|Ph.D Thesis||Department of Biotechnology and Food Engineering|
|Supervisor:||Assoc. Prof. Machluf Marcelle|
Gene therapy can prevent or treat a variety of disorders ranging from allergy to cancer by delivery of the gene of interest into the nucleus of cells. The challenge faced by this approach is the development of a system that will be efficient, safe, simple to manufacture and with minimal side effects. Nowadays methods can be divided into viral and non-viral methods. Viral vectors achieve the highest transfection efficiency; however, substantial concern remains over their clinical safety.
We focused on exploring the potential of a non-viral method, based on the polymer PLGA, a biocompatible polymer with an FDA approval for use in humans.
PLGA was used to formulate particles containing DNA in the nano-range using a modified double-emulsion-solvent-evaporation-technique. PLGA nanoparticles were found to facilitate uptake of DNA into cells without affecting their viability. Transfection of COS-7 cells and Cf2th cells was found to be up to 400-fold higher compared to control. The dependence of protein expression level on plasmid promoter, DNA load and number of washing cycles during preparation was studied. For optimization of the transfection procedure, the dependence of particle incubation time with cells, their concentration, the number of doses and the percent of serum was also studied. These results enlightened part of the complicated process of transfection.
A specific application of gene therapy is vaccination using DNA. Its main advantage over standard vaccination is the ability to activate the cellular immune system. This mechanism may be recruited for the treatment of viral diseases, allergies and cancer.
We have formulated particles in the micro-sized range to eliminate uptake by cells other than antigen presenting cells (APCs). Successful protein expression levels were achieved when APCs such as: J774, RAW and P388 were incubated with PLGA microparticles. The process was optimized with regard to time of incubation with particles, particles concentration and serum percentage. Moreover, the effect of LPS and LLO was studied and found to increase transfection.
The efficacy of our system was tested as a treatment for melanoma. Mice were injected with either microparticles entrapping the pcDNA encoding for the human gp-100 or DNA only. Expression of the tumor antigen couldn’t be detected in the inguinal lymph nodes by means of RT-PCR but inhibition of tumor growth in mice challenged with B16 melanoma cells was detected. Our PLGA particles were shown to carry the potential to deliver DNA efficiently and safely to cells, leading to protein expression and activation of the immune system.