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American Scientist Online - Gene Therapy
see full issue: May-June 1999
07/06/2007 11:51 AM
Volume: 87 Number: 3 Page: 240
DOI: 10.1511/1999.3.240
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 B. Kmiec
This article was published in the May-June 1999 issue of American Scientist.
In the middle of the 19th century, the now-famous monk Gregor Mendel performed his landmark
experiments indicating that certain traits can be inherited, and he postulated a discrete unit of
inheritance that we now call a gene. Since then, scientists have come to appreciate how much of an
individual's constitution is determined by genes, and, in particular, they have focused on the link
between genes and disease. Indeed, over the past 10 or so years, identifying disease-related genes
has become something of a cottage industry within the scientific community.
Any reader of newspapers knows just how fruitful this enterprise has been. Almost daily come reports
about the discovery of a new gene that contributes to some disease or another, be it sickle cell
disease, muscular dystrophy, familial hypercholesterolemia, Alzheimer's or some form of cancer. Right
now, therapies directed toward these conditions can only alleviate the symptoms—the manifestations
of the defective genes. Implicit, and sometimes explicit, in stories about genetic discoveries is the idea
that new therapies can be created that directly address the source of the problem. These gene
therapies seek treatments, even cures, that act at the level of the gene itself.
Most of the gene therapy techniques developed so far are of the gene-addition variety; that is, they
attempt to provide a good copy of a gene to a cell that harbors a bad one. The hope is that the good,
corrective gene will compensate for the bad one and restore the cell to its proper function. Gene
addition has been achieved by a variety of means—not only in test-tube experiments, but in clinical
trials involving real patients as well. Yet, to date, the results of these trials have been disappointing.
Even the most successful clinical trial has fallen short of therapeutic efficacy.
Unfortunately, many of these trials have been widely publicized—and, in some
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cases, oversold—in popular books and magazine articles. Having failed to live up
to the inflated expectations created by such publicity, these disappointing trial
results have left a general impression that gene therapy cannot now or ever
fulfill its initial promise. But these clinical trials may have been conducted before
the technology was fully mature, driven in part by investor demands on
biotechnology companies to rush products to market. Such clinical trials were almost certainly destined
to fall short of the mark.
Many of the fundamental problems with gene therapies have not yet been worked out sufficiently to
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make the technology therapeutically viable. A number of vehicles have been developed to deliver
corrective genes into cells. Some are more effective than others, but none is yet exactly right. The
greater challenge, however, lies in the problem of how to make the therapeutic gene behave reliably
and at clinically beneficial levels.
Making gene therapy a successful endeavor will require careful research to understand why traditional
approaches have not produced the hoped-for results and, in turn, to improve them, while exploring
new ways to deal with genetic defects. In my own laboratory, we are considering the possibility that
inserting an entire gene into a cell and then expecting it to behave as a native gene may be overly
ambitious at this point. It may also be unnecessary. Since the defects in many disease-related genes
are fairly small, my colleagues and I are exploring ways to repair rather than replace them. Our initial
results lead us to be cautiously optimistic that with adequate basic research this or some other
approach will ultimately yield fruit. My own feeling is that pronouncements of gene therapy's imminent
demise are as premature as were those overly optimistic pronouncements of its imminent success. At
its core, the notion of gene therapy or gene correction is scientifically sound.
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.
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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
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expressed, but it does not become integrated into the chromosomes.
An ideal gene-delivery vehicle would be able to enter a
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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.
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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.
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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 genetherapy investigators has also been one of their greatest drawbacks—namely the ability to integrate
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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.
Adenovirus and Others
Considering some of the safety issues surrounding the use of retroviral vectors, investigators have
been casting about for other viruses that can deliver genes to cells without disrupting their normal
chromosomal configuration. There has been much interest in the use of adenoviruses for this purpose.
The bulk of the early work on adenoviral gene therapy was conducted by Ronald Crystal at Cornell
Medical School and James Wilson at the University of Pennsylvania.
Like the retroviruses, adenoviruses deliver their genetic payload to the nucleus, but, except under rare
circumstances, the genes do not integrate into the resident chromosomes. This, of course, relieves
concern about random genetic integration, but it also means that the therapeutic gene is only
transiently active. The adenoviral vectors have to be repeatedly administered in order to maintain a
steady therapeutic dose.
Adenoviruses can infect a broad range of human cells, including those of the lung, liver, blood vessels
and brain. In fact, brain tumors have been treated with adenoviral vectors carrying "suicide genes,"
whose expression leads to cell death only when its product interacts with a specific drug taken by the
patient. These studies generated mixed results.
Adenoviral vectors have also been used in human trials to correct mutations in the cystic-fibrosis
transmembrane receptor (CFTR) gene, which contributes to cystic fibrosis. The success of these trials,
however, has been quite low. For one thing, the host's immune system registers the adenoviral vector
as foreign and eliminates it from the system. In addition, some of the vectors cause an inflammatory
response at the high levels required to achieve therapeutic doses.
One of the most promising vehicles to emerge from recent gene-therapy studies
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is adeno-associated virus (AAV). This virus infects a wide range of cells, including
lung and muscle cells, and it integrates its genes within the host's. In addition, it
can infect nondividing cells and does not elicit an immune response—both of
which are important advantages over retroviral and adenoviral vectors. The work
on this virus has been pioneered by three investigators: Kenneth Berns and
Nicholas Muzyczka at the University of Florida and R. Jude Samulski at the
University of North Carolina at Chapel Hill. Significant advances in the use of AAV
for gene therapy have recently been reported by Mark Kay and colleagues at Stanford University and
Kathryn High and coworkers at the University of Pennsylvania. Both of these research groups used a
modified AAV vector to achieve long-term expression and correction in animals of a gene that
contributes to hemophilia. This achievement required a detailed appreciation for the basic biology of
AAV.
However, as expected, this virus has some drawbacks. First, it can carry only a small genetic payload,
which considerably restricts its usefulness. Second, it, too, carries the risk of disrupting functioning
genes by randomly inserting itself into the chromosomes. Finally, it is somewhat difficult to
manufacture these vectors in sufficiently high quantities. Other viruses under study as potential vector
candidates include Herpes simplex, Vaccinia and even the human immunodeficiency virus.
In addition to viral-based vectors, investigators are continuing to explore nonviral delivery systems.
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One system that holds some promise delivers drugs via liposomes, small vesicles artificially created
from lipids that resemble those making up the membranes of mammalian cells. Because they are
constructed of virtually identical materials, the liposomes can fuse with cell membranes and empty
their contents—which can include drugs or corrective genes—inside the cell. Some of the DNA
delivered by liposomes makes its way into the cell's nucleus.
Targeted Gene Repair
Ultimately, scientists would like to replace a dysfunctional gene with a functional one, within the
normal context of the chromosome, an approach that could skirt the concerns about the number of
genes delivered, the chromosomal location and the level of expression. Right now, homologous
recombination, the only technique that comes close to this, is so inefficient that its success rate is 1 in
10,000. Needless to say, this is not adequate for human use.
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But the idea of completely replacing a bad gene with a good one may be
overreaching, especially when one considers how small are many of the
mutations that contribute to disease. To understand how small, we must first
consider a few basic facts about the composition of genes.
The gene is to inheritance what a word is to language; it is the basic unit of
meaning. In the genetic lexicon, the gene is a length of DNA that codes for a particular protein. The
alphabet used by the genetic language contains only five letters, or nucleotides, named for the bases.
These are adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U). The nucleotides A, T, C
and G are found in DNA. (RNA, a chemical cousin to DNA and an important participant in genetic
decoding, lacks thymine, but has uracil in its place.) The average human gene is a little over 1,000
nucleotides long. In many inherited disorders only one or a few of these nucleotides is incorrect.
For example, sickle cell anemia is the result of a single nucleotide substitution, a single letter
misspelled, in the gene encoding the b-globin strand of hemoglobin. Yet this one-nucleotide
substitution can cause the structural deformity of the molecule and the characteristically distorted
shape of the sickled red blood cell. Over 70 percent of the cases of cystic fibrosis are attributable to
the deletion of three nucleotides in the CFTR gene. Why should the entire gene be replaced when the
error is so minimal? That strategy seems akin to remodeling the whole kitchen to repair a leaky
faucet.
In 1993, while studying homologous recombination in mammalian cells, members of my laboratory
began experimenting with ways to repair damaged genes, rather than replacing them. The cell's own
repair mechanisms are extremely efficient, as evidenced by the simple and continual inheritance of
normal genes through generations of cell divisions. If we could harness the cell's own power of DNA
repair, we reasoned, we might be able to correct mutations.
Normal human chromosomes are actually made up of two strands of DNA complexed to each other in
an interesting way. It turns out that the nucleotides of DNA can bind with each other in a specific
pattern. Except in very rare cases, adenine always pairs with thymine, and guanine always pairs with
cytosine. Each DNA strand carries a nucleotide sequence exactly complementary to the other, such
that every adenine nucleotide on one strand is matched up with a thymine on the partner strand, and
every guanine is matched with a cytosine on the complementary strand. A sequence of GATC on one
strand would therefore bind to the sequence of CTAG on its partner, or so it should be.
Occasionally the wrong nucleotide is inserted into a spot, so that the corresponding nucleotide on the
partner strand cannot properly bind in that position. In that case the mismatched nucleotides form a
bulge. Usually this is not a problem, since the cell contains DNA repair mechanisms that actually scan
the DNA and detect such bulges. When one is discovered, the repair systems work to remove the
incorrect nucleotide and replace it with the correct one. But if the mismatch is overlooked by the cell's
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repair machinery, the error is retained, and the gene remains defective.
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It was our idea to alert these repair mechanisms to the error. The principle is
quite simple. We artificially create a short string of nucleotides, called an
oligomer, that, with one exception, is exactly complementary to the section of
the gene in which the error is located, the exception being at the site of the
error. Here we insert the nucleotide complementary to the one that is supposed
to be in the DNA sequence of the normal gene. The oligomer binds to its
complementary sequence on the DNA, and by design creates a bulge at the site
of the mismatch. This bulge is eventually detected by the cell's internal DNArepair mechanisms. Repair enzymes remove the erroneous nucleotide and
replace it with a nucleotide complementary to the one in that position in the
oligomer, which happens to be the correct nucleotide. This scenario for targeted
gene repair has been experimentally confirmed in my laboratory by Allyson Cole-
Strauss.
Our work builds on earlier studies from Fred Sherman's laboratory at the University of Rochester, who
used a similar technique to change a single nucleotide. But the oligomers used by the Sherman group
were unstable, and the team never extended its work. It turns out that mammalian cells contain
enzymes that either degrade the ends of DNA molecules, or link them in long arrays called
concatamers, which essentially destroys the integrity of the oligomers. We discovered that we could
increase the stability of an oligomer by attaching segments of RNA to each of its ends. Like DNA, RNA
is also composed of strings of nucleotides and therefore can bind to DNA in the same complementary
manner as can another strand of DNA. (RNA contains no thymine. Instead, the uracil in RNA pairs with
the adenine in DNA.)
In the past two years, we have successfully corrected seven chromosomal targets with this approach.
Cole-Strauss and others in my lab have demonstrated the feasibility of using gene repair to correct
the sickle-cell mutation in vitro, and Clifford Steer's laboratory has reproduced and extended those
results in certain animal models. Vitali Alexeev and Kyggeon Yoon have shown that the correction is
maintained through successive generations of cell division, suggesting that gene repair may have longterm benefits. Only continued studies will be able to determine whether this approach will be useful for
human gene therapy.
Clinical Trials
In the clinic, gene therapy has enjoyed few successes and many failures. But within these failures
lessons have been learned. In some cases, scientific rigor was sacrificed in order to bow to financial
pressures to rush gene therapy into clinical trials. In other cases, the goals were too lofty, and the
expectations were unrealistically high.
Limited success in animal models all too often leads directly to clinical trials. But a mouse is not a
small human with four legs, and the positive results in mice do not necessarily portend a positive
outcome in people.
Even at their best, the results of trials with human gene therapy are equivocal. For example, let's
consider a gene therapy trial to treat familial hypercholesterolemia (FH). People with this inherited
condition have dangerously high blood levels of cholesterol, in spite of their body weight or diet. The
condition results from a defective gene that encodes a receptor found on the membranes of liver cells
specific for low- density lipoprotein (LDL), what many call "bad cholesterol." Normally LDL enters liver
cells via this receptor, after which the liver clears the body of LDL. But people with FH have too few
functioning receptor molecules and cannot remove LDL from their blood. As a result, blood serum
levels of LDL are too high in people with this condition, and many FH patients develop coronary artery
disease.
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In animal models, investigators demonstrated some success when corrective
copies of the receptor gene were transferred into liver cells via a retroviral
vector. Blood levels of LDL in the treated animals were significantly reduced and
remained so for over six months. These experiments were done carefully, in
several animal models and with rigorous controls. All the models produced
similar, encouraging results. Based on these results and the fact that patients
with this disease have few good treatment alternatives, a human gene-therapy
trial for FH was approved.
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The experience of one 28-year-old woman represents one of the better outcomes of this clinical trial.
The patient lacked any detectable functioning LDL receptor because she lacked the gene for it. At the
start of the trial, she had 482 milligrams of LDL in each deciliter (mg/dl) of blood, well over twice the
normal level of 160 to 210 mg/dl. Her liver cells were then treated with a retroviral vector containing
the LDL-receptor gene. Within a few days, her serum cholesterol dropped by 180 mg/dl to about 300
mg/dl. With additional cholesterol-lowering drugs, her LDL blood levels stabilized at around 356 mg/dl
and remained there for about two and a half years. These levels, although lower than they were
originally, are still higher than they ought to be.
Herein lies the quandary of human gene therapy: It "sort of" works. This trial demonstrated the
feasibility and safety of gene therapy for treating FH. But the results hardly constitute a ringing
endorsement for this approach as the definitive therapy. In fact, it would be difficult to name a clinical
trial to date that does, a situation that prompted review of the approval process for clinical trials.
Decisions on clinical applications of gene-therapy approaches fall under the
auspices of the federal government. Originally, the Recombinant DNA Advisory
Committee (RAC) was charged with the duty of making suggestions for approval
or disapproval to the director of the National Institutes of Health (NIH). The RAC
was empowered to consider somatic gene-therapy protocols only; that is, the
RAC considered protocols that did not involve the so-called germ line cells, such
as eggs and sperm. In addition to submitting protocols to the RAC, investigators submitted new genetherapy protocols to the Food and Drug Administration for Investigational New Drug (IND) approval.
More than 100 protocols have been approved by the RAC to date.
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In 1996, Dr. Harold Varmus, the director of the NIH, amended the procedure
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because of rising concerns over the rate of failures among approved clinical trial
protocols. The fallout of this effort has been an enhanced oversight role for the
NIH through three mechanisms. First, the office of Recombinant DNA Activities
Advisory Committee (OAC) evaluates protocols. Next, gene-therapy conferences
are held to promote public discussion of scientific merit and ethical aspects of proposals for human
clinical trials. Finally, the public is informed about the progress of ongoing trials. The gene- therapy
policy conferences have already helped to improve the oversight of the approval process. They also
encourage continued review of the ethical aspects of each trial. Although in most cases, increasing
government involvement is viewed as an invasion into scientific enterprises, gene therapy will stand to
benefit by such heightened control.
In spite of the increased government scrutiny of previous gene-therapy protocols, the pressures to
bring gene therapy to the clinic do not seem to abate. This is in part because of the formation of
ventures aimed at commercializing products and techniques. Financial pressures and constraints often
force biotechnology companies to forgo basic research in favor of application-driven development.
Unfortunately, this means that promising but technically difficult approaches may never be adequately
developed for lack of research funding.
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Increases in public funds for research may well be part of the answer to
improving the technology, since it is the basic level of investigation that will in
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the end broaden understanding of gene-therapy techniques and increase the
probability of clinical success. What is clearly needed is the development of
molecular analysis and rigorous testing at the level of basic science, with an eye
towards application in clinically appropriate targets. Only then will gene therapy
have a hope of fulfilling its promise.
Acknowledgments
I wish to thank current and former members of my laboratory for their hard work on gene repair, the
NIH for funding these projects and Michelle Hoffman for editorial advice.
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