Humber College

Cancer Gene Therapy

Submitted to: Peivand Pirouzi Course: REGA 505
Submitted By: Harshani De Silva and Zobia Sharif 12/6/2012

Cancer: An epidemic in disguise Mutations in genes can disrupt a cell by changing the amounts or activities of the proteins that are produce from defective genes. Mutated genes can lead to cells that grow uncontrollably, which can eventually progress to cancer formation (1). In Canada, an estimated 186,400 new cases of cancer (excluding about 81,300 non-melanoma skin cancers) and 75,700 deaths will occur in 2012 (2). There are about 500 Canadians who will be diagnosed with cancer every day, 200 of which will die during that one day (2). Cancer is not a simple disease. It is requires complex series of multiple mutations in which abnormal body cells multiply and spread in an uncontrolled fashion to form a tumor (3). Cancer cells violate the normal cell cycle by ignoring the usual controls of regulation. The current list of known cancer genes includes 70 genes associated with germline mutations and 342 genes associated with somatic mutations (4). During the multi-step process, a series of mutation lead to decreased tumor suppressor gene function and increased proto-oncogene function (4). Protooncogenes code for protein products that normally regulate cell growth, cell division, and cell adhesion (4). Tumor suppressor genes inhibit cell division because the proteins they encode normally prevent uncontrolled cellular growth (4). They are normal genes whose ABSENCE can lead to cancer. What is Gene Therapy? The range of different cancers encountered & the mutations they carry, have led to a variety of strategies for its therapy. Currently, researchers are exploring the profound gene therapy in which normal genes are introduced into cells to reconstitute for missing or inactive proteins (5). It allows a deficient phenotype to be corrected by expressing genes and producing sufficient

amounts of a normal gene product. Gene therapy is bringing about a new era of therapeutic intervention, where doctors can treat diseases by simply injecting genes into the bloodstream.

Vectors in Gene Therapy Gene therapy encompasses several approaches in order to modify faulty genes. These include: insertion of a normal gene to replace a non-functional gene, substituting an abnormal gene for a normal gene through homologous recombination, repairing an abnormal gene through selective reverse mutation and altering regulation of a particular gene (5). However, direct insertion of genes into target cells fail to produce functional gene products. Thus, gene therapy utilizes carriers known as vectors in order to introduce therapeutic genes to target cells. Genetic material is optimally transported by naturally evolved vectors such as viruses (6). Thus, current research on vectors transferring genes into mammalian cells is essentially focused on either viral or non-viral mediated delivery (6) (Figure 1).

These methods differ in specificity, efficiency, stability of gene expression and elicitation of the immune response (6). Though a number of viruses are advantageous for viral mediated delivery

of genes, interest has developed around three main types of viruses; retroviruses, adenoviruses and adeno-associated viruses (6). Viruses have a large capacity for foreign DNA integration into the host chromosomes and most do not cause serious diseases, allowing long term stability (8). For gene therapy, replication deficient viral particles are used to transfect cells (7). Conversely, some draw backs of existing virus vectors are that genes are integrated randomly and target only dividing cell, which disrupts the hosts cell genes (7). Also, some integrated genes only function transiently due to attacks by the hosts immune system (7). Besides virus mediated gene delivery, there are several options for non-viral gene delivery ex. Liposomes and Naked DNA (6). Liposomes are double-layered lipid molecules that are designed to harbor a plasmid containing therapeutic genes with the capability of passing DNA through target cell membranes (6). Some cells are also able to assimilate naked DNA that is injected as a free plasmid (6). Non-viral vectors such as liposomes and naked DNA are non-infectious and are easy to deliver by ex. vivo methods (6). However, non-viral vectors lack specificity for targeting cells and do not provide long term stability, which necessitates repeated delivery (6). Efficient gene delivery is the greatest challenge to successful gene therapy, preventing it from being accessible as an effective tool for healthcare (8). At present, a completely reliable, safe and efficient vector is not available (8). Ideally a perfect vector would target specific cells, insert their genetic material at a safe site in the genome and be regulated by the bodys normal physiological signals. Proficient vectors in gene therapy will be easy to produce at high titres and insert therapeutic genes in a stable way, such that genes do not mutate within the vector or cell. In addition, vectors must be able avoid any adverse immune responses.

Transgene Delivery Method All gene therapy to date has been directed towards somatic cells, whereas germline cell (sperm cells, ova, stem cell precursors of sperm and ova cells) therapy remains controversial (9). In somatic gene therapy, vectors can be delivered to the host by either in vivo or ex vivo methods (9). In direct delivery or in vivo methods, therapeutic genes are delivered directly into hosts targeted somatic cells by injection (9). During the ex vivo or cell based delivery, transgenes are packaged into a vector and delivered into a cell culture (ex. Stem cells) derived from the patient (9). After modifications of the cells outside the body, cells are re-administered or transplanted back into the patient. (9) Gene therapy strategies for cancer treatment: Transgene Products and

Immunomodulation Approaches Tumor Suppressor Genes As loss of tumor suppressor function is commonly associated in many human malignancies, the main strategy for gene therapy in cancer treatment is to introduce tumor suppressor genes to malignant cells (5). This strategy encompasses gene replacement by introducing genes such as the p53, the retinoblastoma gene pRB and p16 INK4A into tumor cells (5). Expression of these genes can lead to apoptosis or cell cycle arrest in the targeted tumor cells (5). Suicide Gene Therapy Suicide gene therapy which is another common strategy, involves the eradication of cancerous cells using genetically expressed toxins (10). This method relies on the delivery of an enzyme encoding gene to targeted cancerous cells followed by the administration of a non-toxic prodrug (10). The enzyme encoding gene is able to convert the non-toxic prodrug to an active cytotoxin, which in turn can kill the cancerous cells (10). An important advantage of suicide

gene therapy is its ability to kill neighboring cells through what is called bystander effect (10). Once activated, the prodrug can diffuse into the neighboring cancerous cells and cause their cell death as well (10). Additionally, dying cells can activate the immune system by inducing natural killer cells and T cells (10). The combination of herpes simplex virus thymidine kinase gene and Ganciclovir drug (GCV) is the most studied gene/drug combination for suicide gene therapy (10). Inhibition of Dominant Oncogenes Many dominant oncogenes are associated with the development of cancers (11). Vectors can be used to inhibit the growth of cancerous cells by expressing genes that will inactivate dominant oncogenes (11). Vectors mediated technologies, such as; triple helix oligonucleotide, antisense and ribozymes, prevent the production of mutated proteins by inhibiting the expression of mutated dominant oncogenes (11). Triple helix forming oligonucleotide gene therapy can prevent transcription by binding to the grooves between double strands of the mutated genes DNA (11). Binding of single stranded pieces of DNA, prevents that segment of DNA from being transcribed into mRNA. This, in effect, will prevent the transcription of the dominant oncogene (11). In antisense gene therapy, RNA strands containing the antisense code of a mutated gene bind to the mutated sense mRNA strand. The complementary binding of the anti-sense RNA prevents the mRNA from being translated into a mutated protein (11). Consequently, it will cause a post-transcriptional block that will remove the dominant negative effects of the oncogenes. Like antisense, RNA ribozyme molecules also bind and targets mRNA transcripts copied from the mutated gene (11). Ribozymes act as molecular scissors that cleave the target mRNA, preventing it from being translated into a protein (11). Each of these techniques also

requires a method (e.g vector mediation) that is specific and efficient, to deliver gene into the targeted cells. Immunomodulatory approaches Researchers are also studying several ways to improve the body's natural ability to fight the cancer (5). An integral component of tumor development is the failure of normal immune surveillance mechanisms to recognize cancer cells as foreign cells (5). Immunostimulatory gene therapy utilizes vectors to introduce genes that activate the host immune system or attempt to bypass cancer inducing cell defects by altering the local immune microenvironment (5). Immunomodulatory strategies use the expression of cytokines, co-stimulatory molecules (B7.1 and B7.2) and tumor specific antigen for cancer gene therapy (5). In such cases, researchers can use an ex. vivo method to insert genes that produce T-cell receptors (TCRs) into the patients white blood cells (6). In the body, white blood cells are able to produce TCRs on the surface of their membranes, heightening their recognition of tumor surface molecules (6). TCRs then activate the white blood cells to attack and kill the tumor cells (6). Vector mediated transfer of genes (ex. cytokines, interferons and interleukins) to white blood cells (T-lymphocyte or dendritic cells), that enhances their anti-tumor activity can be effective in killing tumor cells (5). Similarly, vectors expressing variable regions of tumor-specific monoclonal antibodies can also be used to target tumor associated antigens and kill tumor cells (5). Human gene therapy As mentioned previously, the goal of human gene therapy is to deliver functional genes to reconstitute for defective or absent protein products. In the application of human gene therapy many factors must first be considered. These include: nature of the disease, how to deliver the remedial genes, transcriptional regulations of introduced genes, possibility of additional

physiological problems due to overexpression of these genes and details of treatment effect i.e whether or not treatments need to be repeated (5). Human gene therapy suffered a major setback with the death of 18 year old Jesse Gelsinger, who is the first person (publically identified) to have died in a clinical trial for gene therapy. Jesse suffered from ornithine transcarbamylase (OTC) deficiency, which is a rare metabolic disorder which hinders bodys ability to metabolize ammonia (12). Although the disease is usually inherited and fatal at birth, Jesses condition was due to a spontaneous mutation after conception (12). In September 1999, Jesse participated in a clinical trial headed by the University of Pennsylvania, aimed at developing a possible treatment for infants suffering from OTC deficiency (12). On September 13th Jesse was administered a corrected gene encapsulated in an adenoviral vector (12). He went into coma on September 14th and was pronounced dead 4 days after on September 17th (12). Following administration of the viral vector, Jesse suffered from jaundice, kidney failure, lung failure and eventually brain death (12). This unexpected chain reaction occurred following the administration of the maximum tolerated dose which triggered an overwhelming immune response (12). Investigators concluded Jesse died due to multiple organ failure that was triggered by the adenoviral vector (12). This devastating incidence brought on immediate halt to nearly all gene therapy trials at the time. Gene therapy studies using viral vectors suffered a massive setback as researches opted to switch to non-viral vectors (12). Jesses death also increased regulatory scrutiny for clinical trials for gene therapy studies. Although tragic, this incidence was a shocking reminder to all researchers of the magnitude of risk associated with gene therapy. It reinforced the criticality of choosing appropriate patients for gene therapy trials in accordance with the inclusion criteria. It also reinforced the importance of adequate reporting criteria for adverse effects and disclosing all

relevant information such as adverse effects and preclinical study results in the informed consent forms. Applications of Cancer Gene Therapy in Recent Clinical Trials Case study 1 In a paper published by Xu and colleagues, (Phase I and biodistribution study of recombinant adenovirus vector-mediated herpes simplex virus thymidine kinase gene and ganciclovir administration in patients with head and neck cancer and other malignant tumors) the results of a clinical trial conducted in China on patients suffering from head and neck cancer are presented. Head and neck cancers are common among the Chinese population and these cancers tend to relapse locally and can be resistant to conventional cancer treatment (13). In their study, Xu et al. used suicide gene therapy incorporating the recombinant adenovirus vector-mediated herpes simplex virus thymidine kinase gene (AdV/TK) in combination with the prodrug, Ganciclovir (GCV). The objective of their study was to establish a safety profile, evaluate the humoral immune response and the biologic activity of gene-drug combination, as well as characterize the pharmacokinetics of AdV/TK in cancer patients. The most prevalent adverse effect observed was fever which might be the result of a physical immune response to the viral vector. Reported events of both fever and injection site reactions were easily controllable with minor symptoms. Several other reported adverse events included digestive tract reactions, flu-like symptoms, chills, fatigue, lethargy and diarrhea. However, all these adverse events were expected as they are all frequently associated with adenovirus vector gene therapy. The findings from the clinical evaluations suggest no correlation between the dose of AdV/TK and adverse effects, with the exception for the flu-like symptoms. At a high dose of 1.0*1012 VP, flu-like symptoms were more severe and had a lengthier duration. The researchers also observed that increasing the dose

of GCV prodrug amplified the toxicity but not the response. The study findings indicated a reduction in the local target tumor following treatment, which validated their study objective. The observed treatment effect was independent of the administered dose of AdV/TK. Thus, based on this clinical trial, Xu et al. concluded suicide gene therapy using AdV/TK and GCV is safe in Chinese patients and managed to achieve a local response in the target tumor cells with few adverse effects (13). Case study 2 As mentioned previously, loss of tumor suppressor genes such as p53 can cause malignancies. P53 gene in particular is responsible for cell cycle regulation by means of inducing cell cycle arrest, initiating DNA repair mechanisms, and inducing apoptosis in the case of DNA damage. In another paper published by Tolcher and colleagues (Phase I, Pharmacokinetic, and Pharmacodynamic Study of Intravenously Administered Ad5CMV-p53, an Adenoviral Vector Containing the Wild-Type p53 Gene, in Patients With Advanced Cancer) presented the results from a gene therapy study that delivered a wild-type p53 gene to patients with advanced malignancies (14). Gene delivery was achieved using an adenoviral vector containing the wildtype p53 gene along with a cytomegalovirus (CMV) promoter (Ad5CMV-p53). This clinical trial had promising outcomes as preclinical studies had implied that the administration of Ad5CMVp53 into human tumor cell lines with known p53 mutations can induce p53 protein expression and apoptosis. Results from preclinical studies had also indicated the occurrence of systemic distribution with Ad5CMV-p53 DNA. Systemic distribution is deemed advantageous in gene therapy studies as most cancers are not directly accessible for treatment. The study objectives were to: determine the feasibility of intravenous administration of Ad5CMV-p53 daily for 3 consecutive days every 4 weeks, characterize both the toxicity and clearance of administered

dose, examine evidence of wild-type p53 expression in normal and tumor tissue biopsies and to seek preliminary evidence of antitumor activities in patients. The patients were administered Ad5CMV-p53 at doses between 3x1010 3x1012 Vp. Clinical results indicated a negative impact on blood coagulation factors (decrease in Fibrinogen and increase in Fibrin degradation products) for doses exceeding 3x1012 Vp. Some of the other adverse effects reported included fatigue, nausea, vomiting and fever. As anticipated, Ad5CMV-p53 was detectable in plasma in majority of the patients and Ad5CMV-p53 DNA was detectable in tumor tissue collected from several patients. Based on these results, Tolcher et al. concluded that Ad5CMV-p53 can be feasibly administered at doses up to 3x1012 Vp daily for 3 days every 28 days to patients with advanced cancers. The detection of p53 DNA within tumor sites that are distant from the site of administration, predicted the possibility of systemic gene therapy with Ad5CMV-p53. Systemic delivery of gene vectors can be advantageous for tumors that are not accessible for local site injection. For instance, systemic delivery of Ad5CMV-p53 can target multiple sites that are not accessible for local injection. Tolcher et al. suggested that the rational next stop would be to expand on these results using phase II and pharmacodynamics studies in patients with known p53 mutations. They also suggested examining the effect of Ad5CMV-p53 in combination with a cytotoxic agent to determine the effect of p53 restoration on antitumor activities (14). Future prospects for gene therapy Gene therapy is increasingly being considered for the treatment of conditions such as Leukaemia, Alzheimers disease, breast cancer and Parkinsons disease. Results from several preclinical studies also suggest the possibility of employing gene therapy strategies in the treatment of diabetes and obesity (15). According to the online clinical trials registry (clinicaltrails.gov), there

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are about 123 open gene therapy studies currently underway (16). Almost 2/3 of all gene therapy clinical trials are on cancer treatments (17).

Major Problems Despite some of its promising outcomes, gene therapy still poses several obstacles that might hinder its progression for healthcare. Researches are attempting to develop more efficient delivery systems that would allow vectors to directly insert the desired genes into the targeted cells and precise locations within patients chromosome. Before gene therapy can achieve any long term benefits, therapeutic DNA inserted into target cells must remain functional in a cell that is long-lived and stable (18). The rapidly dividing nature of cells and problems with accurate integration of therapeutic DNA means that patients will need to undergo multiple rounds of gene therapy (18). Researchers must also develop a mechanism that would block or limit the degree of immune hyper-responsiveness observed in patients following gene therapy (18). Emphasis should also be placed on ensuring that transplanted genes are precisely controlled by the bodys normal physiological signals (18). Another concern that must be addressed with the use of gene therapy is the possibility of inserting genes unintentionally into the patients reproductive cells (18). Some Recent Developments in Cancer Gene Therapy Gene therapy trials with genetically modified organisms, presents unique safety and infection control issues that must be addressed, in order to protect the human subject, the general public and the environment. Gene Therapy Discussion Group of The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH)) recognize the rapidly evolving area of gene therapy medicinal products (19). However

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due to the lack the resources in ICH regions, guidance has not been provided (19). Currently, China is the only country to license a cancer gene therapy product, which is called Gendicine (2004) (20). Gendicine is an infectious, replication-incompetent, engineered recombinant human adenoviral particles (rAd/p53), composed of the adenoviral vector and the human wildtype p53 tumor suppressor gene. Gendicine uses an adenoviral vector to carry p53 tumor suppressor genes into tumor cells. In combination with chemo- and radiotherapy, Gendicine has improved treatment efficacy more than 3-fold (20). Some other recent developments in cancer gene therapy include (21): Nanotechnology and gene therapy yielded treatment to torpedo cancer. March, 2009. A combination of two tumor suppressing genes delivered in lipid-based nanoparticles drastically reduces the number and size of human lung cancer tumors in mice during trials conducted by researchers from The University of Texas M. D. Anderson Cancer Center and the University of Texas Southwestern Medical Center. Dual Gene Therapy Suppresses Lung Cancer in Preclinical Test at www.newswise.com (January 11, 2007). Researchers at the National Cancer Institute (NCI), part of the National Institutes of Health, successfully reengineer immune cells, called lymphocytes, to target and attack cancer cells in patients with advanced metastatic melanoma. This is the first time that gene therapy is used to successfully treat cancer in humans. New Method of Gene Therapy Alters Immune Cells for Treatment of Advanced Melanoma at www.cancer.gov (August 30, 2006). Significant progress has been made in the exploitation of various novel apoptotic, cytotoxic genes as therapeutic tools for cancer. Together, these recent advances in cancer gene therapy provide rationales for future clinical testing in cancer patients.

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References 1.. Hui-Wen Lo, Chi-Ping Day, Mien-Chie Hung. Cancer-specific gene therapy. Adv Genet. 2005; 54: 235255. doi: 10.1016/S0065-2660(05)54010-0 2. Canadain Cancer Soceity. [Accessed on Nov. 2012, Last updated: 27 July 2012] Available on: http://www.cancer.ca/Canadawide/About%20cancer/Cancer%20statistics/Stats%20at%20a%20g lance/General%20cancer%20stats.aspx?sc_lang=en 3. Deanna Cross, James K. Burmester. Clin Med Res. Gene Therapy for Cancer Treatment: Past, Present and Future. 2006 September; 4(3): 218227. PMCID: PMC1570487 4. Scitable by Nature Education. Chial, H. (2008) Proto-oncogenes to oncogenes to cancer. Nature Education 1(1). Available on: http://www.nature.com/scitable/topicpage/protooncogenes-to-oncogenes-to-cancer-883 5. Seth, Prem. Vector-Mediated Cancer Gene Therapy. Cancer Biology & Therapy 4:5, 512-517; May 2005. Landes Bioscience. 6. Scholl, S. M., S. Michaelis, and R. McDermott. "Gene Therapy Applications to Cancer Treatment." Journal of Biomedicine and Biotechnology 2003.1 (2003): 35-47. Biological Sciences. Web. 2 Dec. 2012. 7.Gene Therapy Net. com. [Accessed Nov, 2012, Last updated: 2012] Available on: http://www.genetherapynet.com/viral-vectors.html 8. Peel, David. Virus Vectors & Gene Therapy: Problems, Promises & Prospects. Department of Microbiology & Immunology, University of Leicester,1998. 9. Elias, Sherman, and George J. Annas. "Somatic and germline gene therapy." Gene Mapping: Using Law and Ethics and Guides. G. Annas and S. EUas, eds.(Oxford U. Press, New York) pp (1992): 142-157. 10. Learn. Genetics. Genetic Science Learning Center. The University of Utah. [Accessed: Nov. Hung C-F, Chiang AJ, Tsai H-H, Pomper MG, Kang TH, et al. (2012) Ovarian Cancer Gene

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Therapy Using HPV-16 Pseudovirion Carrying the HSV-tk Gene. PLoS ONE 7(7): e40983. doi:10.1371/journal.pone.0040983 11. 2012, Last updated: 2012] Available on: http://learn.genetics.utah.edu/content/tech/genetherapy/gtapproaches/ 12. Stolberg S. The biotech death of Jesse Gelsinger. Published: 1999. [Accessed: Nov 2012] Available on: http://www.gene.ch/gentech/1999/Dec/msg00005.html. 13. Xu F., Li S., Li XL., Guo Y., Zou BY., Liao H., Zhao HY., Zhang Y., Guan ZZ., Zhang L. Phase I and biodistribution study of recombinant adenovirus vector-mediated herpes simplex virus thymidine kinase gene and ganciclovir administration in patients with head and neck cancer and other malignant tumors. Cancer gene therapy. 2009. 16: 723-730. 14. Tolcher AW., Hao D., de Bono J., Miller A., Patnaik A., Hammond LA., Smetzer L., Van Wart Hood J., Merritt J., Rowinsky EK., Takimoto C., Von Hoff D., Eckhardt SG. Phase I, Pharmacokinetic, and Pharmacodynamic Study of Intravenously Administered Ad5CMVp53, an Adenoviral Vector Containing the Wild-Type p53 Gene, in Patients With Advanced Cancer. American society of clinical oncology. 2006. 24(13):2052-2058. 15. Patel P., Current gene therapies. PharmaProjects. Published:2009. [Accessed: Nov 2012]. Available on http://www.pharmaprojects.com/therapy_analysis/genether_future_0409.htm. 16. ClinicalTrials.gov. A service of the US national institute of health. [Accessed: Nov 2012, Last updated, Aug 2012]. Available on http://clinicaltrials.gov/ct2/results?term=%22gene+therapy%22&Search=Search. 17. Adrienne J. Larocque . A Brief History of Gene Therapy. Canadian Health. Published by the Canadian Medical Assocation. Spring 2010. Available on: http://www.canadian-health.ca/.

18) Pfeifer, Alexander, and Inder M. Verma. "Gene therapy: promises and problems." Annual review of genomics and human genetics 2.1 (2001): 177-211.

19) International Conference on Harmonisation of Technical Requirements for Registrations of Pharmaceuticals for Human Use (ICH) 2012. Available on: http://www.ich.org/
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20) Cancer Therapy China. [Accessed on Nov. 2012, Last updated 2011] Available on: http://www.cancertherapychina.com/index.php?option=com_content&view=article&id=84&Ite mid=23.

21) Edelson ML, Adebi MR, Wixon J: Gene therapy clinical trials worldwide to 2007 - an update. J Gene Med 2007, 9:833-842. DOI: 10.1002/jgm.1100

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