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.
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