FEATURE ARTICLE
Gene Therapy
Investigators have been searching for ways to add corrective genes to cells harboring defective genes. A better strategy might be to correct the defects
Eric Kmiec
Gene Addition
Abnormal cell behavior is often the result of an altered gene whose expression is either absent or unregulated. A mutation in just one gene can sometimes cause a cell to malfunction. The mutated gene directs the synthesis of a dysfunctional protein, with the consequence that the cell functions marginally or not at all.
In the case of sickle cell anemia, for example, a mutated hemoglobin molecule actually distorts the red blood cell in which it resides, causing the cell to assume a sickle shape instead of its usual disk shape. The shape change prohibits the cell from adequately performing its designated role of carrying oxygen to the body's organs and tissues.
Another example is presented by muscular dystrophy, which is linked to mutations in the dystrophin gene. This gene codes for the dystrophin protein, which is crucial for the strength and movement of normal muscle tissue. People lacking dystrophin experience the muscle weakness characteristic of the disease.
Finally some genetic mutations do not alter a cell's function as much as they interfere with the cell's normal life cycle, specifically its cell-division cycle. Such mutations can lead the cell to divide uncontrollably, as is the case in certain cancers.
The essence of gene therapy, then, is to deliver to a cell a correct version of a mutated gene, the expression of which will produce the normal protein and hence restore normal cellular function. This has been obvious for some time, but how to achieve this goal has not been. An initial problem centered on how to get a gene into a cell. The chromosomes of a mammalian cell are housed inside a membrane-bounded compartment, called a nucleus. It is not enough for a gene-delivery system to deposit the gene into the cell; the gene must be delivered to the nucleus.
This in itself is not difficult. Scientists have been able to do it for decades. Foreign DNA can be injected into a cell, or its entry can be facilitated by various chemical or electronic means. But these methods are not very efficient, and one requirement for gene therapy is that sufficient amounts of corrective DNA be delivered to enough cells to be therapeutically beneficial.
Under the best circumstances, one would also want the therapeutic DNA to become a permanent part of the host's chromosomes. This would ensure its stability and would mean that the therapeutic gene would be replicated along with the host's chromosomes during each cell division. In contrast, DNA delivered to a cell by physical or chemical means can be placed in the cell's nucleus and can be expressed, but it does not become integrated into the chromosomes.
An ideal gene-delivery vehicle would be able to enter a large number of cells and integrate its DNA into the host's chromosomes. As it happens, some kinds of viruses are perfectly adapted to do just that. And, about 15 years ago, Richard Mulligan and Constance Cepko, who were then at Massachusetts Institute of Technology, along with colleagues at MIT and Harvard, made the important technological leap that initiated the modern era of gene therapy. Specifically they demonstrated that members of the retrovirus family could be engineered to carry foreign genes into mammalian cells and splice them into the host's chromosomes.
To create these gene-delivery vectors, Mulligan and his coworkers essentially gutted the virus of its genes, disposing of those that could be harmful to the host. At the same time, they retained those genes that enable the retrovirus to insert DNA into host chromosomes. By attaching this integrative machinery to the therapeutic gene, they created a retrovirus capable of infecting cells and splicing a corrective gene into chromosomes.
Inserting a gene, however, is only half of the problem. The vector must also contain a mechanism for activating the therapeutic gene, since this is not automatic. Genes have evolved a pattern of expression wherein certain levels of their product are required at specific times in the life cycle of the cell. Hence the corrective action of gene therapy must include a timing and regulatory "device." Such devices are usually found at the start of a gene and constitute the gene's "on" switch, or promoter. But this leads to another problem.
Promoters are often exquisitely complex and sometimes quite large, so placing them into a therapeutic vector is difficult. When constructing their retroviral vectors, Mulligan and his colleagues opted to use promoters native to the virus, rather than the corrective gene's own promoter. In laboratory petri dishes, these vectors sometimes worked quite well, but not always.
In some cases, the therapeutic genes entered the cells as expected but were expressed at unpredictably low levels. Low levels of expression continue to dog gene-therapy efforts, and improving expression levels remains a major focus of research. Recent vectors include portions of the gene's own promoter. This has the added benefit that the therapeutic gene is expressed as naturally as possible—only during the times when its product is needed.
Other constructions attach promoters that can be externally controlled. For example, certain genes have promoters that are sensitive to the antibiotic tetracycline and are activated when the drug is present. A vector was recently constructed by Herman Bujold and colleagues at the University of Heidelberg that pairs a tetracycline-sensitive promoter with a corrective test gene. The test gene would be activated only if the patient ingests tetracycline.
The initial expectation was that cells would have to be removed from the body in order to be treated. This ex vivo approach would necessarily limit therapy to those cells, such as blood cells, that are easily removed and replaced. But more recently, retroviral vectors have been developed that can be infused directly into an organ, such as the liver, or placed into the lung by inhalation. This versatility is one of the great advantages of retroviral vectors.
There are also some considerable disadvantages to retroviral vectors that have made investigators cautious about using them. The same feature that makes the retroviruses so attractive to gene-therapy investigators has also been one of their greatest drawbacks—namely the ability to integrate genes into chromosomes. The problem is that scientists have no control over how many copies of the gene become integrated or where on the chromosome they insert. Since integration appears to be essentially random, the vector's genetic payload may become inserted within another important gene, disrupting or altering its expression. Or a gene may integrate within the regulatory region of a gene responsible for controlling cellular proliferation, thus putting the cell on the path towards cancerous growth. Although these are remote possibilities, they are real and must nevertheless be considered as a potential consequence of retroviral-based gene-delivery vectors.
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