells (MSCs), peripheral blood mononuclear cells (PBMCs), and dendritic cells (DCs), express no or very low levels of CAR Gastrofensin AN 5 free base at their surface and are relatively resistant to Ad transduction (14, 15, 19). Much work has been done with different strategies to promote the entry of Ad5 into CAR-defective cells. These strategies include (i) the genetic modification of Ad capsid proteins to carry cell ligands (2, 15, 20, 28, 49, 50), (ii) pseudotyping Ad5 vectors with fibers from other serotypes (13, 57, 74, 86), (iii) using bispecific adapters or peptides (25, 40), (iv) chemical modification of Ad (9, 42), and (v) tethering on nanoparticles (7). The limitations to these strategies are that modifications of the Ad capsid are susceptible to negatively affecting the virus growth or viability, due to an alteration of virion assembly, stability, the viral uncoating process, and/or intracellular trafficking (13, 51). Other viruses which are gaining popularity as gene transfer vectors are the baculoviruses (BVs). multiple nucleopolyhedrosis virus (AcMNPV) is an insect virus with a large double-stranded DNA genome packaged in a membrane-enveloped, rod-shaped protein capsid (70). Since the 1980s, the BV-insect cell expression system has been highly exploited for the production of recombinant proteins. In the mid-1990s, it was shown that recombinant BVs carrying reporter genes under cytomegalovirus (CMV) or retroviral Rous sarcoma virus promoter efficiently expressed reporter genes in mammalian cells (6, 22, 38, 41, 44, 69), as well as in avian cells (72) and fish cells (45). Since then, BVs have been reported to transduce numerous cells originating from species as various as humans, bovines, and fish (8, 32, 41, 73). As gene transfer vectors, BVs have been found to be rapidly inactivated by human serum complement (23), but exposing decay-accelerating factor (DAF) at the surface of BV by fusion with the baculoviral envelope glycoprotein can overcome this inactivation (33). BVs also have a good biosafety profile due to their incapacity to replicate in mammalian cells (31). Taking advantage of the ability of BVs to transduce a large repertoire of cells of invertebrate and vertebrate origins, including human primary cells, we investigated whether a recombinant AcMNPV could act as a carrier or macroadapter for Ad5 vectors to enter Ad5-refractory cells. To this aim, we pseudotyped AcMNPV virions with the high-affinity receptor for Ad5, the human CAR glycoprotein (BVCAR), to enable the formation of complexes between vector particles of BVCAR and Ad5-green fluorescent protein (Ad5GFP) mediated by Ad5 fiber and CAR interaction. We found that transduction of cell lines which were poorly permissive.
