טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBen-Ari Shunit
SubjectCombined Therapy for the Treatment of Liver Fibrosis
DepartmentDepartment of Medicine
Supervisor Professor Gad Spira


Abstract

Hydrodynamics based transfection (HDI) is a recently described method of non-viral gene transfer that results in a high-level gene expression, primarily in the liver. The procedure, which involves rapid intravenous infusion of plasmid DNA in a large volume of physiological solution, has been reported to date only in mice.

In this research we hypothesized that a combined treatment of halofuginone, a specific collagen α1 inhibitor, and a gene promoting liver regeneration administered via hydrodynamic transfection may help resolving severe cirrhotic rats undergoing partial hepatectomy. In order to test this hypothesis, the method of hydrodynamic transfection had to be adjusted to the rat model. In addition, this method was introduced for the first time in fibrotic animals, and was proved effective. Vascular Endothelial Growth Factor (VEGF) was chosen as a target gene for the combined therapy.

Since HDI does not provide exclusive transgene expression in the liver, construction of a vector, containing an expression cassette of the target gene flanked by liver regulatory elements, was required. Additional calibration had to be done, as any introduction, removal or change in sequence of regulatory elements affects the expression profile of a vector administered through this method.

An attempt was made in the present study, to gain insight into the mechanism of HDI, which remains poorly understood. Electron microscopy images have demonstrated that liver fenestrae fuse to form gaps. The detection of these gaps, led us to hypothesize that rapid penetration of injected plasmid is achieved through these gaps, thereby avoiding DNA disruption. In a preliminary experiment designed to test this hypothesis, it was shown that the gaps are not sufficient for effective transfection, and other factors are involved.