1. Oncolytic viral therapy Presented to Dr. Qamar Saeed Presented by Jawairia Shoukat

2. Definition Use of genetically engineered/naturally occurring viruses that selectively replicate in and kill cancer cells without harming the normal tissues Stimulate immune system  Once cancer  Lytic lysis  Viral using viruses  Therapy for treatment

3. Oncolytic viruses Mechanism

4. Other treatments for Cancer Surgery remove metastasize difficult to remove Chemotherapy toxic drugs severe side effects kill healthy cells Radiations damage surrounding tissues Nanotechnology use carbon/other molecules/shells of cells –deliver drug/radiations

5. Mechanisms of oncolytic viruses

6. Examples of oncolytic viruses  Adenovirus  Herpes simplex virus  Measles virus  Reovirus  Vaccinia virus  Morbilliviruses  Coxsackie virus

7. Advantages of oncolytic viral therapy Advantages Eliminates the population of cells that are often resistant to chemotherapy and radiotherapy Replicates only within tumor cells Increases therapeutic index Minimal toxicity to normal tissues Dual effect of viral Oncolysis and the added effect of the prodrug or immune stimulator

8. Main Challenge of oncolytic viral therapy  Main challenge is patient immune system kill the virus before virus kill the cancer cell Solution: Chemical modification of the coat proteins of the viruses by conjugation of biocompatible polymers, such as polyethylene glycol (PEG) and N-[2-hydroxypropyl] methaacrylamide (HPMA). Deplete macrophages before administration of the virus. To achieve this use chlorinate liposomes. The mechanism of action is very simple: macrophages phagocytose the liposomes and as a result of phospholipase- mediated disruption of the liposomal structure and consequent release of the chlorinate, cell death is triggered

9. Barriers in effective OVT when virus deliver via bloodstream

10. Milestones of oncolytic virus therapy development.

11. Genetically engineered oncolytic viruses  T ‐ Vec  Double ‐ mutated HSV ‐ 1, deletions in the γ34.5 and α47 genes  Human GM ‐ CSF gene inserted into the deleted γ34.5 loci

12. T VEC γ34.5 deletion  Cancer selective replication  Attenuated pathogenicity α 47 deletion  Enhance antitumor immune response  Immediate early response of US11 gene that enhance viral replication GM ‐ CSF  Enhance the antitumor immunity induction

13. Mechanism of T VEC

14. G47∆  Triple ‐ mutated third ‐ generation oncolytic HSV ‐ 1  Insertion of the Escherichia coli LacZ gene inactivating the ICP6 gene.  The ICP6 gene encodes the large subunit of ribonucleotide reductase (RR) that is essential for viral DNA synthesis.  When ICP6 is inactivated, HSV ‐ 1 can replicate only in proliferating cells that express high enough levels of host RR to compensate for the deficient viral RR

15. JX ‐ 594  Genetically engineered vaccinia virus that has a mutation in the TK gene, conferring cancer cell ‐ selective replication, and an insertion of the human GM ‐ CSF gene

16. CG0070  CG0070 is an oncolytic adenovirus. Ad5 adenovirus was engineered so that the human E2F ‐ 1 promoter drives the E1A gene, and the human GM ‐ CSF gene is inserted. E2F ‐ 1 is regulated by the retinoblastoma tumor suppressor protein (Rb), which is commonly mutated in bladder cancer, and a loss of Rb binding results in a transcriptionally active E2F ‐ 1.

17. Naturally occurring oncolytic viruses Reolysin  Reoviruses are double ‐ stranded RNA viruses that replicate preferentially in transformed cell lines but not in normal cells.  Oncolytic properties of Reovirus depend on activated Ras signaling. Reolysin is the T3D strain of Reovirus, which has been most extensively studied among several serotypes as an anticancer agent, and is currently the only therapeutic wild ‐ type Reovirus in clinical development.

18. Summary of major oncolytic viruses under clinical development

19. Example of oncolytic viral therapy Oncolysis of Diffuse Hepatocellular Carcinoma by Intravascular Administration of a Replication competent, Genetically Engineered Herpesvirus1

20. Hepatocellular carcinoma  Malignant tumor of liver.  Greater than 1 million new cases/year  Need new therapeutic approaches for HCC.  Despite curable strategies such as resection or liver transplantation, patients with advanced HCC continue to present a poor outcome.

21. Herpesvirus 1  Replication-conditional HSV-1 mutant  rRp450 Defective in expression of the large subunit of viral ribonucleotide reductase so viruses preferentially replicate in dividing cells rather than quiescent cells permits selective replication  During viral replication, rRp450 also expresses the rat cytochrome P450 2B1 (CYP2B1) transgene, which encodes an enzyme responsible for bio activation of prodrugs, such as cyclophosphamide, into their active cytotoxic metabolites, thus providing a means for intratumoral generation of alkylating metabolites

22. Material and method ( Cell lines and viruses) Cell lines derived from human HCC (Hep-3B,), mouse HCC (Hep 1-6), rat HCC, and the LoVo African Green Monkey kidney (Vero) were obtained Vero cells were infected with rRp450 using an MOI of 0.005 + Media add to the infected Vero cells 1–2 h after infection Plaques were counted 5 days later to determine titers

23. In Vitro Viral Cytotoxicity and Replication Assays. Viral cytotoxicity 5000 cells plated onto 96-well plates & grown for 36 h Cells infected with rRp450 using MOI from 0.0001 to 100 viable cells was determined using a colorimetric 3 Viral replication infecting cells with HSV-1 for 2 h, unadsorbed virus was removed by washing with a glycine-saline solution supernatant & cells exposed to three freeze cycles to release virions and titered on Vero

24. Results Oncolysis of HCC by rRp450

25. Cyclophosp hamide Potentiates the Cytotoxic Effects of rRp450 in Vitro. A B

26. Comparison of Selective rRp450 Replication in HCC and Hepatocytes

27. rRp450 Effectively Treats Diffuse HCC in Vivo

28. FINAL GOAL  The ultimate challenge in the field of oncolytic virotherapy is to produce an “ideal” oncolytic therapy that is:  Highly selective Able preferentially to seek and enter tumor cells while sparing non cancerous tissues and cells  Replication competent Maximally efficient in making copies of itself, amplifying in and destroying tumor cells  Well-tolerated Having minimal side effects in cancer patients, and even more importantly, non pathogenic; in human patients.

29. FINAL GOAL  Carries little to no risk of genomic integration viruses that do not integrate into the host cell DNA (replicate only in the cytoplasm) do not carry this risk, and therefore no risk of mutation.  Systemically administered In metastatic disease, the most clinically relevant approach is to target and eliminate cancer cells, tumors and metastases wherever they are located in the body.