2.6. Attempts to enhance transgene expression in gene transfer

Recently, attempts to enhance transgene expression and to prolong its durability have been made. Apart from reducing the immunogenicity of the vector, the possibilities include suppression of the immune system of the recipient for example by simultaneous administration of corticosteroids or cycklophosphamide with the vector (Zsengeller et al. 1995, Jooss et al. 1996, Kaplan & Smith 1997, Halbert et al. 1998, Cassivi et al. 1999). Cassivi et al. (1999) compared the efficiencies of intratracheal adenovirus-mediated reporter gene retransfection in immunosuppressed and normal rats. They found that adenoviral retransfection 5 weeks after the initial transfection resulted in significantly ­elevated transgene expression and reduced inflammatory responses in lung tissue on days 1, 7 and 14 after retransfection in immunosuppressed animals, while expression was virtually undetectable in control animals after retransfection. A novel immunosuppressant, deo­xyspergualin, has been found to similarly prevent immunological response and to enhance and prolong the expression administered transiently into the lungs during AdV-mediated gene transfer (Kaplan & Smith 1997). Kay et al. (1995) used a T cell receptor-blocking agent, CTLA4lg, during a brief period close to the time of systemic AdV vector administration, which resulted in prolonged transgene expression in the liver. These results suggest the possibility of using systemic immunosuppression in conjunction with viral gene transfer to enable repetitive dosage of vectors and to enhance gene expression.

The role of immune response in gene transfer efficiency was also demonstrated by Kuzmin et al. (1997), who used intravenous systemic administration of AdV vectors. They found that clodronate pretreatment by causing macrophage depletion, and subsequent suppression of the immune system, resulted in improved efficiency and duration of expression.

The transduction efficiency of AdV vectors has been reported to increase when pharmacological agents, such as polycations, e.g. DEAE-dextran, protamine and polybrene, are used in conjunction with AdV administration in vitro and in vivo (Arcasoy et al. 1997, Kaplan et al. 1998), possibly due to an alteration of the charge interaction between adenoviruses and the cell surface. This enabled administration of reduced doses of vectors ­complexed with polycations and resulted in lower levels of AdV-specific antibodies and thereby improved readministration of the vector. It has been suggested that the surfactant may reduce the transfection rate in plasmid-mediated gene transfer into the lungs (­Raczka et al. 1998). In contrast, Jobe et al. (1996) found in their study with rabbits that the administration of surfactant simultaneously with the instillation of AdV vectors resulted in enhanced expression in peripheral airway epithelial cells, but decreased expression in bronchial epithelium, indicating that surfactant facilitates the spreading of vectors into the lower airways.

According to Parson et al. (1998), polidocanol, a nonionic surface-active detergent, administered before adenoviral transfection inreased transfection efficiency in nasal cells, suggesting that adjuvant agents altering cell permeability may enhance adenoviral transfection. Danko et al. (1994) tested etidocaine, mepivacaine, bupivacaine, acetic anhydride, sodium bicarbonate, cardiotoxin and normal saline in plasmid-mediated gene transfer into muscle in vivo and found that bupivacaine increased the transgene expression 4- to 40-fold compared to non-bupivacaine-treated muscle. Haddad et al. (1997) found in their study that overproduction of nitric oxide, NO, or its inhalation reduced the efficiency of AdV-­mediated gene transfer into lungs in vitro and in vivo in the absence of inflammation.

It has been observed in several studies that prolongation of the incubation time between the vector and the target cell increases transfection efficiency (Chen et al. 1994, Zabner et al. 1996, Teramoto 1997, Song et al. 1998, Chia et al. 1998). Chia et al. (1998) carried out liver transplantation in rats using three methods of AdVlacZ vector delivery: by infusion via the portal vein, by infusion via both the portal vein and the hepatic artery, and by clamping the outflow during the infusion and thereafter for up to 18 hours during cold preservation at 4°C. The most effective hepatocyte transduction rate was gained with the clamping technique followed by dual infusion.