Recombinant Baculovirus :

1. Recombinant Baculovirus: A Potential Vector for Cancer Gene Therapy Jenna Kausner November 4, 2010

2. Conventional Cancer Therapeutics • Surgical resection of primary tumor • Chemotherapy • Inefficient- unable to control metastasized tumors and promote long-term survival. • Therefore, cancer is incurable using this conventional methods.

3. Possible Solution? • Gene Therapy • Either in combination with conventional therapeutics • Or as a replacement to these methods • The general goal of cancer gene therapy is to introduce foreign therapeutic genes into cancer cells in order to temporarily suppress or completely destroy the tumor.

4. Different Approaches • Immunogene therapy • Selective prodrug activation • “suicide genes” • Tumor suppressor gene transfer • Antisense techniques • Inhibit activated oncogenes

5. Different Approaches

6. Basic Requirements for Successful Gene Therapy • Safety • Efficiency • Specificity • Expression level of transduced gene • Simplicity of manufacture • Can be difficult to satisfy, depending on therapeutic strategy and cancer type.

7. Delivery Methods • DNA/RNA viral and non-viral vectors. • Mammalian viral-based cancer gene therapy is increasingly popular. • Fairly low efficiency in clinical trials • Safety concerns • Another focus is on replication-competent oncolytic viruses. • Perpetually exist in mammalian cells, safety concerns.

8. Recombinant Baculovirus as a Highly Potent Vector for Gene Therapy of Human Colorectal Carcinoma: Molecular Cloning, Expression, and In Vitro Characterization • Paul et al, primary paper. • They evaluated the insect-cell specific baculovirus as a vector for gene delivery to colorectal cancer cells in addition to other cancer types such as breast, pancreas, and brain.

9. Why baculovirus? • Baculovirus can be a powerful vector to target tumor cells. • It can enter mammalian cells without replicating. • Increased efficiency and eliminated safety concerns with previous vectors • Natural virus promoters are not active in mammalian cells.

10. Gene Construct • A recombinant baculovirus gene was constructed with the Monster Green Fluorescent Protein (MGFP) and the cytomegalovirus (CMV) promoter. CMV Promoter MGFP Gene

11. Two Plasmids Used • phMGFP vector with the MGFP gene and the CMV promoter • pVL1392 baculovirus transfer vector • Both digested with BgIII and Xball restriction enzymes. • The excised PCMV-hMGFP gene was inserted into MCS of transfer vector. • Amplification of recombinant transfer vector in E. coli.

12. Two Plasmids

13. BacMam Virus • Now, the insect baculovirus is able to express its transgenes in mammalian cells, driven by the CMV promoter. • The purified recombinant transfer vector was co-transfected with linear baculovirus DNA in Spodopterafrugiperda (Sf9) insect cells to generate recombinant baculoviruses with the CMV promoter and hMGFP gene.

14. Experiment • Human colon cancer cells (SW480), breast cancer cells (SkBr3), neuroblastoma cancer cells (Neuro2A), hepatocellular carcinoma cells (HepG2), and pancreatic carcinoma cells (PANC-1) were maintained on appropriate mediums with supplemented 10% Fetal Bovine Serum (FBS) as stationary cultures.

15. Experimental Conditions • Several different factors that may affect transduction efficiency • Multiplicity of Infection (MOI)- tested a range of 10 -1,000 • Sodium Butyrate (NaBu) concentration in medium- either 0 or 10 mM. NaBu has various effects on cultured mammalian cells, one of them is to increase gene expression. • Viral Incubation Time- 2-8 hours

16. Transduction • SW480 cells were transduced with baculovirus on 96-well plate. • A plate reader was used to measure the fluorescein readings from time to time to determine the effect of viral incubation time on gene expression. • RNA was extracted from transduced cells and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was performed using MGFP specific primers for a 133 bp product.

17. Results • RT-PCR was also performed on control samples, and the 133 bp MGFP fragment was only found in the transduced SW480 cells. This is evidence that the MFGP gene was successfully transcribed in the cancer cells. • By comparing band intensities from the transduced cells, an increase in MOI and addition of NaBu increased transcription further.

18. Results • This was confirmed by taking fluorescent photographs of the cells with and without phase contrast illumination. • Higher MOI with addition of NaBu shows the most fluorescence.

19. Results

20. Results • Higher MOI, NaBu, and longer incubation time all had positive results on transduction efficiency and maximum transduction efficient occurred when these parameters were combined together. • At an incubation time of 8 hours, the highest efficiency achieved was with MOI 1,000 and 10 mMNaBu.

21. Results Transduction efficiency much higher in SW480 cells

22. Transducing Different Cancer Cells

23. Results • Although a high transduction efficiency was observed, this gradually diminished due to the fact that baculovirus does not naturally integrate with the host organism, so long-term expression was not observed.

24. Longer Incubation Time, Higher Normalized Gene Expression

25. Advantage • This is important in terms of safety because the vector won’t randomly integrate into crucial gene regions and cause unexpected tumerogenesis, whereas mammalian viral vectors would.

26. Disadvantage • However, this is a limiting factor of cancer gene therapy and can be problematic. • It can possibly be solved by using a hybrid baculovirus-adeno associated viral vector for more prolonged expression. • Also, multiple administrations of the viral dose may also overcome transient expression.

27. Conclusion • Baculovirus vectors can be used as safe delivery vehicle for cancer gene therapy, especially colorectal cancer and possibly other cancer types in the future.

28. What now? • The next step is to generate recombinant baculoviruses that can express certain tumor suppressor genes for specific cancer types. • Baculovirus may also be successful in delivering suicide transgenes.

29. References • Paul, A.; Jardin, B. A.; Kulamarva, A.; Malhotra, M.; Elias, C, B.; Prakash, S. Recombinant Baculovirus as a Highly Potent Vector for Gene Therapy of Human Colorectal Carcinoma: Molecular Cloning, Expression, and In Vitro Characterization. Mol Biotechnol [Online] 2010, 45, 129 ff. • Zhang, J.; Russell, F. J. Vectors for Cancer Gene Therapy. Cancer and Metastasis Reviews [Online] 1996, 15, 385 ff. • Gunji, Y.; Ochiai, T.; Shimada, H., Matsubara, H. Gene Therapy for Cancer. Surg Today [Online] 2000, 30, 967 ff.