Suicide gene therapy

Suicide gene therapy is based on the delivery of a gene encoding a cytotoxic protein into tumor cells.
For this, there are two possible strategies:
1. Indirect gene therapy using enzyme-activated pro-drug, which allows the conversion of a pro-drug into a lethal drug into cells.

2. Direct gene therapy using a toxin gene, whose expression can change the stability of the cell membrane and reduce the viability of tumor cells, or correct mutated pro-apoptotic genes, generally tumor suppressor genes that in normal condition induce cell suicide.

1. October 2020 Presenter: Parisa Ghasemiyeh Pharm.D ., PhD. Student of Pharmacotherapy, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran. Suicide Gene Therapy for Cancer

2. Suicide Gene T herapy for Cancer

3. Suicide gene therapy is based on the delivery of a gene encoding a cytotoxic protein into tumor cells . For this, there are two possible strategies: 1. Indirect gene therapy using enzyme-activated pro-drug , which allows the conversion of a pro-drug into a lethal drug into cells. 2. Direct gene therapy using a toxin gene , whose expression can change the stability of the cell membrane and reduce the viability of tumor cells, or correct mutated pro-apoptotic genes , generally tumor suppressor genes that in normal condition induce cell suicide. Suicide gene therapy for cancer

4. Suicide gene therapy 1 2 3

5. G ene-directed enzyme pro-drug therapy (GDEPT ): A two-step treatment designed to treat solid tumors . In the first step , the gene for a foreign enzyme is delivered and targeted in a variety of ways to the tumor where it is to be expressed . In the second step , a pro-drug is administered that is activated to the corresponding drug by the foreign enzyme expressed in the tumor . Enzyme/Pro-drug Systems 1

6. Enzyme/Pro-drug Systems 1

7. Advantages of suicide gene therapy

8. Hurdles of suicide gene therapy

9. One of the most frequently studied therapeutic strategies is based on the transfection of herpes simplex virus thymidine kinase (HSV-TK) gene with ganciclovir (GCV) administration . HSV-TK converts the antiviral drug GCV to a monophosphorylated molecule that is then metabolized to the toxic triphosphate form by cellular kinases, inhibiting DNA synthesis and producing cell death . Enzyme/Pro-drug Systems 1

10. Another system consists in the combined administration of the cytosine deaminase (CD) (enzyme found in bacteria and fungi , but absent in mammalian cells) with 5-fluorocytosine ( 5-FC ). This system relies on the ability of CD to convert the nontoxic 5-FC into 5-fluorouracil (5-FU) , a potent cytotoxic chemotherapeutical , which is then transformed by cellular enzymes , into potent pyrimidine antimetabolites ( 5-FdUMP, 5-FdUTP , 5-FUTP) . CD/5-FC causes inhibition of cell proliferation and cell death . Enzyme/Pro-drug Systems 1

11. Another system employs the conversion of cyclophosphamide and ifosfamide prodrug by human CYP450 isoforms into the unstable metabolites 4-hydroxy forms that decay into phosphoramide mustard and acrolein . These alkylating agents interfere with the DNA and the strand breaks during DNA replication causing cell death . Enzyme/Pro-drug Systems 1

12. An advantage of genetic prodrug activation therapy is the bystander effect , because the prodrug has efficacy not only on transfected cells but also on neighboring non-transfected cells . Enzyme/Pro-drug Systems 1

13. One alternative in suicide gene therapy for cancer is the use of a toxin gene . Advantages: Toxins 2

14. Toxins 2

15. D iphtheria toxin ( Corynebacterium diphtheriae )  protein synthesis inhibition. Diphtheria toxin: inactivates elongation factor 2 ( EF-2 ) by adenine diphosphate ribosylation (ADP-ribose) and inhibits protein translation , thereby triggering apoptosis . A. Bacterial Toxins 2

16. 2. S treptolysin O (SLO) is a membrane-damaging protein secreted by bacteria from the genus Streptococcus. It has been patented that the administration of a recombinant adenovirus encoding SLO induced cell death in transfected cancer cells , by forming pore and permeabilization of the cellular membrane . A. Bacterial Toxins 2

17. 3. P seudomonas exotoxin induces cell killing by apoptosis through adenosine diphosphate ribosylation and by inactivation of EF-2 and thus inhibition of protein synthesis . A. Bacterial Toxins 2

18. 4 . S everal novel E. coli with toxin–antitoxin regulatory mechanisms have been recently discovered. A. Bacterial Toxins 2

19. 1 . MazF is a toxin that is counteracted by the MazE antitoxin . -MazF exhibits sequence-specific ribonuclease activity toward single- or double-stranded RNA regions, -Results in degradation of cellular mRNA. -Causes global translation inhibition. -Thereby effectively inhibiting cellular protein synthesis. -Thus inhibiting cell growth. B. Immunot oxins 2

20. B. Immunot oxins 2

21. 2 . Apoptin , the VP3 protein from chicken anemia virus (CAV), which induces death in tumor cells but not in normal cells . Apoptin is phosphorylated and translocates to the nucleus , enabling its cytotoxic activity . B. Immunot oxins 2

22. The corrective gene therapy of genes involved in cell death and apoptosis , which induce cell suicide, also has been developed . Mutations of p53 gene are the most frequent abnormality identified in human tumors and numerous studies have shown that restoring p53 function can induce apoptosis in cancer cells . In recent years, methods have been patented for the use of this strategy in patient. Pro-apoptotic Genes 3

23. G endicine is a recombinant adenovirus engineered to express wild-type p53 (rAd-p53) , which is designed to treat patients with tumors that have mutated p53 genes. The process of programmed cell death (apoptosis) is regulated by cysteine proteases known as caspases . The initiator of apoptotic pathway is caspase-9 , which is activated when it is caught by Apaf-1 in presence of dATP and cytochrome-c. Caspase-9 has been fused to a CID-binding domain (artificial caspase activated by chemical inducer of dimerization) and is named ‘inducible caspase-9ʹ ( iCaspase-9 ), which induces the apoptotic signaling pathway . Pro-apoptotic Genes 3

24. A system with a nucleic acid sequence encoding a cell death mediator protein ( CDMP) has been patented. The expression of apoptosis promoting activity of the CDMP can be inducible, for example, by iCaspase-9 to activate the apoptotic pathway. Pro-apoptotic Genes 3

25. There are other apoptotic dysfunctions that make cancer cells resistant to treatment and induce tumorigenesis . Bax , a central cell death regulator , is an indispensable gateway to mitochondrial dysfunction and a pro-apoptotic member of Bcl-2 family proteins that controls apoptosis in normal and cancer cells. Other patented strategies include genes involved in others apoptotic and cell death mechanisms such as Smac , caspase 3 , and TRAIL , driven by specific promoters . Pro-apoptotic Genes 3

26. Strategies to improve suicide gene therapy 1 4 3 2

27. In order to be suitable for clinical applications, a vector must meet the following requirements: 1 2 3 4 5 Improved vectors for suicide gene delivery 1

28. S uicide gene delivery systems can be divided into three major groups including: Improved vectors for suicide gene delivery A B C 1

29. Viral vectors Viral vectors are the most efficient vectors for gene delivery . The most common viruses in the field of gene therapy are: Retrovirus A denovirus (Ads ) Lentivirus A deno -associated viruses (AAVs ) Also, a new vector based on sindbis virus , a blood-borne alpha virus transmitted through mosquito bites , was patented. It has been shown that Sindbis virus is able to induce apoptosis in mammalian cells without carrying out cytotoxic genes. Viral vectors A

30. I) Viral vectors Viral vectors A 1 2

31. M any efforts were carried out in the development of nanosystems for gene delivery . Synthetic vectors B

32. Different non-viral delivery systems have been developed, including: Synthetic vectors B

33. Cellular therapy based on the use of stem cells is an emerging therapeutic modality that currently generates great expectations in cancer . These cells have many benefits including: Their renewing ability of themselves through cell division , sometimes after long periods of inactivity. T heir potential to differentiate into many cell types in the body during early life and growth. T heir ability to homing to the tumor microenvironment. Cell-based vectors C

34. Mesenchymal stem cells (MSCs) have been used as carriers for in vivo delivery of various clinically relevant anticancer agents, including interferon , pro-drugs , or replicative adenovirus . The MSCs administered to a subject were able to migrate to the tumor site. These cells may be optimized for use as an off-the-shelf product by using an immortalized MSC line with immunological characteristics to forestall a graft-versus-host response. Cell-based vectors C

35. Cell-based vectors C

36. Moreover, the recent patent “Cancer specific suicide gene for cell based and gene therapy” provides pluripotent and multipotent stem cells that have been modified to contain an inducible cancer-specific suicide gene construct that, upon induction, selectively kills stem- or progenitor-cell-derived cancerous cells . Cell-based vectors C

37. Another invention claimed a method of providing safety of application of pluripotent stem cells in tissue substituting therapy by means of artificial chromosomes carrying bicistronic cassette with suicide gene. This invention makes possible to: Cancer transformation of transplanted cells to zero risk Development of teratomas Clinical efficiency Cell-based vectors C

38. Double-suicide gene therapy is a promising strategy for the treatment of advanced cancer. Co-expression of TK/GCV with CD/5-FC ( CD/TK) offers greater therapeutic efficacy than single-suicide gene therapy and allowed better outcomes to be achieved with lower prodrug concentrations. Double-gene therapy of cancer 2

39. The combination of enzyme-activating and prodrug systems has some drawbacks: Toxic metabolite release. R educed bioavailability . For this reason, a new strategy based on double-suicide gene therapy using gef and apoptin , two killer genes that do not need a prodrug to be effective in tumor cells, was patented. The co-expression of both genes enhances the cell growth inhibition induced by single-suicide gene therapy, taking advantage of the synergistic anticancer effects of both systems in colon carcinoma cells . Double-gene therapy of cancer 2

40. Combining suicide genes with other therapeutic modalities: P romote the therapeutic efficiency and safety M inimize the side effects of different treatments Gene transfer has been proposed as a new strategy to enhance the efficacy of antitumor drugs in the treatment of intractable or metastatic cancers . The co-administration of chemotherapy and suicide genes has resulted in enhanced anticancer effects . Combined suicide gene therapy and classical therapies of cancer 3

41. To increase specificity and safety of suicide gene therapy , the expression of the therapeutic gene needs to be tightly controlled within the target tissue . Targeted suicide gene therapy 4 1 2 3

42. Receptors targets for therapeutic transgene vectors Several cancer-specific receptors may be a molecular marker in the diagnosis of tumor cells and could be a potential therapeutic target. 1

43. Cancer-specific promoters The main objective of suicide gene therapy is to deliver therapeutic genes into target cells safely and without harming surrounding healthy cells . One possibility is the use of tumor-specific promoters overexpressed in cancer cells. They can induce a specific expression of therapeutic genes in the tumor increasing their localized activity . 2

44. There are several cancer-specific promoters employed in gene therapy such as: Cancer-specific promoters 2

45. The design of viral vectors that allow selective tropism for particular types of cells and tissues remains a challenge. To achieve the purpose of targeted killing of tumor cells , in some case the full potential of Adenovirus gene transfer has not been fully realized because of the non-specific in vivo tissue distribution of Adenovirus receptors . Adenovirus receptors are expressed at low levels in some target tissues rendering them difficult to infect. Modification of the tropism 3

46. The patent CN 104328140 A solves this problem modifying adenovirus tropism by integrating the RGD sequence into the adenovirus cilium zone . Thus, along with the expression of the bladder epithelium-specific promoter UPII improved the tropism on the bladder cancer, which lowly expresses the adenovirus receptor and solves the problem of low infection ability of the adenovirus vector. Modification of the tropism 3

47. Recently , a next-generation of Adenovirus viral vectors capable of selective tropism and efficient gene delivery have been developed. The vector allowed selective killing of U87 glioblastoma cells and derived xenografts via the HSV-TK , increasing the potency of GCV by 25-fold . Modification of the tropism 3

48. Conclusion Although suicide gene therapy has been successfully used in a large number of in vitro and in vivo studies, its application to cancer patients has not reached the desirable clinical significance . R ecent preclinical and clinical studies in cancer models prove the enormous potential of this strategy when used in combination with classic therapeutic approaches . Thus, this combination therapy can result in enhanced anticancer effects especially when the different treatments act in diverse pathways and they would reach all the cell types that make up the tumor including cancer stem cells (CSCs), making it a new promising strategy in cancer therapy.

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