therapy is one of the many applications of genetic engineering. It involves
introducing a new gene or modifying an existing gene (itself or its activity)
in cells. It can be used to treat or prevent diseases.
There are two forms of gene therapy:
Germ cell therapy (unethical and not allowed in humans, easily performed in
Somatic cell therapy (currently used to treat cystic fibrosis, severe combined
immune deficiency [SCID], some tumors, etc). This kind of gene therapy can be
applied to the whole body (in vivo
therapy) or to the cells removed from a patient (ex vivo therapy). In the latter, the engineered cells are
returned to the patient.
Gene delivery methods:
Recombinant retroviral or adenoviral vector-mediated: Retroviruses can only
accept up to 7 kb of introduced DNA. They only infect dividing cells. Because
of this, they are used in ex vivo
therapies. They integrate into the host genome. Thus, their effect is long
lasting, but insertional mutagenesis is a potential problem. Retroviruses are
used in the treatment of SCID. Adenovirus can accommodate larger genes and does
not integrate into the host genome. Their effect is transient. The main problem
with adenovirus is the immune response they elicit (used in the treatment of
cystic fibrosis and a1-antitrypsin deficiency).
Liposome-mediated transfer: DNA is encapsulated in liposomes (lipid micels).
This method has no side effects but is less efficient in transferring DNA to
target cells. It does not cause an immune reaction. Liposomes can be used in
vivo and ex vivo and carry any size of DNA fragment.
Microinjection (germ cell therapy): DNA is injected directly into the nucleus
of a fertilized ovum viewed under the microscope. Currently, this method is
used routinely to produce transgenic animals.
Biolistics: This method is used in plants. It involves coating special metal
spheres with DNA and firing them into the plant cell from a special gun.
Biolistics is used as an alternative to Ti plasmid-mediated gene transfer. See Plant
Gene therapy in humans
humans, genetic engineering is only attempted in somatic cells in the form of
gene therapy. Technically, it is possible to modify a fertilized egg at the
four-cell stage (or even clone) but there is no ethical approval for such germ
cell gene therapy at present. The first gene therapy for a human disease was
successfully achieved for SCID by introducing the missing gene ADA into the
peripheral lymphocytes of a 4-year-old girl and returning modified lymphocytes
to her (in 1990). As of January 2000, more than 350 gene therapy protocols have
been approved in USA.
gene therapy uses the same strategies to deliver the genes. This can be
achieved ex vivo or in vivo (in vivo: viral vectors, liposomes; ex vivo: cells
such as tumor-infiltrating lymphocytes (TNF insertion), bone marrow stem cells
as in bone marrow transplantation, or peripheral lymphocytes -as in SCID (ADA
insertion)- are taken out, modified and returned to the patient).
Alternatively, antisense treatment can be tried to prevent the transcripts from
being translated into unwanted proteins (as being tried to combat HIV
infection). Even naked DNA (i.e., not inserted into a carrier virus or
liposome) can be injected directly to the patients to substitute the defective
gene (DNA vaccines). DNA vaccines are plasmids being inserted the desired gene.
Plasmids may directly transfect (animal) cells in vivo. This strategy is
usually used to elicit cytotoxic T cell type immune response using an antigenic
product of a pathogen (e.g., HIV).
popular gene therapy method is using the 'suicide gene'. The gene in question
is thymidine kinase from a herpes simplex virus (HSV-tk) and is delivered to
the target cells (usually cancer cells). When ganciclovir, an otherwise
harmless antiviral agent, is given to patients, HSV-tk-bearing cells convert it
to a toxic substance and the cells die. This is usually used in the treatment
of (brain) tumors, but also used in the treatment of GvHD following BMT. Any
other gene, which would render cancer cells highly sensitive to selected drugs,
can be used as the suicide gene. A logical use of gene therapy in cancer would
be either replacing the missing tumor suppressor gene or blocking the effects
of an oncogene.
tricks that can be used to treat HIV infections include using dominant negative
mutations (generating inactive versions of proteins HIV needs to replicate), or
delivering genes into CD4+ T lymphocytes (target cells for HIV infection) that
would be transcribed to short mRNAs mimicking essential viral control mRNAs to
interfere with the viral regulatory mechanisms.
Other applications of genetic engineering
was constructed by using classical genetic selection to combine genes
originally located on four different plasmids onto one compound plasmid. It is
used to clean up oil spills.
with industrial applications such as Rennin, a protein used in making
cheese, can be produced by recombinant DNA technology. This technology deals
with isolating a gene (or its cDNA) from its natural host and inserting it into
a different (asexually reproducing) species' genome so that it can be copied
every time the new host cell (usually a bacterium) replicates. Before the
advent of genetic engineering it was extracted from the fourth stomach of
cattle. The new technology is known as the 'cheese from microbes'.
Enzymes used in genetic engineering
such as restriction endonuclease (biological scissors), DNA polymerase (for
replication of DNA), reverse transcriptase (to make DNA from RNA) and ligase
(to ligate two DNA fragments) are produced by recombinant DNA technology (by
cloning in high copy number plasmids in bacteria). Other enzymes now routinely
produced by recombinant DNA technology are rennin (an enzyme used in cheese
making mentioned above), lipase (cheese making), a-amylase
(beer making), bromelain (meat tenderizer, juice clarification), catalase
(antioxidant in food), cellulase (alcohol and glucose production), and protease
Proteins for medical applications
such as insulin (previously extracted from pig pancreas, and since 1982
produced by recombinant DNA technology), clotting factor IX (which is lacking
in hemophilia B patients and previously supplied as fresh plasma from volunteer
donors), tissue plasminogen activator (t-PA, used in acute myocardial
infarction), growth hormone (previously extracted from the pineal glands of
cadavers), tumor necrosis factor (TNF), g-interferon
(g-IFN) and interleukin-2 (IL-2) (TNF, g-IFN, IL-2
are used in immunotherapy of cancers), erythropoietin (EPO, to stimulate red
blood cell production) and vaccines (such as HBV, rubella) can be produced by
of valuable (modified) proteins in milk of transgenic animals such as
factor IX and elastase inhibitor a1-antitrypsin (used in the treatment of emphysema) can
be another source for certain protein. The gene for the desired product is
usually combined with a gene coding for a milk protein that is expressed only
in mammary glands. The combined gene is then inserted in fertilized eggs. These
are implanted into recipient females. The desired protein can be harvested from
a female's milk. Because the gene is now in all cells of the animal (including
germ cells), its offspring will also have it. Transgenic and gene knockout
animals are also used for research purposes. The first transgenic animal
was a supermouse with a rat growth hormone gene (1982).
Plants: Plants most commonly used in
genetic engineering are maize, tomato, potato, cotton and tobacco. The main
aims are to induce tolerance to herbicides, resistance to insect pests or viral
disease and to improve crop quality.
tolerance: A gene from a soil bacterium (A. tumefaciens) codes for an
enzyme (PAT) which inactivates the herbicide Basta. When plants are engineered
to contain this gene they are not sensitive to the herbicide any more (already
tried on sugar beet, tobacco and oilseed rape). This allows selective killing
of weeds by herbicides. A plasmid from A. tumefaciens called Ti is used
to integrate the gene into the plant genome (the PAT gene is inserted into the
toxin gene of the plasmid).
to infection by viruses: Genes encoding antisense copies of viral genes were
used with limited success to prevent viral infection. The transfer of a gene
encoding a viral coat protein has been successful with an unknown mechanism.
resistance: Potato plants have been engineered to contain a pea lectin gene.
Lectin interferes with digestion of plants in insects but does not harm the
plant. It is also possible to use a Bacillus thuringiensis toxin called
protoxin as an insecticide. This has been tried in tomato but did not work very
well because of low expression.
improvement: Genetic engineering has also been used to modify plants to create
genetically-modified (GM) foods (also called genetically engineered organisms
‘GEO’ perhaps more appropriately). As tomatoes age, they soften due
to the effects of an enzyme called polygalactorunase which breaks down cell
walls. Its production can be blocked by activating the antisense gene to
inactivate mRNA for its gene. Thus, it is still transcribed but no translation
occurs. These tomatoes can ripen on the plant and are still suitable (hard enough)
for mechanical handling and transport (long lasting tomatoes = FlavrSavr tomato).
The FlavrSavr tomato was the first GM food approved by the FDA to go on the
market in 1994 (now discontinued). Tomato paste from genetically engineered
Tomato Paste; also discontinued) and oil from genetically engineered
oilseed rape were the first two whole foods declared safe in the UK (in 1995)
(see the link for all Transgenic
Crops). Transgenic plants such as soybean and rice can be engineered to
have the essential amino acids they normally lack. Golden Rice is for
example genetically enhanced with beta carotene. Genetic engineering in plants
has also been used to alter pigmentation in flowers, to improve nutritional
quality of seeds and to obtain seedless fruits. See also Plant Improvements: Biotechnology, Transgenic Plants and Genetic
Engineering in Plants (National Geographic, May 2002).
Drawbacks and potential dangers of genetic engineering
Gene integration into the right place: Position effect or tissue/cell-specific
expression of genes in gene therapy depend on the effects of enhancers and
Immune response against the vector used for gene delivery
Spread of the gene conferring antibiotic resistance to the vector
Escape of the introduced virus or bacteria to the environment (to avoid this a
modified strain of E.coli is used in recombinant DNA applications which cannot
survive in nature but is useful in the laboratory. The laboratories using this
technology are also under stringent control to avoid any contamination of the
Acceleration of the evolution of resistance to the toxins or antibiotics
biotechnology is achieving what classical biotechnology could not have in such
a short time and across a species barrier. There is no need for generations of
classical breeding schemes any more and hybridization between different species
can be achieved (like inserting a human gene into a mouse germ cell).
Nuclear transfer in frogs resulting in viable juveniles up to the tadpole stage
in the 1950s (Elsdale et al. J Embryol Exp Morph 1960;8:437)
Nuclear transfer in sheep using donor nucleus from early morula stage (8-16
cell) embryos (Willadsen SM. Nature 1986;320:63)
Nuclear transfer in sheep using a cultured embryonic (totipotent) cell's
nucleus (March 7, 1996 - Nature)
Cloning of Dolly the sheep by nuclear transfer from an adult's udder cell (Febr
27, 1997 - Nature) with a success rate of 0.3% (1 in 277 attempts)
Cloning of Polly the Sheep by transfer of a transgenic nucleus of a fetal
fibroblast cell - (Dec 19, 1997 - Science) [an alternative to pronuclear
injection in creation of transgenic animals]
Successful use of nuclear transfer in mice (Cumulina, success rate 2-2.8%) and
cows in 1998 followed by cloning of many other mammals (cattle, goats, rabbits,
cats, pigs, mules, horses). The most recent example is the cloned horse who was
delivered by her dam twin in 2003 (see below).
is the ultimate phase in creating an animal with a desired trait. It follows
the same path as selective breeding, artificial insemination, egg
transplantation and in vitro fertilization. In genetics, cloning means creation
of genetically identical animals (mammals) by means of nuclear transfer. In
nuclear transfer, the chromosomes of an unfertilized oocyte (egg) are removed
(enucleation) and a nucleus from a mature (diploid) cell is placed into the
egg. Note that no fertilization takes place and it is the presence of a diploid
nucleus inside an egg, which initiates cleavage divisions. It has been
established that an unfertilized oocyte is a better recipient than a zygote.
This is asexual reproduction of an animal, which would normally reproduce
sexually. A clone resembles its (single) parent, the animal from which the
donor cell was taken. This technology can be used either to create genetically
identical animals or to introduce a genetic modification to the mammal. Another
application is the production of undifferentiated cells which can then be
induced to differentiate to any organ or cell type. The success rate is
invariably very low. It may take hundreds of tries to succeed in nuclear
transfer. It is, however, highest in sheep. This is probably because of the
fact that transcription of the embryonic genome does not begin until the 8-16
cell stage in sheep, whereas it is the late 2-cell stage in mice.
the previous cloning experiments, embryonic cells were used as the source of
nucleus and the nucleus was transferred to a fertilized egg whose own nucleus
was removed (in mice in 1980s). In the creation of Dolly, however, the nucleus
of an adult -differentiated- cell was used. The key to the new procedure was
synchronization of the cell cycle of the adult cell with that of the egg. The
Dolly experiment showed
that for successful cloning, an -undifferentiated- embryonic cell nucleus was
not the only choice. Dolly was created from a mammary gland (udder) cell of a
six-year old ewe. This showed that any adult cell still has the potential to
start from the undifferentiated state and give rise to new differentiated cells
(reversibility of differentiation). To achieve synchronization, the (donor)
mammary cell is deprived of nutrients to stop its cell cycle (Go phase).
The nucleus is then implanted into the egg, and an electrical current is
applied to simulate fertilization which initiates egg activation which normally
occurs at the time of sperm penetration (in parthenogenesis, egg activation is
achieved by physical or chemical stimuli). The egg recombined with a new
nucleus begins dividing and proceeds to become an early embryo (blastocyst).
This is then implanted into the uterus of an ewe (surrogate mother). The lamb
that is born is a clone of the donor. At present, dedifferentiation can only be
achieved by forming an embryo from the donor cell and culturing the embryo to
the stage when it has a few hundred still undifferentiated cells. Then, the
cells would be separated and grown in culture. Cloning, when it becomes more
practical to do, has the potential to make copies of a bred line of cattle,
sheep or other economically important animals without having to do artificial
selection. Another application is to clone genetically modified pigs to use
their organs for xenotransplantation. The modification is required to prevent
has to be remembered that the cloned animal may not be fully identical to the animal who donated the nucleus. This is because of some
epigenetic phenomena (methylation patterns) and cytoplasmic (extra-nuclear)
inheritance. The low success rate in nuclear transfer may be due to a random
loss of correct imprinting. Differentiated female cells, for example, contain
one active X chromosome, but in early female embryos both X chromosomes are
active. It is also a concern that imprinting that results in functional
differences in the male and female genome may complicate the reprogramming of the
genome after nuclear transfer. This may be why out of the 277
mammary-gland-cell nuclei that were fused with enucleated eggs, only 29
developed to the blastocyst stage and only one of those resulted in a live
birth, Dolly, in surrogate mothers. Similarly, it took scientists 87 tries to
successfully clone the first cat ‘CopyCat’ in 2001. Cloning of a
horse in 2003 surprised many scientists because the surrogate mother was the
donor of the egg. Therefore she gave birth to her own clone (Nature,
This time Italian researchers had to try more than 800 fusions. Most failed to
develop from the cleavage stage to blastocysts and at early implantation.
Interestingly, the failure rate was higher in male embryos (8 of 467 males vs
14 of 286 females, meaning three times high failure in male embryos; P =
0.01), which is probably the first concrete documentation of the same
phenomenon known to occur in human pregnancies (see Dorak
MT et al, 2002 for references).
of the totipotency of the first four cleavage cells in the early embryo, a
woman who is suffering from a mitochondrial DNA disease can still have a
healthy baby by having the nucleus of her embryo implanted into a donor oocyte
whose own nucleus has been removed. This is nuclear transfer (from an embryonic
cell) but not cloning. Another development in recent years in reproductive
physiology is the feasibility of ICSI (intracytoplasmic sperm injection) that
enables sterile man to have a child. The bioethical implications of these applications
are discussed elsewhere (see Bioethics). One important and usually neglected piece in discussion of the ethics
of cloning is what will happen in the possible outcome of having a disabled /
malformed cloned offspring. Are the scientists who have done the cloning going
to be held responsible? It appears that cloned mammals usually have a
collection of disorders called large offspring syndrome (LOS). Most cloned
mammals so far have also developed deformities and arthritis (like Dolly).
There is still a long way to go to optimize the conditions of cloning if it
will ever be a common practice.
Suggestion for further reading
Wilmut I: Cloning for medicine. Scientific American 1998 (Dec):30-35
A Student’s Guide to
Biotechnology - Debatable Issues. Greenwood Press, 2002
Rhind et al. Human Cloning: Can it
be made safe? Nature Reviews Genetics 2003 (PDF)
M.Tevfik Dorak, B.A. (Hons), M.D., Ph.D.
Last updated on Dec 7, 2004