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Gene therapy — a novel form of drug delivery
MOLECULAR MEDICINE
cy. Meeting this challenge will require better methodsfor delivering genes, the development of animal models
GENE THERAPY — A NOVEL FORM OF DRUG
of disease that adequately mimic diseases in humans,
DELIVERY
and well-designed clinical trials to assess the safety andbiologic activity of these products in patients.
The development of novel vectors or vehicles for gene
delivery warrants serious attention. Most gene therapyto date has involved the use of viruses as carriers of the
GENE therapy is a novel form of drug delivery that gene. The carrier virus may be a retrovirus that lacks
enlists the synthetic machinery of the patient’s
virtually all viral genes except those required for in-
cells to produce a therapeutic agent. Using the body to
fecting mammalian cells. Indeed, if properly prepared,
treat its own disease overcomes the need to manufac-
these viruses are so defective that after they infect the
ture highly purified proteins. It also eliminates the need
appropriate target cell, they cannot replicate or infect
for repeated parenteral administration of proteins (as in
other cells. The use of retroviruses is also advanta-
hemophilia) or drugs (as in hereditary hypercholester-
geous because they are easily generated, the infected
olemia) and reduces the difficulties of complying with
viruses can be extensively characterized in tissue cul-
exogenous-drug regimens. Applications of gene thera-
ture before being injected into patients, and the stable
py are not limited to rare inherited diseases but extend
integration of the virus into the chromosome ensures
potentially to common acquired disorders, including
its retention by the cell. The risk that retroviral inte-
cancer, heart disease, and the acquired immunodefi-
gration into the cellular genome could inactivate host
ciency syndrome. Gene therapy is thus likely to have
tumor-suppressor genes or activate proto-oncogenes
broad implications for the future practice of medicine.
appears to be minimized by reducing the efficiency of
This introductory article presents an overview of
infection so that at most one virus infects a cell. As
gene therapy. Succeeding articles will discuss specific
vectors, retroviruses have two major limitations. First,
advances and the obstacles to various kinds of gene
they can harbor genes of only limited size, approxi-
therapy for hereditary and acquired diseases.
mately 7 kb; although most therapeutic genes of inter-
The targets of gene therapy are either intracellular or
est can be accommodated, some are too large. Second,
extracellular (Fig. 1). In the first example shown in Fig-
retroviruses infect only dividing cells, whereas most
ure 1, the gene delivered to the cell affects the cell itself
tissues in adults consist primarily of nondividing cells.
by replacing a defective or missing gene (Fig. 1A) or by
For this reason, retroviruses are generally used for ex
providing a product that kills or inhibits the growth of
vivo gene delivery — i.e., the injection of cells that
cells (Fig. 1B). In the second example, the cell releases
were genetically engineered by infection outside the
the product of the new gene, which can act locally on
body, rather than by in vivo infection with the retrovi-
neighboring cells (Fig. 1C) or enter the circulation for
delivery to distant cells (Fig. 1D).
Adenoviruses are t he second most commonly used
Although easily comprehended in principle, gene
viral vector for gene delivery. These viruses have the
therapy is not so simple in practice. While gene therapy
same size constraints as retroviruses, but they have cer-
is likely to become a major part of the medicine of the
tain advantages. Adenoviruses readily infect nondivid-
future, it is by no means a component of the clinician’s
ing cells and can be produced in large numbers, making
armamentarium at present. Clinical experience with
infection of tissues in an adult more efficient. Moreover,
gene therapy over the past five years has shown that in
these viruses remain extrachromosomal, thereby reduc-
most cases, toxicity is not a serious problem. As with
ing the chance of disrupting the cellular genome. A lim-
recombinant DNA, the birth of gene therapy appeared
itation of adenoviruses is that if delivered in vivo they
to open a Pandora’s box of potential complications and
infect all tissues, including the germ line, and thus
possible toxic effects. However, most of the early wor-
could affect subsequent generations. Another problem
ries now appear unwarranted. Indeed, it has been sug-
is that they are not as defective as retroviruses and
gested that clinical tests of gene therapies may no long-
could more readily yield infectious virus in the body.
er require special consideration by the Recombinant
A third problem is that the current generation of ad-
DNA Advisory Committee to be performed, but should
enoviral vectors is immunogenic, which reduces the
require the same rigorous evaluation as other novel
length of time available for the expression of the gene
medical treatments. Exceptional review would thus be
and makes repeated administration of the vector im-
limited to unprecedented applications or issues — for
example, the use of new viral vectors, gene therapy for
There are also nonviral methods of gene delivery.
fetuses, or approaches that involve altering the germ
Liposomes, or lipid vesicles, which combine readily
line. Currently, the main challenge is achieving effica-
with cell membranes, are being used to deliver genes,sometimes as aerosols. Liposomes are also being testedin conjunction with viruses to enhance localized deliv-
From the Department of Molecular Pharmacology, Stanford University School
ery of genes. Direct injection of naked DNA plasmids
of Medicine, Stanford, CA 94305-5332, where reprint requests should be ad-dressed to Dr. Blau.
is possible, but for unknown reasons this method ap-
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Copyright 1995 Massachusetts Medical Society. All rights reserved.
Example 1: Gene affects
Product kills or inhibits the growth of cells
Sample application: CFTR gene is delivered
Sample application: Tumor cells receive tumor
necrosis factor or TK gene followed by
Example 2: Gene affects neighboring or distal tissue
Sample application: The implantation of cells
or injection of DNA leads to the production of
genetically altered myoblasts or injection of
growth factors that stimulate angiogenesis.
plasmid DNA results in muscle that secretes
Figure 1. Four Types of Gene Therapy.
CFTR denotes cystic fibrosis transmembrane conductance regulator, and TK thymidine kinase.
pears to function primarily in heart and skeletal mus-
therapy, cells express the genes continuously. As a re-
cle. The disadvantage of this method is that relatively
sult, the production of the therapeutic protein cannot
few cells take up the DNA (1 to 3 percent), and thus
be modulated. In diseases such as hemophilia regula-
only small amounts of the encoded protein are pro-
tion of the new gene is not critical, but such diseases
duced. Therefore, the primary and most important cur-
are the exception. In most other diseases, gene regula-
rent use of DNA plasmids as gene vectors is in vaccine
tion is desirable; indeed, constitutive expression of the
development, since the small amount of protein pro-
introduced gene may be detrimental or even life-threat-
duced by the few cells that take up the plasmid can elic-
ening. To overcome this problem, yeast-gene or bacteri-
al-gene regulatory systems have been adapted for use in
An important aspect of gene-delivery systems is the
mammalian cells. These inducible systems appear ad-
ability to regulate the expression of the introduced
vantageous because they affect the expression of intro-
gene. With the vectors that are now approved for gene
duced but not resident genes. There is no toxicity, and
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Copyright 1995 Massachusetts Medical Society. All rights reserved.
the gene-inducing agent can be administered orally. For
a number of existing viral and plasmid vectors. Some
example, it is possible to regulate the expression of a
desirable properties might include the ability to incor-
gene of interest, such as one encoding a hormone, by
porate large genes, the absence of immunogenicity, the
means of hybrid microbial and mammalian proteins
potential to direct the vector to certain cell populations
and microbial DNA regulatory elements that respond
— possibly by incorporating elements that recognize
to either the antibiotic tetracycline or the progesterone
specific membrane components and enhance uptake —
antagonist mifepristone (RU 486). In principle, these
the ability to incorporate elements to limit the expres-
new regulatory systems allow genes to be turned on and
sion of the delivered genes to particular types of cells,
off and the level of the therapeutic protein varied over
and the ability to turn on or off and modulate the levels
time. The complex goal of regulating genes through the
of gene expression in response to either nonmammali-
use of endogenous biologic signals, such as glucose lev-
an exogenous regulators or endogenous signals such as
els in the case of diabetes, is also being pursued but will
Animal models of human genetic diseases have been
Perhaps the ideal vector will be completely synthetic,
invaluable for testing and comparing different vectors
a composite of DNA-sequence components derived from
for gene delivery. They provide a convenient means of
Table 1. Gene-Therapy Protocols That Have Been Approved for Clinical Trials by the Recombinant DNA Advisory
Inherited diseases*
Cystic fibrosis transmembrane conductance regulator
ficiency due to adenosine deaminase deficiency
Acquired diseases
HIV-cleaving ribozyme; antisense (complementary
RNA sequence) to HIV TAR (transactivation-response element) along with transdominant nega-tive Rev (regulator of HIV gene expression)†
Herpesvirus thymidine kinase followed by ganci-
clovir treatment (toxic only to cells expressing herpesvirus thymidine kinase)
Antisense RNA to c-fos and c-myc RNA
Antisense RNA to insulin-like growth factor-1 RNA
Multidrug resistance (MDR-1) for chemoprotection
B7 cofactor to induce T-cell costimulation
Cytokines to induce immune response to tumor (in-
terleukin-2, 4, and 7; granulocyte–macrophage
Interferon-g to induce immune response to tumor
*The approach used is shown in Figure 1A.
†HIV denotes the human immunodeficiency virus.
‡The approach used is shown in Figure 1C.
§The approach used is shown in Figure 1B.
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Copyright 1995 Massachusetts Medical Society. All rights reserved.
evaluating in vivo the plasma and tissue concentrations,
problem is to test gene therapies in well-defined ani-
time course, and distribution of the expressed proteins.
mal models of cancer in which specific mechanisms
However, mouse models created by disrupting the re-
can be established — for example, those in which ei-
sponsible gene often do not manifest the disease in the
ther an oncogene is introduced or a tumor-suppressor
same way as humans with the equivalent genetic defect.
For example, the mdx mouse model of Duchenne’s mus-
A number of clinical trials of gene therapy have been
cular dystrophy harbors the same defective gene as the
completed or are under way. They can provide informa-
patient but has only transient muscle weakness. Similar-
tion that cannot be gleaned from preclinical tests in an-
ly, the mouse model of cystic fibrosis does not have life-
imals. The clinical trials listed in Table 1 were ap-
threatening pulmonary disease, although there is some
proved by the Recombinant DNA Advisory Committee
accumulation of mucus in the lung. The mouse model of
and are designed to meet the major goals outlined in
the Lesch–Nyhan syndrome does not have the charac-
Figure 1. In the ex vivo approach, cells are genetically
teristic profound neurologic symptoms, including self-
engineered and characterized in tissue culture before
mutilating behavior, or gout, but has reduced dopamine
being implanted in the body. In the in vivo approach,
levels. However, these models manifest at least some of
the gene is delivered and expressed in situ within the
the properties of the human diseases, and an under-
body. A current limitation of clinical trials is that very
standing of how they circumvent the problems found in
few viral vectors have been approved for use in hu-
their human counterparts may be informative. Nonethe-
mans. Thus far the trials have not shown convincingly
less, animal models that more closely mimic human dis-
that gene therapy is effective in treating disease in hu-
ease would be invaluable for preclinical tests of efficacy.
mans. For this reason, Dr. Harold Varmus, Director of
Mouse models of acquired human diseases such as
the National Institutes of Health, has initiated a major
cancer also have their limitations. The cancers in ani-
evaluation of the field to ensure that future efforts will
mals that are used to test novel therapies are frequent-
ly transplantable tumors that grow rapidly in the host,
Despite the tremendous attraction of and interest in
with nearly 100 percent of the cells undergoing division
gene therapy, the field is still in its infancy. Quick cures
— a situation quite atypical of most human tumors.
for genetic or acquired diseases are not to be expected.
These animal tumors grow readily in culture and
quickly develop into cancers in vivo with a highly re-
ECOMMENDED
producible phenotype. By contrast, genetic models of
Friedmann T. A brief history of gene therapy. Nat Genet 1992;2:93-8.
cancer in mice that resemble the disease in humans
Gossen M, Freundlieb S, Bender G, Müller G, Hillen W, Bujard H.
Transcriptional activation by tetracyclines in mammalian cells. Sci-
more closely pose difficulties, because to assess the ef-
ficacy of treatment in them often requires a very large
Mulligan RC. The basic science of gene therapy. Science 1993;260:
sample and long-term studies. The solution to this
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Copyright 1995 Massachusetts Medical Society. All rights reserved.
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Minimally-interventional therapeutic procedures in the spine:an evidence-based reviewIoannis Karnezis FRCS(Orth)Orthopaedic and Spinal Surgeon,Director, Back Care network (Athens)invasive interventional techniques for theclinical trials al articles were categorisedpracticing clinician from the principles ofclinical practice) as all clinical decisionsThe aim of the present article is to study