Baculovirus as a gene delivery and expression vector in mammalian cells
The interior homeostasis of a solid organ, such as a kidney, is greatly altered during the surgical removal, storage and transplantation process from donor to recipient. Consequently, normal organ function may be delayed or prevented following transplantation. The main injury that organs have to withstand often occurs subsequent to the restoration of blood flow; this injury is known as ischaemia reperfusion-injury (I/R injury) and is associated with hypoxia, free radical formation and organ failure. Viral gene therapy, such as BacMam gene transfer technology (Baculovirus-based expression in mammalian cells) is an attractive option for reducing I/R injury due to its high safety profile compared to mammalian viruses. However, limiting factors, including the requirement of high-doses of viral vector and low transgene expression levels are still a hurdle for the successful use of this technology. Therefore, the initial objectives of this study were to develop and validate a number of methods for concentrating BacMam along with the use of a number of chemical compounds to improve BacMam gene delivery in kidney cells. Amongst all the approaches screened, high-speed (for small volumes) and low-speed (for large volumes) centrifugation methods were the most effective for concentrating BacMam virus (4x109 and 1x1010 pfu/ml, respectively). Transduction of HEK cells was improved between two- to 10-fold in the presence of sodium butyrate or hydroxyurea alone and significantly improved egfp (enhanced green fluorescence protein) expression (~40-fold) when both chemicals were used in combination. Subsequently, in the second part of this study an in vitro culture model was developed and evaluated for assessing ischaemic injury in kidney cells using antimycin A in combination with 2-deoxyglucose. It was shown that sod-2 and bcl-2 overexpression in injured kidney cells improved the effect of I/R injury by enhancing the recovery of ATP post-reperfusion Furthermore, to examine the possibility of reducing injury associated with cold preservation, an ex vivo hypothermic perfusion system of porcine kidney was established and validated using reporter and protective genes. Importantly, for the first time, BacMam has been used to transfer ex vivo a protective gene (sod-2), into kidneys which appeared to provide protection against hypothermic preservation-associated injury by improving the recovery of cellular ATP.
Networked Digital Library of Theses and Dissertations