Germline gene therapy:
is a process of producing changes in the genome of early embryo which is then
incorporated in the whole cells of the body and then transfer to the next
generation. Advance in science technologies turned this fanciful science
fiction into hypothetical and practical possibility. It has some ethical
concerns such as its possible use for eugenics.
The germline gene therapy was first used in animal experiment for the
study of gene function to make models for the study of human diseases.1
Transgenic animals provides useful tools
for genetic study but human germline therapy is a doubtful procedure with some
clinical usefulness and it may not be
cost chasing considering the dander postured by its relative usefulness for
eugenic improvement.2 From a medical viewpoint the uses of this technology in
the treatment of genetic disease are limited and more effective substitute
procedures such as preimplantation diagnosis followed by selective
reimplantation will probably available.
History and Ethical
initial debate about the human gene therapy was began in 1980s which aroused
strong opposition and some claims that it involve playing God and against the
natural law. A background paper was submitted to the Canadian parliament which
stated that genetic manipulation of human activity is simply not an acceptable
activity. Then debate focus more on the useful aspects of this treatment.3 In
1980 U.S government found that 84% of American supported the genetic
manipulation of human cells to cure genetic diseases.
The major arguments which are elevated in the favors of germline gene therapy derives
from the ethical principle of
beneficence for the newborn which
are up to 2% suffers from genetic defect and some miserable diseases such as
LeschNyhan syndrome. Germline gene therapy in some cases is the only way to
prevent disease. The Ecological arguments was also against the human germline
gene therapy it clains that human gene pool is a product of thousands and
millions years of carefully balanced evolution and human germline gene therapy
will weakend it in unpredictable way. For example due to germline gene therapy
loss of unrealized heterogeneous advantages.
It is also claim that germline gene therapy would may also use for the
improvement of human genome for eugenic purposes. In 1987 U.S government found
that 44% of Americans scientists changing the makeup of human cells to improve
human intelligence and physical characteristics. In response to this it was
argued that germline genetic alterations are different classical eugenics in
that they remove the defective gene rather than human gene and multifactorial
traits like intelligence may never prove agreeable to manipulation.4 The way
to grab this problem as suggested by Inuyama Declaration of the Council for
International Organizations of Medical Sciences, is to assume agreement only in
cases where genetic involvement would seem clearly acceptable to the affected
Human gene therapy
steps are involves in human gene therapy protocol;
1. Isolation of totipotent embryonic
cell at an undifferentiated stage:
fertilized ovum recover by laparoscopic flushing before implantation or in
vitro fertilization. Both of these strategies are possible in human but are
expensive and take months or year for repeated attempts.5 It is preferable
morally to manuplate gametes instead of embryo but this is not technically
2. Determination of the genetic state
of the embryo:
diagnosis is already available on experimental basis. If normal embryo can be
separated from abnormal embryo the simplest option is selective reimplantation
which removes the need for genetic manipulation. In some cases step 2 is
skipped in which all the embryos will be suffered and it is not necessary step
in improvement procedure.
3. Expansion of embryonic stem cells
step is difficult to attain in human because success rate is low and
maintenance of such culture is also difficult, it is expensive and require
skilled labour. Normal karyotypes have
limited lifespan in culture X chromosome are lost in culture and only male
embryo could be successfully treated.6
4. Transfer of genetic material into
of cells are transfected to get only one targeted recombinant. The avalible
method for transfection have limited efficiency which kill some of the targeted
cells and cause undesired DNA
integration and recombinant event.
5. Selection of cells which have
stably taken up the transfected gene:
frequency is about 1 per 10,000, the antibiotic resistant marker for
transformed selection was also added in transfected DNA after a week of growth
in a presence in drug selection. Normal gene is added to replace the defective
gene by recombination of genome.7
6. Targeted gene replacement:
step cure the genetic truly, deleterious DNA base pair are replace with normal
sequence in chromosomal position for the proper function of gene product. For
mammalian cells over 1000 homologous recombination events are require for the
replacement of target gene. Distinguishing targeted gene replacement events is
difficult because cell have taken up the selectable marker attached to the
transfected DNA. 8Scientist now form a transfection vector which increase the
proportion of targeted recombinant but there is no way currently available to
induce high level of homologous recombination
which remain at the terribly low rate of around 1 per 10 million cells transfected.
7. Marker removal:
Unused marker genes in the locality of the
modified locus may effects the neighbouring genes in undesired ways and it
should not the part of the every cell in the person or pass to next generation.
So the molecular scientist developed the strategies to remove the marker gene
and remaining genome completely unaltered except the target sequence change.
This needs another round of drug selection and reexpansion of culture.
8. Confirming genomic integrity:
embryonic cell lines with desire embryonic changes has been established. Indirect mutations are commonly induced in
target regions treated by homologous recombination. It is necessary to confirm
the genomic integrity of cells line before implantation in the embryo. This
step is expensive and difficult. Months or years of growth in culture are
required to reach this point and the entire cell should be alive for nuclear
9. Nuclear transfer:
Wilmut cloned sheep from cultured cells arrested in G0 phase of cell cycle make
this step hypothetically possible in humans. Unfertilized ova will need to be
recovered from superovulated woman or donor. The nucleus of the cell was remove
and replace with nucleus of donor cell and embryo grow in the cuture which contain modified human genome this step is
also expensive and difficult.
into the mother:
of the embryo lost roughly this is the limiting factor in human in vitro
fertilizations. Following natural abortion is normal and only 15% in vitro
fertilization have chances of successful pregnancy. To increase the chance of success many
embrtos are reimplanted at a time and would may result in twins or triplets.
for gene transfer:
techniques are used for gene transfer some are elaborate here;
are mixed with DNA solution and connected with electric current. Current pulses
creates pores in cell membrane and allow the DNA to enter the cell.
Electroporation can work with any type of cell which resisted DNA uptake by any
other technique. Electroporation allow the integration of gene into the
chromosome of host cell.
2. DNA Microinjection:
It is use to deliver the foreign gene into the
mouse embryo at the early stage of development DNA is mostly injected in the
male pronucleus of fertilized mouse egg.
Embryo is then implanted into the fertilized foster mother. The foreign
gene is inserted into the every cell’s chromosome and present in every cell of
the mature organism. The animal with foreign gene is called transgenic animal.
This gene is than transfer into the next generation as a normal gene.9
It is a technique in which two cells are fuse which have some common traits. it
is basically use for the production of monoclonal antibody. These monoclonal
antibody cell lines are formed by fusion with animal myeloma cells which could
not produce antibodies.10 The antibodies obtained from this culture was pure
and use in agricultural and medicinal purposes. Diagnostic kits and human
vaccines are based on monoclonal antibodies.