Cancer occurrs by the production of multiple mutations in a single cell that causes it to proliferate out of control. Cancer cells often different from their normal neighbors by a host of specific phenotypic changes, such as rapid division rate, invasion of new cellular territories, high metabolic rate, and altered shape. Some of those mutations may be transmitted from the parents through the germ line. Others arise de novo in the somatic cell lineage of a particular cell. Cancer-promoting mutations can be identified in a variety of ways. They can be cloned and studied to learn how they can be controlled.
Several methods such as surgery, radiation, and chemotherapy have been used to treat cancers. The cancer patients who are not helped by these therapies may be treated by gene therapy. Gene therapy is the insertion of a functional gene into the cells of a patient to correct an inborn error of metabolism, to alter or repair an acquired genetic abnormality, and to provide a new function to a cell.
Two basic types of gene therapy have been applied to humans, germinal and somatic (1). Germinal gene therapy, which introduces transgenic cells into the germ line as well as into the somatic cell population, not only achieve a cure for the individual treated, but some gametes could also carry the corrected genotype. Somatic gene therapy focuses only on the body, or soma, attempting to effect a reversal of the disease phenotype by treating some somatic tissues in the affected individual.
One of the most promising approaches to emerge from the improved understanding of cancer at the molecular level is the possibility of using gene therapy to selectively target and destroy tumor cells, for example, the loss of tumor suppressor genes (e.g. the P53 gene) and the over expression of oncogenes (e.g. K-RAS) that have been identified in a number of malignancies. It may be possible to correct an abnormality in a tumor suppressor gene such as P53 by inserting a copy of the wild-type gene; in fact, insertion of the wild-type P53 gene into P53-deficient tumor cells has been shown to result in the death of tumor cells (3). This has significant implications, since P53 alterations are the most common genetic abnormalities in human cancers. The over expression of an oncogene such as K-RAS can be blocked at the genetic level by integration of an antisense gene whose transcript binds specifically to the oncogene RNA, disabling its capacity to produce protein. Experiments in vitro and in vivo have demonstrated that when an antisense K-RAS vector is integrated into lung cancer cells that over express K-RAS their tumorigenicity is decreased (4).
Despite the promise of such approaches, a number of difficulties remain to be overcome, the most important of which is the need for more efficient systems of gene delivery. No gene transfer system is 100% efficient, unless germ-line therapy is contemplated. During the past two decades, there have been major advances in our understanding of how cancer develops, proving that cancer has a genetic basis (2). A series of genetic abnormalities that accumulate in one cell may result in a pattern of abnormal clonal proliferation. Our growing understanding of the genetic basis of cancer offers new opportunities for the molecular prevention and treatment of cancer. There has been a substantial growth in gene therapy, especially in the field of oncology since the first experiment in human gene therapy began in 1990 (5), with the aim of treating adenosine deaminase deficiency. By the end of 1993, there were 45 approved trials by US Recombinant DNA Advisory Committee, 30 of which are for the treatment of cancer. This is, in part, became tumor cells can be manipulated ex vivo, while the affected tissues from individuals with other genetic diseases often cannot.
More than 100 clinical applications of gene transfer into human patients for both therapeutic and cell-marking purposes have now been approved in the USA and a number of other countries. Trials for cancer gene therapy that have been approved in the USA have involved malignancies that are considered incurable. This clinical situation, which is unlike many genetic diseases for which life expectancy is measured in years rather than weeks or months, has been considered more appropriate ethically for untested technologies. For these reasons, applications of gene therapy to cancer will continue to be the fastest growing area of human gene therapy.
Gene therapy for the treatment of cancer has a wide variety of potential uses. There are several potential strategies for gene therapy in the treatment of cancer.
Strategies of gene therapy for cancer
Approaches to ex vivo gene transfer
(1) Genetically engineered tumor cells
Various groups are investigating the production of autologous cellular vaccines for the treatment and prevention of cancer. This is most commonly attempted by surgically removing tumor cells from the patient, growing them in tissue culture and inserting immunostimulatory genes in vitro. These cells are then reinjected into the patient in an effort to induce a significant systemic immune response that will both destroy tumor cells and protect the patient against a recurrence of the tumor. Treating cells that produce cytokines has been shown to result in systemic immunity in mice. Alteration of syngeneic tumors with the genes that encode IL-1 b, IL-2, IL-4, IL-6, TNFa, GM-CSF or r-interferon (6, 7) results in immunological destruction of the tumor cells in vivo.
In human gene therapy trials, patients are injected with either autologous or allogeneic genetically modified tumor cells. These trials involved the insertion of retroviral vectors carrying the gene that encodes either IL-2, TNFa or GM-CSF into melanoma, colorectal renal cell carcinoma, neuroblastoma or breast cancer cells in vitro. One modification of this technique is the insertion of the gene for either IL-2 or IL-4 into autologous fibroblasts, which are then mixed with irradiated tumor cells from the patient and reinjected. This approach has the advantage that growing fibroblasts in vitro is much easier than culturing tumor cells from a large number of individuals. Besides modifying tumor cells to produce immune activating cytokines, another strategy is to block the production of insulin-like growth factor-1 (IGF-1). Many tumors such as breast cancer produce high levels of IGF-1. Insertion of an antisense gene that stops production of IGF-1 in the tumor allows immunological rejection of the genetically altered tumor after reimplantation (8). Destruction of the tumor is mediated by cytotoxic T lymphocytes. The precise mechanism by which IGF-1 mediates tumor protection in vivo remains unclear.
(2) Genetically engineered T lymphocytes
T lymphocytes have the capacity to hone in on tumor tissue. This property has been used to deliver cytokines directly to tumor masses for human gene therapy. The secretion of cytokines locally at the tumor site by the effector T lymphocytes will enhance their anti-tumor activity and avoid the side-effects that result from the systemic administration of cytokines. For the trial of TNF-modified tumor infiltrating T lymphocytes, T lymphocytes are difficult to transduce with retroviral vectors and tend to downregulate expression of the cytokine gene carried by the vector (9).
These two problems of poor gene transfer efficiency and poor cytokine expression have so far limited the application of this approach, and have shifted the emphasis from modification of T lymphocytes toward the genetic alteration of tumor cells, which are much easier to grow in culture and more readily engineered.
(3) Insertion of a sensitivity gene
Gene therapy uses the genes to activate a relatively nontoxic pro-drug to form a highly toxic agent. The most widely studied system uses the thymidine kinase gene of the Herpes simplex virus (HSVtk). The HSVtk gene confers sensitivity to the anti-herpes drug, ganciclovir (GCV), by phosphorylating GCV to a monophosphate form (GCV-MP). Phosphorylation to the triphosphate form(GCV-TP) by cellular kinases results in inhibition of DNA polymerase, and leads to cell death. In this procedure, GCV kills tumor cells which express KSVtk, and the adjacent cells that lack the gene are also destroyed. This is termed the bystander effect phenomenon. To use the bystander effect to kill human cancer in vivo, the irradiated ovarian tumor cells that contain the HSVtk gene will be injected into the peritoneal cavity of patients, who will be given GCV. These HSVtk-expressing cells will destroy bystander tumor cells in vivo.
(4) Protection of hematopoietic stem cells
Protection of hematopoietic stem cells (HSCs) from the toxic effects of chemotherapy by using the gene that confers multiple drug resistance type 1(MDR-1) is another possible strategy for human cancer therapy. The MDR-1 gene (10) will be isolated from tumor cells, where it functions to pump chemotherapy drugs (including daunorubicin, doxorubicin, vincristine, vinblastine, VP-16, VM-26, taxol and actinomycin-D) from within the cell. Transfer of a retroviral vector carrying the MDR-1 gene into bone marrow stem cells and their subsequent reintroduction will protect stem cells in vivo from the effects of large doses of taxol.
Genetic alteration of cancer cells in situ
(1) Liposome-mediated gene transfer
The genetical modification of tumors in situ involves the direct injection of liposomes containing an allogene that encoded HLA-B7, a foreign antigen that is transiently expressed on the cell surface and includes an immune reaction against the altered tumor cells. Anti-tumor immune response is significantly increased when some of the tumor cells express foreign antigens on their cell surface. The transient expression of immunostimulatory genes in tumors might have potential as a treatment and as a vaccination against certain malignancies.
(2) Retrovirus-mediated gene transfer
In vivo gene transfer using murine retroviral vectors has been applied to the treatment of brain tumors (11). In this process, murine fibroblasts that are actively producing retroviral vectors, so-called retro viral vector producer cells or VPCs, are implanted directly into growing tumors. The gene transferred by the retroviral vectors into the surrounding tumor cells is the HSVtk gene. The HSVtk gene should integrate only into the proliferating tumor cells because retrovirus-mediated gene transfer is limited to mitotically active cells. This technique resulted in transfer of the gene for HSVtk into 30-60% of brain tumor cells and was capable of mediating complete tumor destruction in 80% of patients.
More than 50% of the cancers can be eliminated completely, At least 10% of cells in a tumor contain HSVtk, adjacent tumor cells that do not contain HSVtk are destroyed through the bystander effect. No associated systemic toxicity or evidence of systemic spread of the retroviral vectors is seen with this form of in vivo gene transfer. So far, however, it is not clear whether this gene delivery system will suffice to eradicate the larger, infiltrative human tumors.
Two protocols for in vivo gene transfer for cancer therapy have been approved for clinical trials. Both entail the direct injection of a supernatant containing a retroviral vector (RV) into tumor deposits. One group will inject two different RVs into endobronchial non-small-cell lung cancers (4). The vectors will carry genes that target the genetic mechanisms responsible for the malignancy: for example, if the lung tumors are deficient in expression of the P53 tumor suppressor gene, this gene will be used. In lung cancers that overexpress the K-RAS oncogene, a vector containing an antisense K-RAS gene will be used. Experiments in vitro have demonstrated that the introduction of both such vectors can result in decreased tumorigenicity. Another group will inject a RV containing a vector that encodes r-interferon directly into melanoma deposits.
Civic, religious, scientific, and medical group have all accepted, in principle, the appropriateness of gene therapy for cancer of somatic cells in humans for specific cancer. Somatic cell gene therapy is seen as an extension of present methods of therapy that might be preferable to other technologies. It is considered that patients should not be subjected to unreasonable risk of harm, excessive discomfort, or unwanted of privacy, and that they should receive special care, monitoring, and consideration.
Scientists and physicians acknowledge the fact that somatic-cell gene transfer is the only form that is technically feasible and ethically acceptable for human use, and gene therapy will revolutionize medicine. The development of gene transfer strategies for human cancer is limited as much by our imagination as by current technology. It is thought that a large number of projects about gene therapy for cancer will be needed to provide very important information on the safety of the biologies, efficiency of gene transfer and efficacy in humans.
It has been suggested that the researches in preclinical and clinical studies will be necessary to evaluate the therapeutic benifit of such gene-based therapies for cancer. For example, the main advantage that retroviral vectors have over vaccinia-based vectors is that they are non-replicating, minimizing the concerns regarding the use of a freely replicating virus in immunocompromised patients. Disadvantages of retroviral gene delivery system are the complexity and high cost of the transfer procedure. People are concerned with the safety of retroviral delivery system associated with the use of viral vectors. All the evaluations of these therapeutic benefit of these methods await imminent clinical trials.
The gene therapy tools for cancer currently in use are blunt, and will remain so until solutions are found to the technical problems posed by the need for persist transgene expression, specific targeting of foreign genes to the appropriate tissues, site-specific integration of retroviral vectors and adeno-associated vectors, stable gene transfer into post-mitotic cells, and the need to overcome the immune response to the gene transfer vector and the transience of gene expression from non-integrating vectors.
The Scientific advisory panel to the National Institutes of Health' public upbraiding of gene therapy for cancer was mingled with the message that gene therapy is "not a failure," but a technology, which likes most innovative technologies, progresses slowly. They also suggested that human experiments should be set up so that both positive and negative results give us an answer, but so far, in human gene therapy experiments, there is no certain answer.
Perhaps the biggest hurdle to better results with gene therapy for cancer are the gene transfer vectors, which ferry the gene of interest into target cells. Although most of these vectors are retrovirues, none is ideal, or perhaps even nearly ideal. It was also believed that the low frequency of gene transfer as a major shortcoming in virtually all of the existing trials and other failings were a lack of suitable controls in experiments. Therefore, a suitable control is necessary for experiments.
The public thinks that researchers and media have oversold the current research in the field of gene therapy for cancer, leading to an inaccurate perception of its success. This problem will threaten the field and future public support of the field. One immediate problem is the public's inflated expectations about gene therapy for cancer may lead some patients to relax compliance with treatment plans or alternative reproductive choices in the belief that a gene treatment is close at hand.
Other genetics experts argue that the time has come to re-evaluate the approach taken by most gene therapists, and perhaps even to redirect their effects. They are also a bit concerned that they were not fulfilling the promise of gene therapy for cancer in any obvious way at this point, and they suggested that more basic aspects of gene therapy research for cancer will need to be emphasized. Some of patients with cancer made a reproductive decision based on the feeling that gene therapy for cancer is "right around the corner".
Currently , NIH spends an estimated $200 million of its $11 billion annual budget on gene therapy, while industry pumps an estimated $200 million annualy into the field. As of June 1995, the majority of the 106 approved clinical protocols involved proposed therapies for cancer. There are now 600 Americans enrolled in 100 clinical trials. Yet after all the tests and all the hype, there is still no unambiguous proof that gene therapy for cancer has cured, or even helped, a single patient. Gene therapy, because it is a modification of the human genome, generates a level of concern different from other therapies and therefore is deserving of continued public scrutiny.
No one denies that gene therapy for cancer holds extraordinary promise or that it will eventually yield results. But critics have grown increasingly concerned that the initial excitement led to a premature rush to get unproved gene therapies for cancer out of the laboratory and into human patients. Researchers are still not sure which are the best methods to transport genes into cancer cells. Nor have they figured out how to stop a person's own immune systems from rejecting what are, in effect, microscopic transplants of foreign material.
Even more troubling are signs that financial considerations may have replaced scientific rigor in determining how and when to use gene therapy for cancer. Some critics charge that businessmen are pushing researchers too hard in order to get a quick return on their investment, and that some doctors have been too hasty, launching clinical trials early in the hopes of "cashing in" when a large drug company buys their firm.
A number of therapies such as drug and chemotherapy treatments have been developed over the years in an attempt to treat patients with cancer, most of them can only ameliorate the symptoms rather than effect a cure. The possibility of gene therapy opens a new area of therapeutics and hope for individuals afflicted with these cancer. But several technical hurdles must be overcome before successful and complete cures are possible for the cancers, and technologies must continually be improved upon if the cancers are to be treated. Like all medical therapies, certain gene therapy will ameliorate some, but not all, symptoms of a particular cancer. It seems likely that no single vector system will be appropriate for treating all cancers or will cure all cancers.
Scientists think that clinical trials of gene therapies for cancer has been made possible by two major technological advances: the ability to clone genes that constitute the genetic basis of cancinogenesis or that have therapeutic potential and the development of an increasing number of gene transfer methods. Increasing numbers of cytokine and sensitivity genes will present new opportunities for the selective destruction of cancer cells, but the primary factor hampering the wide spread application of gene therapy for human cancer is the lack of an efficient method of delivering genes in situ. Therefore, developing strategies to deliver genes to a sufficient number of tumor cells to induce complete tumor regression or restore genetic health remains a challenge. However, as these obstacles are overcome, gene therapy will become a standard part of the practice of oncology.
Before gene therapy can become the strategy of choice in a wide variety of clinical settings, improvements in the efficiency of gene transfer into target cells and in the maintenance of expression from the relevant transferred gene must occur. The problem of efficient gene transfer will require not only further research to improve delivery systems and vector constructions but also a parallel effort to understand the biology of the target cells. Germline therapy would change the genetic pool of the entire human species and future generations would have to live with that change. Because of these ethical problems, a number of technical difficulties would make it unlikely that germline therapy would be tried on human in future.
For the assessment of gene therapy for cancer, the central question is: what kinds of morbidity and what mortality rates are associated with the cancer? If the kind of cancer is severe, one can proceed to consider existing therapies and their effectiveness. If conventional methods provide reasonable control for the cancer victims, the cancer may not be a good early candidate for gene therapy. On the other hand, if no effective therapy methods exist or if the therapy methods can not be used with some categories of patients, gene therapy then may be chosen for the patients.
Personally, I recommended that researchers should abandon the "hype" surrounding the gene therapy for cancer and refocus on technical problems limiting its success. I believe that gene therapy for cancer still has "extraordinary potential for the long term" treatment of human cancer. An emphasis for gene therapy for cancer should be put on improving the delivery of genes to target cells and on their "expression" and clinical trials using gene therapy for cancer should continue. But it should be noted that public expectation are too high, leading to the "mistaken and wide-spread perception that gene therapy is further developed and more successful than it actually is. Gene therapy for cancer is still a young field with much in front of it, there is no established efficacy of any gene therapy for cancer today. Even the widely heralded success of gene therapy to treat adenosine deaminase deficiency is somewhat suspect.
Like the development of all innovative medical therapies, gene therapy as an alternative therapeutic mode involving preclinical studies in animal models and tissue cultures. The risk assessment process must seek to determine and evaluate the probable safety and effectiveness of the new technique. From a safety standpoint, one advantage of the gene therapy approach that uses bone marrow cells is that the cells are treated outside the body, in vitro. If a particular experiment goes awry, the cells are simply not returned to the laboratory animal or, in the clinical context, to the patient. But other safety questions are matters of continuing concern. The new genes will probably be carried into the stem cells by carefully designed retrovirues from which much of the native genetic information has been deleted. These defective retroviruses will function as vectors for the new gene. It is possible, but not likely, that the retroviral vectors will recombine with undetected viruses or endogenous DNA sequences in the cells and so become infectious. It is also possible that the vector and gene combinations will activate previously dormant proto-oncogenes or disrupt essential, properly functioning genes. Efficiency is still not clear whether sufficiently high levels of expression can be achieved in primates to offer a reasonable hope of clinical benefit.
In the more distant future, the prospects for, and possible approaches to, human gene therapy for cancer are not yet clear. But if the link between gene therapy and bone marrow transplantation can be broken, gene therapy for cancer may become available to a wider circle of patients with cancer. At that stage, if it is ever reached, medicine will stand at the threshold of a new era in treating a lot of cancers that afflict our species.
Furthermore, it is not just the immune system that scientists must outwit. They also have to get the cells that are targeted for treatment to open their molecular locks and allow the foreign genes inside, so the basic principles necessary to make gene therapy for cancer successful are only beginning to be defined.
I suggest that for gene therapy for cancer we have to test it in the lab on animals, try it in humans and then go back to the lab. It should be a cyclic process. Unless we do clinical trials, we are never going to learn. The best outcome of human gene therapy for cancer would be a single treatment that would correct enough cells to provide a permanent cure for the patient's cancer. This kind of complete success is unlikely in the beginning stages of human gene therapy for cancer but will remain the long-term goal of research scientists working in this field.
Gene therapy for some cancers will be possible in the foreseeable future. My view is that knowledge in several areas should be obtained from studies in animals before gene transfer in human beings is ethically justified. Besides, experiments in animals should demonstrate that the new gene can be put into the target cells and remain in them, that the new gene can be regulated appropriately , and that the presence of the new gene does not harm cells of human body.
1. Biotechnology Industry Organization. Biotechnology in Perspective. "Gene Therapy - An Overview." Washington, D.C. Biotechnology Industry Organization, 1990. Obtained from the WWW 10/14/96: http://www.gene.com/ae/AB/IWT/Gene_Therapy_Overview.html
2. Bishop, J.M. 1991. Molecular Themes in Oncogenesis. Cell 64, P.235-248
3. Harris, C.C. and Hollstein, M. 1993. Clinical implications of the p53 tumor-suppressor gene. New Engl. J. Med. 329, p. 1318-1327.
4. Zhang, Y. Et al. 1993. Retroviral Vector-mediated Transduction of K-ras Antisense RNA into Human Lung Cancer Cells Inhibits Expression of the Malignant Phenotype. Hum. Gene Ther. 4. P. 451-460.
5. Blaese, R. M., Culver, K.W., and Anderson, W.F. 1990. The ADA Human Gene Therapy Clinical Protocol. Hum. Gene ther.1 p. 331-362.
6. Fearon, E.R. et al 1990. Interleukin-2 Production by Tumor Cells Bypasses T Helper Function in the generation of an Antitumor Response. Cell 60. P.397-403.
7. Tepper, R.I., Pattengale, P.K., and Leder, P. 1989. Murine Interleukin-4 Displays Potent Anti-tumor Activity In Vivo. Cell 57. P. 503-512.
8. Trojan, J. Et al. Treatment and Prevention of Rat Glioblastoma by Immunogenic C6 Cells Expressing Antisense Insulin-like Growth Factor I RNA. Science 259. p. 94-97.
9. Hwu, P. Et al. 1993. Functional and Molecular Characterization of Tumor-infiltrating Lymphocytes Transduced with Tumor Necrosis Factor-r cDNA for the Gene Therapy of Cancer in Humans. J. Immunol. 150. p. 4104-4115.
10. Sorrentino, B.P. et al. 1992. Selection of Drug-Resistant Bone Marrow Cells in Vivo After Retroviral Transfer of Human MDR1. Science 257. P. 99-103.
11. Oldfield, E.H., Culver, K.W., Ram, Z., and Blaese, R.M. 1993. Gene Therapy for the Treatment of Brain Tumors using Intra-Tumoral Transduction with the Thymidine Kinase Gene and Intravenous ganciclovir. Hum. Gene Ther. 4. P. 39-69.