One of the unexpected offshoots of the baculovirus expression system has been its development as a vehicle for the delivery of genes to mammalian cells. These vectors are otherwise known as BacMams. Although baculoviruses are unable to replicate in mammalian cells, they can enter them via receptor-mediated endocytosis. In Frederick Boyce’s original paper, the Rous sarcoma virus promoter was used to express beta-galactosidase in the HepG2 human liver cell line. Further work showed that over 70% of cells in primary cultures of rat hepatocytes displayed expression of beta-galactosidase after exposure to the virus. Although in this study other cell lines did not appear to be able to express the reporter gene, the subsequent use of other promoters, such as that from cytomegalovirus, seems to have overcome this problem. BacMams not only permit the delivery of genes to mammalian cell cultures, they also offer the opportunity for somatic gene therapy. A recent paper where flashBAC™ was used to derive recombinant BacMams has demonstrated that it may be possible to treat whole kidneys with the long term goal of improving the success of organ transplantation in humans.
Ischemia with subsequent reperfusion (I/R) is a serious problem in organ transplantation. After removal of the organ from a donor blood flow is restricted. This leads to a hypoxic state in which there is prolonged insufficient flow of nutrients and oxygen to the organ. In consequence, cell death programs may be activated, seriously affecting organ viability. In kidneys, renal hypoxia injury has been linked to a number of cellular biochemical, physiological and morphological alterations. These include increased reactive oxygen species and severe depletion of intracellular ATP, which activate apoptotic pathways. Therefore, I/R injury is a major factor in graft survival in kidney transplantation.
In the paper by Hitchman et al (2017) flashBac ULTRA was used to produce a BacMam that expressed a manganese superoxide dismutase (sod-2), a major antioxidant in mitochondria. Production of the recombinant protein was demonstrated in cell culture and could alleviate simulated ischemia. The virus was also perfused into a porcine kidney model to show that ATP levels in treated kidneys were maintained at a higher level than in control samples. This is the first time that a BacMam has been used to deliver sod-2 to kidney cells ex vivo and demonstrates that the baculovirus system for gene expression in mammalian cells has expanded its possibilities still further. It should be feasible to deliver other protective genes to kidneys or other organs to improve their viability between donor and recipient. However, the use of such recombinant BacMams in animal or human trials remains some years away.
This study was also a good demonstration of the use of flashBAC™ to produce recombinant BacMams rapidly and with high transduction efficiencies. It is important to note that BacMams do not infect mammalian cells as they are unable to produce more virus particles in cultures derived from non-insect cells.
References
Hitchman, E., Hitchman, R. B. and King, L. A. BacMam delivery of a protective gene to reduce renal ischemia-reperfusion injury. Human Gene Therapy. January 2017, ahead of print. doi:10.1089/hum.2016.100.