Sunday, July 12, 2015

What is gene therapy? How might it help cancer patients?




How this therapy works: Success with gene therapy depends on multitudes of factors: how an altered gene is introduced into the body, how efficiently the modified (recombined) gene is able to restore or resuscitate normal function, how specific the targeting of a particular region is, how sustained the response of the body is, how low the immunological response of the host is, and the extent to which the other associated risks are eliminated. Gene therapy is ideal only for diseases that are caused by mutations of a single gene.



In general, it is impossible to introduce a gene directly into cells, and therefore, special carriers called vectors are used to aid in the transfer of genes. Most commonly used vectors are viruses. Viruses have the remarkable capacity to recognize specific cells and integrate themselves into those cells. Specific classes of viruses called retroviruses have been used extensively in many clinical trials involving gene therapy, although studies using other types of viruses, such as adenoviruses, adeno-associated viruses, lentiviruses, poxyviruses, and herpesviruses, also abound. The reasons for using viruses as vectors are multifold. First, viruses possess a relatively simple genome (genetic makeup). Only a handful of genes exist in a viral genome (as opposed to the approximately 30,000 genes, for example, in the human genome), and therefore, it is relatively easy to handle. Second, viruses have evolved ways of surpassing natural barriers that cells put forth, gaining access across membranes into the cytoplasm as well as to the nuclei of the cells. Third, viruses can be engineered with relative ease to stop replication, gain integration into host deoxyribonucleic acid (DNA), and exit without causing destruction of the host cell. Fourth, viruses have remarkable specificity to infect certain types of cells. This particular characteristic affords greater reliability and hope for better outcomes. However, there are various complications associated with the use of viral vectors, such as host immune response, and therefore, use of nonviral vectors is preferred in certain cases. Numerous types of genes are delivered using viral vectors.



Enhancing host immune responses: Different approaches have been attempted to use gene therapy as a treatment option for patients. Of these, major focus has been on improving the body’s immunological responses to cancer. The body’s immunological system consists of populations of different kinds of cells (broadly called white blood cells or lymphocytes) whose function is to fight against multitudes of foreign bodies that invade the cells. In cancer patients, however, this immunological response is highly compromised either because of deficiency or improper functioning of lymphocytes. Immune therapy for cancer generally falls under a few specific categories: injection of cytokines (special proteins secreted by the immune system), addition of lymphokine-associated killer cells (LAK cells, which are special immune cells) to existing treatment options, addition of activated peripheral lymphocytes, and infusion of antigen-presenting cells to increase the antigen-recognizing capacity of immune cells. The gene therapy approach employs the idea of enhancing the capacity of normal lymphocytes to become aggressive combatants of cancer by using any of these techniques. Specific retroviruses are manipulated to incorporate certain proteins called T-cell receptors (a kind of lymphocyte), which when introduced into the body engender the required confirmation for lymphocytes to destroy cancer cells. Some ongoing clinical trials use this approach to treat melanoma.


T lymphocytes exert their immunogenicity through secretion of specific proteins called cytokines. Human cells possess different kinds of cytokines, most of which have been used in gene therapy studies. The major advantage of using cytokines in gene transfer studies is that they help not only in tumor destruction but also in eliciting immune memory. Most known cytokines such as interleukins (ILs), interferons (IFNs), granulocyte-macrophage colony-stimulating factors (GM-CSF), and tumor necrosis factor-alpha (TNF-α) are used in immunotherapy and gene therapy experiments. In fact, cytokines constitute about 25 percent of the genes used in experiments in gene therapy around the world. Preclinical studies using IL-2, IL-12, IL-24, GM-CSF, TNF-α, and IFN are extremely promising, and all these compounds are used in clinical trials. Cytokines can be delivered using both viral and nonviral modes of entry into the cells. They can be administered directly into tumor cells in vivo (inside cells) or can be engineered with lymphocytes ex vivo (outside cells) and then implanted in stem cells. There are some sporadic reports of vectors themselves contributing to antitumor effects in studies involving cytokine gene transfers. However, gene therapy experiments using cytokines face similar challenges as those of other comparable experiments, where determination of optimal doses of cytokines and elimination of immunosuppressive agents still hamstring progress.



Introduction of normal tumor-suppressor cells: Other approaches to gene therapy include replacement of mutated (altered) or deleted genes with healthy copies of genes. For example, a tumor-suppressor gene called TP53 is mutated in a wide array of tumors. The TP53 protein, under normal conditions, suppresses the activation of genes contributing to uncontrolled cell division and proliferation. Replacement of normal, wild type TP53 genes using a retroviral TP53 expression vector is used in gene therapy trials.



Inhibition of oncogenes: An alternative strategy is to alter the oncogene or cancer-inducing gene directly. Genes belonging to the RAS family of oncogenes, such as HRAS, NRAS, and KRAS, and others such as
MYC
are examples of oncogenes. These genes are activated by mutations or modifications that change the composition of their protein products, resulting in the development of cancer. A common approach to inactivate such oncogenes uses a method called antisense technology. A ribonucleic acid (RNA) sequence complementary to that targeted for inactivation is introduced into the cell, then binds and thereby blocks translation of that RNA. Thus, inhibition is ensured at a level before the protein is formed, reducing its opportunities to induce tumor development.



Insertion of suicide genes: A novel approach of gene therapy is to transplant what are called suicide genes in the body; these help convert generally nontoxic substances called prodrugs into physiologically active forms, thereby triggering death of cancer cells. The popular gene and prodrug combination is the herpes simplex virus (HSV) thymidine kinase (HSV-tk)/ganciclovir (GCV). Ganciclovir is a prodrug that is inactive in its dephosphorylated (no phosphate group attached to the protein) form. When it is phosphorylated by HSV-tk introduced through an adenovirus, it attains the capability of inducing so called “death pathways” and triggering cell death. The effect is accentuated by a process called “by-stander effect,” whereby surrounding cells receive the toxic metabolites and join the race of combating tumor cells. This strategy has yielded very promising results in clinical trials.



Aiding antiangiogenesis: Tumors require a copious supply of oxygen for sustenance and access to blood vessels for spread. The process of formation of new blood vessels is called angiogenesis. It is a multistep process that includes proliferation of endothelial cells, cellular migration, membrane degradation, and reorganization of cell cavity or lumen. Some factors that aid in angiogenesis are growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and hepatocyte growth factor (HGF). Gene therapy experiments focus on introducing viruses containing inhibitors of some of these growth factors and attempt to block angiogenesis in tumor cells.



Interaction with other treatment techniques: A commonly employed gene therapy strategy is to insert genes that will facilitate or augment some existing therapeutic intervention. In the case of patients undergoing radiotherapy or chemotherapy, for example, introduction of certain beneficial genes could afford resistance to multiple drugs or protection of bone marrow. Tumor cells have the capacity to efflux drugs, and this capacity is a challenge to overcome in therapeutic procedures. The strategy is to use drug-resistance genes for overcoming drug efflux. A gene that has been approved in protocols for breast or ovarian cancer treatments is multiple-drug-resistance gene (MDR1). This gene is inserted into normal bone marrow to select for cells that are particularly resistant to the specific chemotherapeutic agent in a treatment regimen.



Other approaches: The use of small inhibitor ribonucleic acid (siRNA) technique for silencing oncogenes is gaining momentum in gene therapy studies. Experiments have begun using viral vectors incorporating siRNA in animal models to examine their efficacy in targeting and silencing specific genes. Attempts have also been made to introduce a forty-seventh, artificial chromosome with relevant beneficial genes through a large viral vector.



Using nonviral vectors: The use of viral vectors for delivering specific genes poses some palpable problems such as interaction of the introduced virus with other viruses that it might encounter in the body and nonspecific target stimulation. Therefore, use of nonviral material to transfer genes is preferred in certain cases. Some examples of nonviral vectors are cationic liposomes, polyethylenimines, DNA-liposome complexes, and synthetic polymers. Liposomes are lipid particles possessing the innate ability to traverse cell membranes. The strategy behind gene therapy is to harness this property to deliver desired DNA to cancer cells via lipid-DNA complexes. Naked DNA or plasmids and transposable elements called transposans are other examples of nonviral vectors. Electrotransfer of DNA has been used in experiments as a physical method of transfer of DNA. In this process, the transfer of genetic material is achieved by local application of electric pulses after introducing DNA into the extracellular medium.


Nanomaterials are temperature-sensitive polymers capable of binding and complexing with DNA to form nanorods. The stability of nanorods at physiological temperatures and their capacity to expand when heated make them attractive alternatives to deliver and trap genetic materials. Once inside tumor cells, they disintegrate and make the delivery complete and competent. Recent experiments have used both naturally occurring polymers like chitosans and synthetic, biocompatible compounds called propyleneimines for DNA-binding experiments.



Side effects: Major side effects for gene therapy include host immune responses to foreign genes introduced into the body. Immune responses can include inflammation, allergic reactions, and, rarely, death. It is worth mentioning that gene therapy studies have reported minimal side effects and encouraging responses. The challenge lies in improving efficiency and producing sustainable responses.



Progress and perspectives: Due to its ability to alter genes, which are the basic units of heredity and variation, gene therapy encounters multitudes of social and ethical concerns. The ability to alter genes confers the ability to change the genetic makeup and ultimately the genetic composition of the human population. It might become difficult then to determine what is legitimate and ethical to manipulate. Some of the issues concerning germ-line gene therapy and genetic enhancement are serious concerns and need to be tackled. In the United States, there are stringent procedures and regulations for conducting gene therapy studies and clinical trials. Various government organizations, including the Food and Drug Administration (FDA) and the Recombinant DNA Advisory Committee (RAC), need to provide approval for protocols. However, as with any other approach, the benefits of gene therapy in curing life-threatening diseases have to be weighed appropriately against any possible misuses or abuses of this approach.



George, L. Gene Therapy. Woodbridge: Blackbirch, 2003. Print.


Gerl, R., and D. L. Vaux. “Apoptosis in the Development and Treatment of Cancer.” Carcinogenesis 26 (2005): 263–70. Print.


Gillham, Nicholas W. Genes, Chromosomes, and Disease: From Simple Traits, to Complex Traits, to Personalized Medicine. Upper Saddle River: FT Science, 2011. Print.


Lattime, Edmund C., and Stanton L. Gerson. Gene Therapy of Cancer: Translational Approaches from Preclinical Studies to Clinical Implementation. 3rd ed. San Diego: Academic, 2014. Print.


Panno, J. Gene Therapy: Treating Disease by Repairing Genes. New York: Facts On File, 2005. Print.


Percorino, Lauren. Why Millions Survive Cancer: The Successes of Science. Oxford: Oxford UP, 2011. Print.


Podajcer, O. L., M. V. Lopez, and G. Mazzolini. “Cytokine Gene Transfer for Cancer Therapy.” Cytokine and Growth Factor Reviews 18 (2007): 183–94. Print.


Roth, J. A., and R. J. Cristiano. “Gene Therapy for Cancer: What Have We Done and Where Are We Going?” Journal of the National Cancer Institute 89 (1997): 21–39. Print.

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