Contents
12.1 Introduction . . . 227
12.2 Principles of Treatment . . . 228
12.3 Method of Delivery . . . 229
12.4 Potential Side Effects . . . 230
12.5 Special Considerations . . . 230
12.6 Future Perspectives . . . 231
References . . . 231
12.1 Introduction
Gene therapy, also known as gene transfer, is a novel approach of altering the genetic basis of normal or cancerous cells in order to modify their function. The manipulation of genes may help fight cancer and prevent genetic diseases as well as in- crease the body’s ability to receive intense treatment that would be impossible otherwise. The first gene therapy clinical trial was conducted in 1990 on two children with adenosine deaminase (ADA) deficien- cy and today is being evaluated with the hopes of curing diseases such as cancer, cystic fibrosis, and hemophilia.
One of the major goals of gene therapy is to supply cells with healthy copies of missing or altered genes.
Gene therapy remains in its infancy, with limited suc- cess up to this point, and is viewed by many in the same way that chemotherapy was viewed in the 1950s. To date, more than 200 cancer patients have re- ceived genetically modified cells (Brenner, 2002).
Many of these trials involved removing the patients’
tumors, infecting them with the new genes, and then irradiating the cells to kill the gene therapy vector so that it could no longer spread or cause illness. Other experiments have involved genetically modifying the hematopoietic stem cells so that the blood-forming cells could withstand a much greater intensity of chemotherapy.
Finally, many patients have had their tumors di- rectly injected with gene therapy vectors containing suicide genes that, when picked up by the dividing tu- mor cells, would allow them to be selectively killed.
Throughout these experiments, minimal procedure- related toxicity has been seen in most patients,
Gene Therapy
Kathleen E. Houlahan · Mark W. Kieran
although severe reactions to this approach, including death, have been observed in rare cases.
12.2 Principles of Treatment
To date, four major strategies have been adopted for incorporating gene transfer into cancer therapy:
1. The tumor cell itself is modified, either by repair- ing one or more of the genetic defects associated with the malignant process, by introducing a gene that triggers an antitumor response, or by deliver- ing a pro-drug metabolizing enzyme that renders the tumor sensitive to the corresponding agent (Brenner, 2002). An example of this approach is adding back key regulatory genes that have be- come dysfunctional in the tumor, such as p53, whose normal function is to prevent abnormal (cancer) cells from dividing. Oncogenes are genes that typically stimulate tumor formation when ab- normal, and inserting new genes that turn off or counteract these oncogenes might therefore also stop tumor growth. There are, of course, some in- herent limitations with this approach. Tumor cells that don’t pick up the vector will not be killed or affected. Others will react to the therapy by alter- ing (mutating) genes not treated by the inserted gene that allowed them to escape.
Over the last 50 years, it has been discovered that tumors have an almost unlimited ability to rapidly and effectively develop resistance to what- ever modality we treat them with. A form of gene therapy known as suicide gene therapy uses the delivery of the herpes simplex virus thymidine ki- nase (TK) gene by a replication-defective adenovi- ral vector injected directly into the tumor. Ganci- clovir, a nucleoside analog, is then administered intravenously to the patient. The expressed TK phosphorylates the ganciclovir within the tumor cells. The resulting nucleotide analog is a potent inhibitor of deoxyribonucleic acid (DNA) synthe- sis and causes the death of the dividing tumor cells (Hurwitz et al, 2002). Normal cells throughout the rest of the body that did not pick up the vector will be exposed to ganciclovir, but because this pro-
drug cannot be activated unless TK is expressed, they will not be affected. Furthermore, by virtue of a phenomenon called the “bystander effect,” if the gene is picked up by a few cells and causes their death, some of the adjacent cells will die as a result of the localized destruction.
2. The immune response to the tumor is modified by altering the specificity or effector function of im- mune system cells (Brenner, 2002). Tumors are able to grow in part because the immune system fails to recognize them as abnormal. Two strate- gies that have been implemented are to insert genes into the tumor cells, acting like large neon signs that help the immune system “see” the tu- mor, and to introduce genes into the immune cells so that they are better able to detect the tumor. For example, expression of major histocompatibility antigens (MHC) on a cell is an excellent way to help the immune system reexamine not just the MHC protein but also other proteins on the cell surface. Once aware of these new “tumor anti- gens,” the immune system can then aggressively go after all of the tumor cells, including those that failed to pick up the vector. On the other side, transferring the immune cells themselves with genes that wake up the immune system (cytokines such as IL-2 and GM-CSF, for example) are other ways of getting a previously subdued immune re- sponse up and running.
3. The drug sensitivity of normal host tissue is de-
creased by delivering cytotoxic drug-resistant
genes to marrow precursor cells, thereby increas-
ing the therapeutic index of chemotherapy (Bren-
ner, 2002). Certain alkylating drugs such as CCNU
and BCNU damage the DNA of both tumor and
normal cells by adding methyl groups onto the
DNA, preventing the cells from properly duplicat-
ing it at the time of cell division. An enzyme called
MGMT reverses this damage. As a result, one ap-
proach has been to infect normal blood-forming
stem cells with the MGMT gene. When a patient
receives a standard dose of chemotherapy, the ob-
served myelosuppression may be significantly re-
duced if the blood-forming genes express the
MGMT protein, making the therapy better tolerat-
ed and allowing for more frequent or higher doses
of therapy to be administered against the tumor. A potential concern with this approach relates to the consequences if a tumor cell inadvertently picks up the “resistant” gene vector because it, and all of its progeny, would be highly resistant to this kind of therapy.
4. Normal or malignant cells are marked (tagged) so that the efficacy of conventional therapies can be monitored more closely (Brenner, 2002). Humans have between 50,000 and 100,000 genes (National Cancer Institute, 2004). A gene, which is a stretch of sequence of the DNA molecule, is the basic unit of heredity. Genes determine traits such as height, hair, and eye color, and carry the instructions that allow the cells to produce specific proteins and en- zymes. Each protein has a certain function in the body. The pattern of active and inactive genes in a cell and the resulting protein composition deter- mine the type of cell and its capabilities. Genes that protect against cancer are referred to as tumor suppressor genes. Mutated genes that are capable of causing cancer when expressed at the wrong place or time are known as oncogenes. Although the last 50 years of cancer-directed therapy (i.e., radiation and chemotherapy) have targeted cells that divide, researchers are now realizing that can- cer therapy may be more effective if therapy is di- rected at the abnormal genes and their signaling pathways.
Gene therapy, or gene transfer, is defined as the trans- fer of genetic material, including complementary DNA, full-length genes, RNA, or oligonucleotides, into somatic or germline cells (Albelda et al., 2002).
Gene therapy changes the cell function dependent on the selection of the gene. A simple explanation of gene therapy is to take a virus that has evolved to ef- fectively penetrate cells and remove the parts of the virus that code for propagating the infection and the genes that cause illness. The virus acts as a Trojan horse, easily able to penetrate dividing cells. Once the bad part of the gene is removed, the gene of interest is inserted, the cell is infected, and the cell begins to translate it into a protein. While viruses are a com- mon “vehicle,” a large number of different structures are now being evaluated, although the principle for
all of them is the same. As previously mentioned, more than 200 patients have received genetically modified cells. Although most of these patients re- ceived irradiated gene-modified tumor cells that would be expected to have little safety risk, more than 60 children have received genetically modified hematopoietic progenitor cells with no adverse events attributable to the gene transfer process. There are more than 100 clinical protocols approved thus far, most open only to patients with advanced malignan- cy in whom highly experimental therapies such as this can be justified (Brenner, 2002).
12.3 Method of Delivery
Gene therapy has two methods of delivery that are es- sential in the process of successful gene transfer. The first essential mode of delivery is transferring the gene in the cell. The second essential mode of deliv- ery is transferring the cell into the patient. In most gene therapy clinical trials, cells from the patient’s blood or bone marrow are removed and grown in the laboratory. The cells are exposed to the virus that is carrying the desired gene. The virus enters the cells, and the desired gene becomes part of the cells’ DNA.
The cells grown in the laboratory are extensively washed to remove any unincorporated virus and are then returned to the patient by injection into a vein.
This type of gene therapy is called ex vivo (outside the body) gene transfer because the gene is trans- ferred into the patient’s cells while the cells are out- side of the body.
In the last few years researchers have developed several methods of delivering the gene into the cell, including transfer via viruses, plasmids, tumor cells, and immune system cells. The most successful mode of delivery is the use of virus as a vector to deliver the gene into the cell. This likely results from the millions of years that viruses have used to evolve specifically to infect human cells and insert their DNA into ours so that they can make new virus and continue to sur- vive. However, other viral and nonviral vectors that can achieve similar functions are also being tested.
Retroviral, adenoviral, and vaccinia are examples of
viral vectors. An example of a nonviral vector is plas-
mid DNA (naked chunks of DNA). Examples of the viral vectors are the adenovirus (AV), the adeno-as- sociated virus (AAV), the herpes virus (HV), and the AIDS virus. The use of these natural vectors, all of which demonstrate significant ability to infect hu- man cells, requires specialized knowledge in terms of how to inactivate them from causing human disease before reinjecting them into patients.
The most widely used vector system in cancer gene therapy has been the replication-incompetent recombinant adenovirus. Adenoviruses are respira- tory tropic viruses with a double-strand DNA genome and a naked protein capsid coat. Recombi- nant vectors have been created by genomic deletion of viral gene functions involved in replication and provision of these functions in trans by a packaging cell line (Zhang, 1999). The deleted gene regions then can be replaced with expression cassettes containing the desired gene under the control of general or tu- mor-specific promoters. This vector system offers a number of advantages, including high-efficiency transduction in a wide range of target cells (including nondividing cells) and high expression levels of the delivered transgene (Yeh and Perricaudet, 1997). Oth- er vectors are plasmids, which are small particles of raw DNA. The advantage of using plasmids is that there is less chance of the patient becoming infected by a virus particle that regenerates the ability to be- come infectious. The disadvantage of using plasmids is that they are often limited to skin cancer, where they can be delivered directly and easily to the pa- tient’s cancer cells. The most common viruses used as vectors in pediatric oncology gene therapy are AAV and HV.
12.4 Potential Side Effects
It would be unrealistic not to expect genetic therapies to produce side effects. Gene therapy continues to re- quire informed use in controlled clinical studies, with a clear consideration of the risks and potential bene- fits. Current vector systems may need to be modified, and additional efforts are required to better under- stand the biology of the diseases that are candidates for therapeutic genetic intervention. Together this in-
formation will enable risk classifications for specific vectors and transgenes, as well as assessment of the risk factors that are unique to each clinical trial. With this approach, the therapeutic potential of somatic gene transfer may be realized through the application of appropriate prevention strategies (Williams and Baum, 2003).
Some of the potential side effects of gene therapy include viruses infecting more than one type of cell, being inserted in the wrong location in the DNA (for example, in the middle of a critical gene), and possi- bly causing cancer or other cellular damage. Other concerns include the possibility that the transferred genes could be “overexpressed,” producing so much of the missing protein as to be harmful, resulting in inflammation or immune reaction (National Cancer Institute, 2004).
12.5 Special Considerations
Given the considerable risk and unproven nature of this approach, most institutions have a number of local and national regulatory bodies that oversee the design and conduct of gene therapy trials. In the USA, gene therapy protocols must also be approved by the U.S. Food and Drug Administration (FDA), which regulates all gene therapy products. In addi- tion, trials that are funded by the National Institute of Health (NIH) must be registered with the NIH Recombinant DNA Advisory Committee (RAC) (Na- tional Cancer Institute, 2004).
There have recently been two documented cases of
children diagnosed with severe combined immunod-
eficiency disease (SCIDS) who developed leukemia
after participating in a gene therapy trial. In both cas-
es the retrovirus was the vector, and the gene wrong-
fully inserted itself into a piece of DNA that can cause
leukemia. As a result of those cases, gene therapy and
clinical trials face intense scrutiny, with the recom-
mendation from the NIH that gene therapy should be
used only as a last resort.
12.6 Future Perspectives
Gene therapy is a new approach to treating children with cancer, and despite some recent setbacks, it holds significant promise for the future. Scientists have made great progress in the last decade in under- standing the molecular architecture of viruses used for gene therapy, and based on these advances, more sophisticated vectors have been designed. The progress in hand is hopeful; however, there remains a need for continued advancement in this field.
In addition to identifying disease-specific risk fac- tors, there are three ways to limit the possible delete- rious side effects of genetic interventions. The first is to develop vectors with improved safety profiles, in- cluding a reduced propensity for insertional “geno- toxicity.” The second is to define “safe integration sites” in the genome and to design integration vectors that are targeted to these sites. The third is to reduce the number of vector-exposed cells (and thus vector integrations) that are infused into the patient; for ex- ample, by correcting a very small number of stem cells ex vivo and genetically characterizing them be- fore they are infused back into the patient (Williams and Baum, 2003)
One exciting possibility for the future is to harness homologous recombination that will either target vi- ral vectors or directly correct genetic defects. Homol- ogous recombination enables the introduction of for- eign genetic material at specific locations on the chromosome. For instance, it would enable the re- placement of a defective gene by inserting a normal copy of the gene into the locus (Pellman, 2004). How- ever, it is important to remember that most advances
in medicine proceed incrementally, and gene transfer is already being used successfully to complement conventional therapies for malignant hematologic disorders. The benefits of this new technology can only increase as current limitations are progressively, albeit slowly, surmounted (Brenner, 2002).
Gene therapy is still in the early phases despite the fact that it has been an area of research and clinical trials since 1990. It is hoped that although barriers and challenges remain, gene therapy will someday be FDA-approved and offer successful cancer treatment as well as reduce the impact that the disease has on pediatric patients.
References
Albelda S, Wiewrodt R, Sternman D (2002) Gene therapy for lung neoplasms. Lung Cancer 23:265
Brenner M (2002) The applications of gene transfer to pedi- atric malignant disease. In Pizzo P, Poplack Principles and Practice of Pediatric Oncology, 4th edn (pp 453–461) Philadelphia: Lippincott Williams & Wilkins
Hurwitz R, Shields C, Shields J, Chevez-Barrios P, Hurwitz M, Chintagumpala M (2002) Retinoblastoma. In Pizzo P, Poplack Principles and Practice of Pediatric Oncology, 4th edn (p 841) Philadelphia: Lippincott Williams & Wilkins National Cancer Institute. Questions and answers about gene
therapy. http://cis.nci.nih.gov (accessed January 1, 2004) Pellman D. Dana-Farber Cancer Institute, Boston. Personal
communication. January 8, 2004
Williams D, Baum C (2003) Gene therapy: new challenges ahead. Science 302:400–401
Yeh P, Perricaudet M (1997) Advances in adenoviral vectors:
from genetic engineering to their biology. The FASEB Jour- nal 11:615–623
Zhang WW (1999) Development and application of adenoviral vectors for gene therapy of cancer. Cancer Gene Therapy 6:113–138
