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Medicinal
Applications
of
Genetic
Engineering |
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Medicinal
Applications
of
Genetic
Engineering -Among the medical applications of genetic engineering are the production of hormones, vaccines, interferon; enzymes, antibodies, antibiotics and vitamins, and in gene therapy for some hereditary diseases.
1. Hormones. The hormone insulin is currently produced commercially by extraction from the pancreas of cows and pigs. About 5% of the patients, however, suffer from allergic reactions to animal-produced insulin because of its slight difference in structure from human insulin
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Human insulin genes have been implanted in bacteria which, therefore, become capable of synthesizing insulin. Bacterial insulin is identical to human insulin, since it is coded by human genes. Diabetics have been receiving bacterial insulin in test programmes, and it appears to be as effective as insulin from animal sources.
Insulin
is
secreted
by
the
beta
cells
of
the
islets
of
Langerhans
in
the
pancreas.
The
beta
cells
first
synthesize
a
preproinsulin
molecule
of
109
amino
acids.
The
first
23
amino
acids
of
preproinsulin
serve
as
a
signal
for
the
passage
of
the
molecule
through
the
cell
membrane
these
are
cleaved
to
produce
proinsulin
which
consists
of
86
amino
acids.
The
central
part
of
the
proinsulin
molecule
is
cut
out
by
enzymes,
leaving
two
chains
which
comprise
insulin.
One
chain
consists
of
20
amino
acids
and
the
other
of
30
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Preproinsulin
(109
amino
acids)
↓
Pro
insulin
(86
amino
acids)
↓
Insulin
↓
20
amino
acids and 30
amino
acids
- Beta
Chaintakura
and
his
associates
synthesized
two
DNA
strands
coding
the
two
chains
of
human
insulin.
The
strands
were
then
separately
attached
to
bacterial
DNA.
Two
different
hybrid
proteins
in
two
different
bacteria
were
synthesized.
The
two
short
pieces
were
cut
off,
purified,
and
then
joined
together
to
produce
insulin.
Gilbert
and
Villa
Komaroff
obtained
rat
insulin
from
a
hybrid
protein
composed
of
a
part
of
bacterial
penicillinase
and
proinsulin.
They
first
isolated
beta
cell
mRNA
and
made
DNA
copies
from
it.
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The
DNA
(proinsulin
gene)
was
then
inserted
in
the
middle
of
the
bacterial
penicillinase
gene
in
the
plasmid
pBR322,
which
served
as
a
cloning
vehicle.
(Penicillinase
is
secreted
through
the
bacterial
cell
membrane).
The
proinsulin
gene
was
inserted
in
the
penicillinase
gene
between
the
two
cleavage
sites
of
the
restriction
enzyme
Pst.
The
recombinant
plasmids
were
put
into
E.
coli
cells
and
cloned.
The
clone
synthesizes
hybrid
protein
consisting
of
most
of
the
penicillinase
and
proinsulin.
Most
of
the
penicillinase
and
the
middle
segment
of
the
proinsulin
were
cut
away
by
trypsin
to
produce
biologically
active
insulin.
The
radioactive
antibody
test
was
used
to
locate
from
clones
containing
insulin
DNA
any
clone
that
was
synthesizing
a
hybrid
protein.
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A
plastic
disc
coated
with
an
antibody
against
insulin
or
penicillinase
was
applied
against
the
clones.
Insulin
penicillinase
present
in
any
of
the
clones
became
fixed
to
the
antibody/on
the
disc.
Radioactively
labelled
anti
insulin
antibody
was
then
applied
to
detect
the
presence
of
proteins
with
insulin
shapes.
One
clone
gave
positive
results
to
the
anti
insulin
antibody
for
discs
coated
with
anti
insulin
as
well
as
anti
penicillinase.
This
proved
that
a
hybrid
protein
consisting
of
penicillinase
+
insulin
was
being
synthesized
in
the
clone.
The
clone
was
propagated
in
liquid
culture,
and
the
hybrid
protein
was
located
outside
the
membrane.
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In 1977 Herbert Boyer created E.coli capable of synthesizing somatostatin, the human growth hormone of the brain hypothalamus. Formerly 500,000 sheep brains were required to produce 5 mg of the hormone. Boyer's bacterium can produce the same quantity of the hormone for the cost of about $ 2.50. somatostatin is a small hormone consisting of 14 amino acids coded by a DNA sequence of 42 bases., Itakura and his co-workers chemically synthesized a DNA molecule with the ,required base sequence for coding somatostatin
A bacterial gene was cut open with a restriction enzyme, and the DNA sequence for the 14 amino acids, followed by a stop signal was inserted. A hybrid protein was synthesized from which the somatostatin part was cutoff chemically and purified
The hormone thymosin alpha-I, which may prove to be effective against brain and lung cancer, has been produced by genetically engineered microorganisms. Beta-endorphin, one of the pain killers produced by the brain, has also been produced by gene-splicing techniques
2. Vaccines. Injecting an animal with an inactivated virus stimulates it into making antibodies against viral proteins. These antibodies protect the animal against infection by the same virus by binding to the virus. Phagocytic cells then remove the virus. Vaccines are manufactured by growing the disease-producing organism in large amounts. This process is often dangerous or impossible. Moreover, there are difficulties in making the vaccine harmless. Recombinant DNA techniques permit the insertion of specific genes into bacteria so that they produce only the proteins against which the antibody response is required
The foot and mouth disease is a viral disease affecting cattle, goats, pigs and deer. It causes blisters on the feet, tongue and mouth,lameness, loss of weight, and, in dairy cows, reduced milk production. The disease is highly contagious, and often an entire herd of cattle must be destroyed to prevent it, even if only one animal is affected.
Although vaccines are available against the disease, there is a slight risk that they can actually cause the disease because they sometimes contain live viruses. Genentech Inc., in collaboration with the U. S. Dept. of Agriculture scientists, has now 'produced a safe effective vaccine against the foot and mouth disease. The virus causing the disease consists of a nucleic acid core covered with a coat of four proteins. One of these proteins.VP3, creates immunity without causing infection, in test animals. The VP3 gene was isolated from the virus and spliced into a plasmid from E.coli. The offspring of this bacterium now produced VP3. Vaccines against malaria and hepatitis may also be- produced in the near future.
3. Interferon. Interferons are virus induced proteins produced by cells infected with viruses. They appear to be the body's first line of defence against viruses. The interferon response is much quicker than the antibody response. Interferons are anti-viral in action. One type of interferon can act. against many different viruses, i.e. it is not virus specific. It is, however, species specific. Interferon from one organism does not give protection against viruses to cells of another organism
Interferon provides natural defence against such viral diseases as hepatitis and influenza. It also appears to be effective against certain types of cancer, especially cancer of the breast and lymph nodes. Natural interferon is collected from human blood cells and other tissues. It is produced in very small quantities. Each daily injection costs upto $ 150. If bacterially produced interferon is found to have no harmful reaction, it may become available for as low as $1 per injection. In 1980 Biogen S. A. produced the first recombinant. DNA induced interferon like human protein. Genentech is offering several types of interferon, one of which is undergoing clinical tests
Weissmann and his associates have produced interferon by recombinant DNA methods. Messenger RNA for interferon was taken from WBC stimulated by viral infection. Complementary DNA transcribed by the mRNA was converted into double stranded DNA, which was then inserted into vector DNA and cloned. Some 20,000 c1ones were examined for the presence of interferon
This was done as follows. Plasmid DNA from the clones was hybridized to mRNA from the WBC. mRNA was then isolated from the DNA-RNA hybrids and injected into frog's eggs to test whether it directed the synthesis of interferon. Once the desired clones were obtained they were tested to see whether they produced interferon. Interferon DNA was inserted into the penicillinase gene of bacteria, and the product was found to be biologically active.
4. Enzymes. The enzyme urokinase, which is used to dissolve blood clots, has been produced by genetically engineered microorganisms
5. Antibodies. One of the aims of genetic engineering is the production of hybridomas. These are long lived cells that can produce antibodies for use against disease
6. Gene therapy for treating hereditary diseases. The earlier gene transplantation experiments were concerned with transplanting genes in vitro into isolated cells or into bacteria. Gene transplantation experiments have now been extended to living animals. In 1980 Ruddle and his c01leagues inserted into adult mice a gene conferring resistance to a specific drug.
They isolated genes from two viruses and cloned them in large quantities. 1,000 to 20,000 copies of the, genes were then microinjected by capillary tubes into the nuclei of living, freshly fertilized mouse eggs kept in laboratory dishes. The eggs were then transferred into the uteri of female mice, where they underwent development. DNA was extracted from the tissues of 150 new born mice. Parts of the viral genes were found in two of the mice
The experiment demonstrates that it may be possible in the future to alter the genetic material in the human egg. This could lead to the elimination of inherited diseases like haemophilia, Tay-Sachs disease and phenylketonuria. There are, however, still a number of problems to be solved before this is achieved
It is not known how the transplanted genes will function in their host cells. Will they be inactivated or modified as a result of interaction with the genetic machinery of the host cell? Will the genes become attached to host cell chromosomes, or will they float freely in the cell? Will genes that are, activated only in particular cell types behave in the same Way after transplantation? And, finally, will the transplanted genes be inherited? In 1980 Kline and Salser and their associates isolated genes that code for an enzyme resistant to methotrexate
This drug is used to treat leukemia and other types of cancer, as, it destroys rapidly dividing cells. It, however, affects both malignant and healthy cells, like the cells of the bone marrow that produce R. B. C. Its effectiveness is, therefore, restricted because of the damage it causes to bone marrow. Kline and Salser added methotrexate resistance conferring genes to mouse bone marrow cell culture.
These cells, along with cells without methotrexate resistance genes, were injected into mice whose bone marrow had been destroyed. The mice were then treated with methotrexate. After two months it was found that the newly developed bone marrow consisted mostly of cells carrying the gene for methotrexate resistance. If methotrexate resistance can be introduced into human bone marrow, cancer patients can be, given more intensive treatment with methotrexate
The technique could also be used to cure blood diseases like thalassemia and sickle-cell anaemia, which result from defects in single genes. Non-defective genes could be transferred into the bone marrow along with the methotrexate-resistance genes. Treatment with methotrexate would then destroy the cells containing defective genes and would permit the non-defective cells to form bone marrow
Kline and Salser have extended genetic engineering techniques to humans, in an experiment that has become controversial. Two terminally ill patients suffering from beta zero thalassemia, a fatal inherited blood disease, were used for the experiment. In this disease one of the protein components of haemoglobin is missing. Genes that could produce the missing protein component were first cloned. Small amounts of bone marrow cells were taken out from the patients, and the cloned genes were put in the marrow. The marrow was then put back into the patients
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