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Human Gene Therapy
Introductory article
Article Contents
Panos A Ioannou, The Murdoch Childrens Research Institute, Parkville, Victoria, Australia
. The Promise of Gene Therapy
Human gene therapy is the treatment of human diseases with DNA fragments comprising
whole genes or parts of genes.
. Ex Vivo Gene Therapy with Retroviruses
. In Vivo Gene Therapy
. Nonviral Gene Delivery
The Promise of Gene Therapy
. Homologous Recombination
Studies in the first half of the twentieth century demonstrated that bacteria were able to exchange genetic
material, resulting in permanent and heritable changes in
the properties of the recipient strain. The subsequent
understanding of the molecular basis of inheritance in the
second half of the twentieth century naturally led to the
concept of using deoxyribonucleic acid (DNA) fragments
comprising whole genes to overcome the effects of genetic
defects in various human diseases. The experiences with the
transfer of genes between bacteria (transformation)
encouraged the development of a fairly simplistic view of
gene therapy for human diseases: a single introduction of a
normal copy of a defective gene into affected cells should
result in long-term, stable production of the missing
protein, leading to a complete cure! The promise for
100% cure seemed to be very different from anything else
available in the armamentarium of twentieth-century
medicine and caught the imagination of many scientists,
as well as the attention of the news media and of the
investors. Rather than treat disease by repeated administration of pharmacological agents, gene therapy offered
the prospect of complete cure after a single application and
with no side effects! This concept has formed the premise
for much of the gene therapy research that has been
conducted over the last 30 years, and underlies much of the
‘hype’ that has been associated with it.
Gene therapy was originally envisaged as a method for
delivering normal copies of genes for the treatment of
patients with various rare genetic diseases. This concept
has gradually changed to include the use of gene fragments,
including PCR (polymerase chain reaction) fragments as
well as chimaeric and antisense oligonucleotides. Furthermore, the increasing understanding of the molecular
mechanisms underlying cancer development and other
somatic gene diseases is opening day by day many more
applications for gene therapy. This is indicated by the fact
that gene therapy trials for cancer now comprise about
70% of all gene therapy trials. These developments have
further excited interest in the potential of gene therapy to
transform the quality of life of most human beings in the
twenty-first century.
On the other hand, it did not take very long for gene
therapy research to come up against some serious
obstacles. By the early 1980s it was clear that most human
genes were much more complex in organization than
. Summary
. The Problems of Gene Therapy
anything encountered in bacteria. While bacterial genes are
composed of continuous coding sequences and adjacent
regulatory elements, the coding sequences of human genes
(exons) were found interspersed with noncoding intervening sequences (introns), ranging in size from a few base
pairs to hundreds of thousands of base pairs. By what
wondrous mechanisms could coding sequences placed
apart over such distances be brought accurately together,
again and again, to make continuous coding sequences in
the mature messenger ribonucleic acids (mRNAs), so as to
ensure the faithful production of the thousands of proteins
needed by each cell? Studies on the regulation of the
expression of the b-globin locus have also revealed not only
the presence of regulatory elements in the introns and the
untranslated sequences immediately adjacent to each
globin gene, but also regulatory elements conferring tissue
and developmental specificity as far as 50 kb (50 000 base
pairs) away from the target genes.
The early concept of gene therapy could thus not be
applied directly to the treatment of human diseases, as
there are still no methods for packaging and efficiently
delivering DNA fragments in the 100–300 kb range, which
is the size of most human genes with their natural
regulatory sequences. The discovery of the technique to
reverse transcribe mRNA back into complementary DNA
(cDNA) seemed to offer a way out of the difficulty.
Minigenes produced in this way could be readily cloned
and manipulated in bacteria before delivery into human
cells. Furthermore, such minigenes were easily accommodated in various viral vectors, thus facilitating efficient
delivery into different cell types. However, such synthetic
minigenes carried few if any of their natural regulatory
elements and, not surprisingly, failed in most cases to
approach normal levels of expression under physiologically relevant conditions. With the exclusion from the viral
vectors of most of the essential regulatory elements that
underlie the developmental, tissue and locus specificity of
gene expression, the goal of gene therapy proved much
more elusive than was ever anticipated.
The task of identifying one by one individual regulatory
elements followed by the inclusion and testing of combinations of these elements in the context of synthetic minigenes
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Human Gene Therapy
in viral vectors has proved very slow work. Thus, although
the sequence of the intact b-globin locus has been known
for about 15 years and numerous elegant studies have
examined the regulation of the expression of the different
globin genes, we still do not have a detailed picture of
globin gene expression under various physiologically
relevant conditions, while it has proved very difficult to
achieve a therapeutic level of globin expression with any
viral constructs. Our understanding of the mechanisms
underlying the developmental, tissue and locus specificity
of expression of all the other human genes remains of
course at an even lower level. Although the completion of
the sequencing phase of the Human Genome Project has
now revealed the sequences of the regulatory elements for
most genes, it will probably take research during most of
the twenty-first century before a good understanding of the
regulatory mechanisms for most human genes is achieved.
The development of effective viral gene therapy vectors is
of course conditional upon such an understanding for each
gene of interest. In contrast, the delivery of large DNA
fragments carrying intact functional loci and/or the direct
correction of mutations in genomic DNA are not conditional on such detailed knowledge and should enable the
faster exploitation of knowledge of the human genome for
the effective therapy of genetic and somatic gene diseases.
The development of several high-quality, large insert
genomic bacterial artificial chromosome (PAC/BAC)
libraries for the Human Genome Sequencing Project is
for the first time opening the real possibility of using intact
human genes for the treatment of gene diseases. These
resources have already proved extremely popular for gene
mapping, isolation and sequencing. Similarly, the use of
fully sequenced genomic fragments from PAC/BAC
libraries for functional studies is already shifting the
attention of many researchers to packaging, delivery and
long-term maintenance of intact human genes in human
cells as independent minichromosomes or episomes, or by
targeted integration into specific sites in the genome using
homologous or site-specific recombination mechanisms.
At the same time, it is also likely that new methods will be
developed for the targeted correction of mutations by
enhancing the endogenous mismatch repair and/or homologous recombination mechanisms of the cells. The high
fidelity of these mechanisms should reduce the risks
associated with random integration of viral vectors in the
human genome, while enabling the corrected gene to
function under the control of the endogenous regulatory
mechanisms.
Finally, it should be noted that no drugs have been
developed so far to treat human diseases by modifying the
expression of specific genes so as to complement other
defective genes, or by modifying the way specific mutations
interfere with gene expression. However, the completion of
the Human Genome Sequencing Project in combination
with DNA chip expression profiling is expected to lead to
an unprecedented degree of understanding of genetic
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networks under different physiological states, thus facilitating the identification of genes playing key regulatory
roles in health and disease. The coupling of this knowledge
with technologies for high-throughput screening for agents
acting on the mechanisms that underlie the developmental,
tissue and locus specificity of gene expression should
facilitate the development of drugs that modify the
expression of specific genes in a tissue-specific manner, or
that are able to suppress or reverse the effects of specific
types of mutations. Although such approaches may not
involve delivery of any DNA fragments and would not be
strictly speaking in the realm of gene therapy, they could
provide a basis for the effective and safe management of
most genetic and somatic gene diseases.
Ex Vivo Gene Therapy with Retroviruses
Viruses have naturally evolved efficient mechanisms to
deliver their nucleic acids into eukaryotic cells. Typically, a
few particles per cell are sufficient to lead to productive
infection for most viruses. Many different viruses are now
known, ranging in the type of nucleic acid they use (RNA
or DNA), the size of their genomes and their species and
host cell specificity. Many of these viruses, including
murine retroviruses (MMLV, Moloney murine leukaemia
virus), lentiviruses, adenovirus, adeno-associated virus
(AAV), Vaccinia virus, Herpes simplex type 1 virus
(HSV1), have been intensively studied as potential gene
delivery systems.
Murine retroviruses were the first to be studied as gene
delivery vectors and still remain very popular because of
their high transduction efficiency, their ability to infect
rapidly dividing cells and their efficient integration into
genomic DNA. The maximum size of the therapeutic insert
with murine retroviruses is about 8 kb, thus precluding the
use of most genes with their natural regulatory elements.
This is also true for most of the other viral vectors.
Lentiviral vectors, despite the obvious safety concerns, are
also being used increasingly in gene therapy research,
because of their ability to integrate therapeutic genes into
nondividing or quiescent cells, a property that may be
particularly useful for therapeutic gene delivery into
haematopoietic stem cells and neuronal cells.
In retroviral vectors the therapeutic gene is inserted
between the long terminal repeats (LTRs), in place of the
viral genes that are needed for replication and packaging.
The packaging of the recombinant retroviral vector is
carried out in a packaging cell line that provides all the viral
proteins that are required for the formation of mature viral
particles. A major concern in this approach has been to
minimize the chances of generating replication-competent
retroviruses by recombination between the therapeutic
vector and the helper sequences in the packaging cell line.
The latest packaging systems have been designed to make
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Human Gene Therapy
the production of wild-type virus through homologous
recombination highly unlikely. However, cells have a
remarkable ability to join together DNA molecules
through nonhomologous recombination mechanisms,
and thus the generation of modified replication-competent
retroviruses remains a small but not negligible possibility.
Retroviral vectors are best suited for ex vivo gene
delivery to cultured cells (e.g. cultured haematopoietic
stem cells). After transduction, the recombinant retroviral
construct is efficiently integrated into the chromosomes of
the recipient cells. However, the random nature of the
integration events could lead to a number of undesirable
complications:
. The expression of the therapeutic gene may be affected
by the overall organization of the chromatin in the
region of integration, or by nearby regulatory sequences,
leading to variability of expression and/or silencing.
. One or more integration events may take place near a
gene involved directly or indirectly in the cellular
processes leading to cell division. Modification of the
expression of such genes by the LTR elements or other
regulatory sequences in the recombinant vector may
lead to tumorigenesis. Since there have not been
systematic long-term studies to assess the magnitude of
this risk, this needs to be kept constantly in mind as gene
therapy reaches the stage of clinical effectiveness with
these vectors.
. The ability of retroviral and lentiviral vectors to achieve
efficient integration may be linked to equally efficient
mechanisms for replicational excision and re-integration
at secondary sites in the presence of complementing
functions, thus enhancing considerably the risk of
complications.
. An integration event may take place near one or more of
the numerous retrovirus-like elements that are scattered
in the human genome, with the potential to activate a
dormant element into a new type of transmissible virus.
. Another cause for concern is the potential of interaction
between an ongoing viral infection and a recombinant
viral vector, to produce a virus with a new potential. This
is of particular concern in gene therapy trials on HIVinfected patients, where the high rate of virus production
and the high rate of spontaneous mutagenesis are
already challenging all the tools of modern medicine.
Ex vivo gene therapy allows in principle the possibility of
careful evaluation of the effectiveness and safety of the
procedure before returning the cells to the patient,
although other considerations may necessitate the return
of the cells to the patient before a thorough evaluation can
be carried out. It is hoped that advances in stem cell
research will enable long-term maintenance of human stem
cells in culture after gene delivery, to allow a thorough
evaluation to be carried out. Similarly, the possibility to
direct de-differentiation and re-differentiation of various
types of stem cells is opening new avenues for the
combination of ex vivo cell and gene therapy approaches.
In Vivo Gene Therapy
The potential for ex vivo gene therapy is currently limited to
haematopoietic stem cells. This approach is not applicable
to most other cell types, either because the tissue cannot be
cultured or because the tissue forms too large a part of the
body to be effectively repopulated after gene therapy of
stem cells in culture. Besides these practical limitations to
the application of ex vivo gene therapy for genetic diseases,
in vivo gene therapy is a necessity for cancer and other
somatic gene diseases.
Besides the difficulties encountered with ex vivo gene
therapy as discussed above, in vivo gene therapy also faces
the additional challenge of delivering enough therapeutic
vector to the target tissue without toxicity to other tissues.
While in some situations the correction of a defect in a
proportion of the cells in a tissue may be sufficient to
restore health, in the case of cancer most, if not all, cells
have to receive the therapeutic gene. Thus a high degree of
specificity during in vivo gene delivery is essential. This may
be achieved by one of the following methods.
. Altering the glycoprotein coat of the recombinant vector
so that it can only get into cells carrying the appropriate
receptor. Such modifications can be carried out either by
insertion of a peptide on the glycoprotein coat, to alter
its cell specificity, or by chemical modification (e.g.
chemical attachment of lactose to the surface of MMLV
vectors facilitates transduction into hepatocytes via the
asialoglycoprotein receptor).
. Inclusion of a tissue-specific promoter to drive a
therapeutic gene may ensure that the gene is expressed
only in certain tissues, although the vector may be
delivered to a wider range of tissues.
Nonviral Gene Delivery
Nonviral approaches to gene delivery date back to the
middle of the last century, when Avery, MacLeod and
McCarthy showed in 1944 that genes were transferred
between bacteria by nucleic acids. Calcium phosphatemediated transfection was developed in the early 1970s as
the first nonviral technique for eukaryotic cells and is still
the method of choice for the production of recombinant
viral vectors. The size limitations and safety concerns of
viral-based gene delivery approaches have spurred on
efforts to develop safer, nonviral vectors, without limitations on insert size. A variety of nonviral approaches are
now available, although the efficiency of the most effective
nonviral techniques in stable gene expression is still less
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Human Gene Therapy
than that of transduction with retroviral vectors. The key
differences between viral and nonviral approaches seem to
lie not so much in getting DNA across the cellular
membrane but in the efficiency of transport into the
nucleus and the stable integration of the therapeutic genes.
Viruses have evolved over a long period to go through these
steps very efficiently, while nonviral delivery systems have
paid little attention to these aspects of gene delivery.
Microinjection
This procedure is labour intensive, as DNA (or RNA) is
injected into the nuclei of individual cells under a light
microscope. The method is proving particularly useful in
the generation of transgenic animal models with large
genomic fragments from YAC, PAC and BAC clones.
About 10% of the surviving embryos normally carry the
microinjected transgene integrated randomly into the
genome. Since such clones carry most genes as intact
functional units, transgenic animals generated in this
manner can be very useful in unravelling the mechanisms
that underlie developmental, tissue and locus specificity in
gene expression.
Electroporation
Electroporation usually involves the application of high
voltage to a mixture of DNA and cells in suspension,
although techniques for the electroporation of DNA into
muscle and other tissues have also been described. The high
voltage opens small holes in the cell membrane, through
which the DNA enters into the cytoplasm. In bacteria this
process is very efficient, resulting in over 10 billion clones
per microgram of DNA for small plasmids. Although the
efficiency decreases considerably for large plasmids, the
process is efficient enough to make the generation of highquality genomic PAC and BAC libraries (clone size range
100–300 kb) a rewarding endeavour.
Electroporation of mammalian cells is much less efficient
because starting numbers of cells are much smaller and the
DNA is delivered naked into the cytoplasm, where it can be
subject to degradation. It is possible that complexing the
DNA with agents that may protect it from degradation and
facilitate its uptake into the nucleus will overcome some of
these limitations.
Liposomes
Liposomes are formed by a variety of amphiphilic lipids.
Chemical synthesis of a large variety of liposomes has
allowed gene delivery in vitro and in vivo in various cell
types and tissues, under conditions that overcome most of
the safety concerns with viral vectors. Major advantages of
the liposome technology over viral vectors include the
defined chemical composition of the liposomes and the
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absence of any obvious size limitation in the DNA
fragments that can be packaged. Thus, as the liposome
technology improves, it should be possible to package and
deliver intact genomic loci, if not whole chromosomes, into
specific tissues with good efficiencies.
Cationic liposomes interact with DNA electrostatically
to form condensed particles, which can then be taken up by
cells. Once inside the cells, the DNA is slowly released and
some of it makes it to the nucleus where it can be transiently
expressed. In a very small proportion of cells the DNA is
eventually integrated into the chromosomes, leading to the
establishment of stable cell lines. A large variety of cationic
liposomes are commercially available and the effectiveness
of these seems to vary greatly with different cell types and
the conditions for lipofection. In general, lipofection uses
105 –106 small plasmid molecules per cell. Thus the
efficiency of lipofection is several orders of magnitude
lower then transduction with viral vectors. The difference
between the two systems may largely be due to differences
in the uptake of the DNA from the cytoplasm into the
nucleus, rather than the delivery into the cytoplasm.
Cationic liposomes are generally unsuitable for in vivo
delivery, since they tend to interact nonspecifically with
many tissues. In contrast to cationic liposomes, neutral
liposomes entrap DNA inside the liposome particles.
Although such liposome formulations have generally been
less efficient than cationic liposomes, they are potentially
more useful in vivo, since they can stay in circulation for
much longer and can thus be targeted more effectively to
various tissues. The compaction of DNA with peptides
before encapsulation to facilitate nuclear uptake may
eventually enable the in vivo delivery of large genomic
fragments to specific tissues.
Naked DNA injection
A variety of tissues show transgene expression after
delivery of naked DNA, with expression in striated muscle
persisting for long periods after a single injection. The
naked plasmid DNA appears to persist primarily as free
plasmid, with no significant integration into the host
genome after intramuscular injection. The efficiency of
DNA uptake and expression in striated muscle is affected
by various parameters, including age of animals and
species. Plasmids up to about 20 kb in size have been
successfully expressed after naked DNA delivery to
muscle. A major limitation of this approach is the low
percentage of expressing myofibres. However, expression
of the erythropoietin gene under such circumstances seems
to be sufficient to bring about changes in the haematocrit
levels of the treated animals. Similarly, injection of DNA
into muscle can be used to induce the production of
antibodies to the encoded protein.
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Human Gene Therapy
Ballistic DNA injection
The ‘gene-gun’ was originally developed for the introduction of DNA into plant cells, but it has since been modified
to transfer genes into mammalian cells both in vitro and in
vivo. The technique is restricted to skin, muscle or other
organs that can easily be exposed surgically. The most
exciting application of ballistic plasmid DNA injection is in
DNA-based immunization. In contrast to immunization
with foreign antigens, which generates only an antibodymediated immunity, DNA-based immunization is more
likely to result in a cell-mediated immune response and
thus it may be more effective in immunization against a
variety of viruses.
Homologous Recombination
The ideal form of gene therapy involves the direct
correction of the underlying genetic defect, enabling the
mutant gene to recover its activity under its normal
regulatory mechanisms. This type of repair may be
achieved through homologous recombination, a process
that involves the exchange of genetic information between
two similar DNA sequences, with or without the involvement of mismatch repair. In mammalian cells this process
allows the introduction of specific changes into the genome
by interaction of the endogenous sequences with homologous sequences in DNA constructs that are delivered into
the cells by a variety of vectors.
A major obstacle to successful gene therapy through
homologous recombination is the low efficiency of the
targeting process. A large number of different proteins are
involved in homologous recombination. The mechanisms
are best understood in bacteria, where inducible homologous recombination systems have recently been developed. Similar studies in eukaryotic cells have yielded only
marginal improvements. Further advances in our understanding of the mechanisms of homologous recombination
will no doubt open novel possibilities for the therapy of
genetic diseases.
Correction of mutations directly in the genome may also
be achieved through the enhancement of the endogenous
mismatch repair mechanisms. In order for this process to
become operational, it is necessary to induce heteroduplex
formation over the site to be corrected. Short PCR
fragments, homologous sequences delivered with the
AAV vector and specially designed chimaeric RNA/
DNA oligonucleotides have all been used to induce
mismatch repair correction, with variable results.
Site-specific recombination mechanisms are well known
in bacteria and a small number of such systems have been
adapted to eukaryotic cells. Each bacterial integrase has a
large recognition site and thus there are only a limited
number of potential endogenous recognition sites in the
human genome. As more and more integrases become
characterized, it may be possible to have a whole spectrum
of different integrases for targeting therapeutic constructs
into specific regions of the genome.
The Problems of Gene Therapy
Gene therapy research has gone through many ups and
downs in recent years. The hype driven by the concept of
gene therapy as an ‘elixir’ for genetic diseases, and by the
rush of investors to make a quick profit, has given way to a
more careful assessment of potential risks and benefits. The
death of a patient taking part in a gene therapy trial in
September 1999 has renewed safety concerns over viral
vectors and has rekindled the debate on the ethical aspects
of gene therapy. At the same time, the apparent successful
therapy of a number of patients with severe combined
immunodeficiency (SCID)-X1 disease, after ex vivo retroviral transduction of bone marrow cells, has given new
momentum to the field. These recent developments
underscore the potential of gene therapy in treating human
diseases but also demonstrate that there are still some
major obstacles to overcome before gene therapy becomes
an essential tool of modern medicine.
The main challenges still facing gene therapy research
are the following.
What sequences to deliver?
Use of cDNA sequences in therapeutic vectors can hardly
ensure the regulated expression of the cDNA under a
variety of physiologically relevant conditions. While some
cDNA constructs may have useful applications, the
availability of most genes as intact functional units in
genomic DNA fragments from PAC/BAC libraries should
encourage efforts at delivering intact functional loci. The
delivery of an intact functional locus should overcome
expression problems, as it should come under the
endogenous regulatory mechanisms, even if it is integrated
at a different site. Similarly, the direct correction of defects
in genomic DNA by the delivery of short PCR fragments
or chimaeric oligonucleotides will also restore the ability of
the gene to function appropriately under a variety of
physiologically relevant conditions.
How to deliver?
Viruses have evolved a number of complex mechanisms
that endow them with extraordinary efficiency and
specificity in delivering their genomes into the nuclei of
different cell types. Similarly, some viruses have very
efficient mechanisms for integration into the genome, while
others can maintain their genomes extrachromosomally.
The design of viral vectors for various gene therapy
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Human Gene Therapy
applications has concentrated primarily on taking advantage of some of these properties while deleting potentially
harmful viral DNA sequences from the final constructs.
Thus most retroviral, lentiviral and AAV gene therapy
vectors retain only the corresponding LTR sequences.
However, in a number of viral vectors, some of the viral
proteins and mRNAs from the packaging cell lines that
may be included in the viral particles may precipitate a
variety of short-term acute reactions in the recipient host
cells. These short-term acute reactions and the continuing
need for a careful assessment of the long-term risks to
individual patients and to society further support the
development of alternative gene delivery systems.
The development of nonviral gene delivery systems is
aiming to take advantage of our increasing understanding
of the mechanisms for the compaction of DNA in various
physiological states, as well as for the packaging and
delivery of therapeutic sequences across the cellular and
nuclear membranes. The incorporation of additional
mechanisms for the maintenance of the therapeutic DNA
as independent minichromosomes or episomes, or for its
targeted integration through homologous or site-specific
recombination mechanisms, should enable nonviral gene
delivery to become a safe and indispenable tool of medicine
in this century.
Target cell specificity
Delivery of therapeutic genes to a specific tissue and/or cell
type in vivo represents one of the major hurdles in gene
therapy for somatic gene diseases. While the design of viral
vectors has often taken advantage of the natural tissuespecificity of different viruses, various strategies have also
been developed to modify such targeting specificity, so as
to modify the types of cells that are susceptible to
transduction by each viral vector. Some of these approaches are readily adaptable for use in nonviral delivery
systems. Since many cDNA constructs do not display a
high degree of cell specificity in the expression of the
cDNA, a high degree of cell specificity in the delivery is
desirable to reduce unwanted effects on other cell types. In
contrast, since expression of intact fucntional loci is highly
cell-specific, it is anticipated that there will be less need for
cell targeting during delivery of intact functional loci.
Fate of DNA in cells
Retroviruses integrate very efficiently into the genome of
dividing cells and this has been one of the main reasons for
their development as gene therapy vectors. Lentiviruses
appear better in this respect, since they are also capable of
gene delivery and integration in nondividing cells. The only
elements that appear to be needed for integration are the
LTR sequences and the viral integrase. It has generally
been assumed that integration is not reversible. However,
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the recent demonstration that lentiviral vectors can be
mobilized to integrate at additional sites in the course of
HIV infection is of particular concern, since it highlights
the potential for interaction between viral vectors and
other ongoing viral infections in patients.
It is clear that the random integration of viral vectors
into patients’ chromosomes cannot provide a safe
approach for the widespread use of gene therapy to
alleviate human disease. The enhancement of homologous
or site-specific recombination mechanisms to facilitate
targeted integration of therapeutic genes into specific sites
in the genome, or their maintenance in independent
artificial minichromosomes or episomes, may provide a
safe alternative approach.
Summary
Gene therapy is emerging as one of the most potent tools of
medicine for the treatment of genetic and somatic gene
diseases. Although the problems associated with the
design, delivery and fate of therapeutic constructs into
patient cells have been grossly underestimated and oversimplified, leading to unrealistic and overoptimistic
expectations, the completion of the sequencing phase of
the Human Genome Project is expected not only to
catalyse the development of effective therapies for many
diseases in the first half of this century but also to provide a
solid basis for the biological emancipation of mankind by
the end of the twenty-first century.
Further Reading
Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, et al. (2000) Gene
therapy of human severe combined immunodeficiency (SCID)-X1
disease. Science 288: 669–672.
Evans JT and Garcia JV (2000) Lentivirus vector mobilization and
spread by human immunodeficiency virus. Human Gene Therapy 11:
2331–2339.
Felgner PL (1997) Nonviral strategies for gene therapy. Scientific
American 276: 102–106.
Fox JL (2000) Gene-therapy death prompts broad civil lawsuit. Nature
Biotechnology 18: 1136.
Friedmann T (1997) Overcoming the obstacles to gene therapy. Scientific
American 276: 96–101.
Friend SH (1999) How DNA microarrays and expression profiling will
affect clinical practice. British Medical Journal 319: 1306.
Kresina TF (ed.) (2001) An Introduction to Molecular Medicine and Gene
Therapy. New York: Wiley-Liss.
Michael A (1996) Financing gene therapy beyond phase II. Gene Therapy
3: 1035–1038.
Morgan RA and Blaese RM (1999) Gene therapy: lessons learned from
the past decade. British Medical Journal 319: 1310.
Orford M, Nefedov M, Vadolas J et al. (2000) Engineering EGFP
reporter constructs into a 200 kb human globin BAC clone using GET
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ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Human Gene Therapy
Penn SG, Rank DR, Hanzel DK and Barker DL (2000) Mining the
human genome using microarrays of open reading frames. Nature
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Thompson L (2000) Human gene therapy. Harsh lessons, high hopes.
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Walther W and Stein U (eds) (2000) Gene Therapy of Cancer: Methods
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Zhao S (2001) A comprehensive BAC resource. Nucleic Acids Research
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