The concept of gene therapy to treat human disease originally developed as a potential treatment for inherited monogenic disorders.[1,2] In theory, a disease caused by the absence or mutation of a single gene, such as cystic fibrosis or Gauchers disease, could be treated and potentially cured by inserting a normal copy of the mutant or deleted gene into a renewable population of host cells, such as bone marrow stem cells. While conceptually simple, this strategy of gene replacement therapy is proving to have practical complexities that make its clinical implementation more difficult than had been anticipated. Currently available vectors have been unable to sustain high enough levels of gene expression over long enough periods of time.
A major focus in gene therapy has been the treatment of cancer. Approaches have included transferring cytokine genes to stimulate an antitumor immune response, delivering genes that express prodrugs to tumors, and transferring genes to protect stem cells during high-dose chemotherapy. This review will focus on restoring the function of tumor suppressor genes. In the context of cancer, transient gene expression that triggers cancer cell death may be enough to mediate a therapeutic effect. Expression of the transgene in only a fraction of tumor cells also may not be limiting because these cells may alter the growth of adjacent cells.
The gene families implicated in carcinogenesis include dominant oncogenes like ras and tumor suppressor genes like p53.[4-6] Proto-oncogenes (normal counterparts of oncogenes) ordinarily participate in such functions as signal transduction (relaying information from the outer cell membrane to the nucleus) and gene transcription. An abnormality (point mutation, amplification, translocation, or rearrangement) in only one of the two alleles of a proto-oncogene is sufficient to convert it into an active oncogene, that is, to dysregulate its function and lead to malignant transformation.
In contrast, a single normal tumor suppressor gene in a cell is typically sufficient to perform the normal function of the gene, but loss of function of both alleles, by mutation or deletion or a combination thereof, leads to dysregulation of cellular growth. In addition, some tumor suppressor gene mutationsfor example, some p53 mutationsact in a dominant negative manner, that is, a mutation in one allele can lead to production of a mutant p53 protein that binds to, and thereby inactivates, the structurally normal protein encoded by the opposite allele. Viral proteins also may bind and functionally inactivate p53.
All cancers are thought to contain multiple abnormalities in a variety of genes that control various aspects of cell growth and development. Thus, correcting all the genetic abnormalities in the cancer cell might seem necessary to reverse the malignant process. Correction of all genetic abnormalities would be an impossible task, however, particularly since some of these abnormalities have not yet been identified. Moreover, individual patterns of expression would need to be assessedfor each gene in every patient. Fortunately, correcting a single genetic abnormality is enough in some cases to induce tumor cell death by apoptosis.[8-10]
A number of in vitro studies that used cultured cancer cell lines demonstrated that eliminating the expression of a single dominant oncogene (ras) or adding a normal copy of a tumor suppressor gene (p53 or the retinoblastoma [Rb] gene) to cells that had deleted or mutated copies of these genes reduced or even abolished critical aspects of the malignant phenotype, such as tumorigenicity in animals or anchorage-independent growth.[10-12] Because of this, the problem of simultaneously correcting multiple functional genetic defects in the cancer cell did not appear as daunting.
Restoring normal gene function to every cancer cell, which is beyond the capabilities of the vectors currently available for use in gene therapy, was also thought to be necessary at one time. However, transduced cells expressing a toxic transgene are now recognized to alter the growth of adjacent nontransduced cells. This has been termed the bystander effect.
While multiple genes offer potential targets for gene therapy in several common malignancies, our group has focused on the goal of replacing normal p53 function. Many of the identified tumor suppressor genes and proto-oncogenes encode proteins that are components of a network that converges on the protein encoded by the Rb gene.[13,14] Phosphorylation of the Rb protein increases transcription of other genes and protein synthesis, leading to cell growth. Phosphorylation of Rb is controlled by a multimolecular complex of proteins, containing cyclins and cyclin-dependent kinases as well as a potent inhibitor of most cyclin-dependent kinases, termed p21.[15,16] In turn, one of the many functions of the p53 protein is regulating p21 function.[17,18] Thus, p53 plays a central role in regulating the cell cycle because it indirectly regulates the function of Rb. When p53 function is normal, this pathway is tightly regulated; however, when p53 mutates or is absent, uncontrolled cell growth reflects lost control of the pathway.
Moreover, p53 plays a central role in other metabolic pathways, including, importantly, control of apoptosis. In response to various toxic insults to cells, such as exposure to ionizing radiation or chemotherapy, normal cells either pause in their cell cycle long enough to repair DNA damage or, in other cases, undergo apoptosis; repair and programmed cell death both prevent the damaged DNA from being passed along to the cells progeny. When normal p53 function is absent, however, damaged DNA is much more likely to be passed along.
Nonsmall-cell lung cancer is a logical target for novel therapeutic strategies for several reasons. It is the leading cause of cancer death in the United States, and the median survival of patients who present with stage III or IV disease, as the majority of patients do, is measured in months. For most patients who are not potentially curable by surgery, other standard therapies are relatively ineffective. Radiotherapy, which offers the best chance for locoregional control of disease in patients with surgically unresectable disease, is successful in only 20% of cases, and local primary failure or recurrence may be the only site of failure in up to one third of patients.
Mutation or inactivation of p53 occurs in a high proportion of nearly all common human cancers, including nonsmall-cell lung cancer.[21-23] In view of the key role of p53 in cell-cycle regulation and apoptosis and the role of defective p53 function in carcinogenesis, attempting to replace p53 represents a logical gene replacement strategy. My colleagues and I have therefore studied this approach in patients with nonsmall-cell lung cancer. Our initial approach has been based on injecting the primary tumor with a vector expressing wild-type p53, with the aim of improving the locoregional control of nonsmall-cell lung cancer.