Enhancement Of Tumour Regression In Vp3-Based Gene Therapy In Combination With Immunomodulators
Shia, John Kwong Siew (2009) Enhancement Of Tumour Regression In Vp3-Based Gene Therapy In Combination With Immunomodulators. PhD thesis, Universiti Putra Malaysia.
The VP3 gene of Chicken Anemia Virus has the potential to be an effective anti-cancer therapy. In vitro studies showed that several types of transformed cells transfected with recombinant VP3 genes become apoptotic within 72 hours post-transfection. The apoptotic activities were confirmed by TUNEL assay, and the apoptotic activities were mainly found in the nuclei of transfected cells. Expression of VP3 gene also triggered apoptosis in tumour mass in immune-competent mice. Following an injection of 100μg recombinant plasmid containing the VP3 gene into a tumour mass, the tumour tissue started to regress from 47.9 ± 5.2 mm3 on day-1 to 0.8 ± 0.4 mm3 on day-11 post-injection and completely resolved by day-13 post-injection. Meanwhile, in control group, the tumour mass measured 54.1 ± 5.2 mm3 (day-1), increased to 589.0 ± 0.4 mm3 on day-11 post-injection and 808.8 ± 0.4 mm3 by day-13 post-injection. In a different experiment, selected cellular (pVIVO-IL12/GM-CSF) or humoral (pBoost/IL4/IL13) immune modulator was injected together with the recombinant plasmid containing VP3 gene. In the presence of cellular immune modulator, the tumour sizes were significantly decreased from 48.6 ± 7.7 mm3 on day-1 post-injection to 0.6 ± 0.4 mm3 on day-9 post injection. The tumour mass was totally resolved by day-11 post-injection. The most rapid and complete regression of tumour mass as determined on day-11 post-injection, suggested that an enhanced effect of tumour regression is attributable to the effect of IL12 and GM-CSF. A delay in tumour growth was also noticed in the treatment group receiving a single dose of pVIVO-IL12/GM-CSF plasmid only (100 μg/mouse). It is suggested that the anti-cancer mechanism is being induced following expression of IL12 gene in the tumor cells. This in turn will activate Th1 and NK cells. Meanwhile, an expression of GM-CSF will cause mobilization of granulocytes and macrophages. Combination of these cellular activities may produce more antigen presenting cells for the recognition of tumour-associated antigens that facilitate elimination of tumour cells. The treatment group receiving humoral immune modulating factors (IL4/IL13) in addition to VP3 gene therapy showed reduction in the size of tumour from 48.5 ± 5.3 mm3 on day-1 post-injection to 4.1 ± 1.6 mm3 on day-9 post-injection. The tumour was totally resolved by day-11 post-injection. However, its anti-cancer effect was still mediocre compared to VP3 gene therapy incorporated with IL12/GM-CSF. The flow cytometry analysis on the clusters of differentiation (CD) for the 3 treatment groups (Group 1: recombinant VP3 gene only; Group 2: recombinant VP3 gene + cellular immune modulator; and Group 3: recombinant VP3 + humoral immune modulator) further supported the rationale of choosing the particular cellular and humoral immune modulators for effective anti-cancer therapy. The percentage of CD4 cells detected in the thymus of tumour –bearing mice showed that cellular and humoral immune modulators enhanced the proliferation of CD4 lymphocyte population in the thymus. Meanwhile, the highest percentage of CD8 count was most distinguished in group 2 (39.02%) on day-10 post-injection. An increase in CD8 cell count was probably due to the enhanced immune-stimulation by IL12 and GMCSF expressed by pVIVO-IL12/GM-CSF recombinant plasmids against tumour antigens. The distribution of CD4 and CD8 lymphocyte populations in the spleen of treated mice has a similar trend as in primary lymphoid organs (thymus). Again, the percentage of CD8 count was most distinguished in group 2 (38.00%) on day-10 post-injection. It is suggested that, the increase in CD8 cell counts could be due to the increase in mature cytotoxic T-cells produced by the thymus against tumour antigens, as a result of immune-stimulation of GM-CSF. These CD4 and CD8 cells may then be detected in the secondary lymphoid organs. The percentage of CD19 lymphocyte populations was only prominent in Group 3 (26.71%) on day-10 post-injection. This suggests that the increase of CD19 lymphocytes was due to the immune-stimulation by IL4/IL13. In conclusion, recombinant VP3 gene is best combined with cellular immune modulators (IL12/GM-CSF) for a more effective anti-cancer gene therapy.
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