HumanGermline gene therapy:Introduction:Itis a process of producing changes in the genome of early embryo which is thenincorporated in the whole cells of the body and then transfer to the nextgeneration. Advance in science technologies turned this fanciful sciencefiction into hypothetical and practical possibility. It has some ethicalconcerns such as its possible use for eugenics. The germline gene therapy was first used in animal experiment for thestudy of gene function to make models for the study of human diseases.1 Transgenic animals provides useful toolsfor genetic study but human germline therapy is a doubtful procedure with someclinical usefulness and it may not becost chasing considering the dander postured by its relative usefulness foreugenic improvement.
2 From a medical viewpoint the uses of this technology inthe treatment of genetic disease are limited and more effective substituteprocedures such as preimplantation diagnosis followed by selectivereimplantation will probably available.History and Ethicalissues:Theinitial debate about the human gene therapy was began in 1980s which arousedstrong opposition and some claims that it involve playing God and against thenatural law. A background paper was submitted to the Canadian parliament whichstated that genetic manipulation of human activity is simply not an acceptableactivity.
Then debate focus more on the useful aspects of this treatment.3 In1980 U.S government found that 84% of American supported the geneticmanipulation of human cells to cure genetic diseases. The major arguments which are elevated in the favors of germline gene therapy derivesfrom the ethical principle of beneficence for the newborn whichare up to 2% suffers from genetic defect and some miserable diseases such asLeschNyhan syndrome. Germline gene therapy in some cases is the only way toprevent disease. The Ecological arguments was also against the human germlinegene therapy it clains that human gene pool is a product of thousands andmillions years of carefully balanced evolution and human germline gene therapywill weakend it in unpredictable way. For example due to germline gene therapyloss of unrealized heterogeneous advantages.
It is also claim that germline gene therapy would may also use for theimprovement of human genome for eugenic purposes. In 1987 U.S government foundthat 44% of Americans scientists changing the makeup of human cells to improvehuman intelligence and physical characteristics. In response to this it wasargued that germline genetic alterations are different classical eugenics inthat they remove the defective gene rather than human gene and multifactorialtraits like intelligence may never prove agreeable to manipulation.
4 The wayto grab this problem as suggested by Inuyama Declaration of the Council forInternational Organizations of Medical Sciences, is to assume agreement only incases where genetic involvement would seem clearly acceptable to the affectedfuture generations.Human gene therapyprotocol:Manysteps are involves in human gene therapy protocol;1. Isolation of totipotent embryoniccell at an undifferentiated stage:Naturallyfertilized ovum recover by laparoscopic flushing before implantation or invitro fertilization. Both of these strategies are possible in human but areexpensive and take months or year for repeated attempts.5 It is preferablemorally to manuplate gametes instead of embryo but this is not technicallypossible.2. Determination of the genetic stateof the embryo:preimplantationdiagnosis is already available on experimental basis.
If normal embryo can beseparated from abnormal embryo the simplest option is selective reimplantationwhich removes the need for genetic manipulation. In some cases step 2 isskipped in which all the embryos will be suffered and it is not necessary stepin improvement procedure.3. Expansion of embryonic stem cellsin culture:Thisstep is difficult to attain in human because success rate is low andmaintenance of such culture is also difficult, it is expensive and requireskilled labour. Normal karyotypes havelimited lifespan in culture X chromosome are lost in culture and only maleembryo could be successfully treated.
64. Transfer of genetic material intoembryonic cells:Millionsof cells are transfected to get only one targeted recombinant. The avaliblemethod for transfection have limited efficiency which kill some of the targetedcells and cause undesired DNAintegration and recombinant event.5. Selection of cells which havestably taken up the transfected gene:Thisfrequency is about 1 per 10,000, the antibiotic resistant marker fortransformed selection was also added in transfected DNA after a week of growthin a presence in drug selection.
Normal gene is added to replace the defectivegene by recombination of genome.76. Targeted gene replacement:Thisstep cure the genetic truly, deleterious DNA base pair are replace with normalsequence in chromosomal position for the proper function of gene product. Formammalian cells over 1000 homologous recombination events are require for thereplacement of target gene. Distinguishing targeted gene replacement events isdifficult because cell have taken up the selectable marker attached to thetransfected DNA.
8Scientist now form a transfection vector which increase theproportion of targeted recombinant but there is no way currently available toinduce 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 themodified locus may effects the neighbouring genes in undesired ways and itshould 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 geneand remaining genome completely unaltered except the target sequence change.This needs another round of drug selection and reexpansion of culture.8. Confirming genomic integrity:Anembryonic cell lines with desire embryonic changes has been established. Indirect mutations are commonly induced intarget regions treated by homologous recombination. It is necessary to confirmthe genomic integrity of cells line before implantation in the embryo.
Thisstep is expensive and difficult. Months or years of growth in culture arerequired to reach this point and the entire cell should be alive for nucleartransfer.9. Nuclear transfer:Wilmut cloned sheep from cultured cells arrested in G0 phase of cell cycle makethis step hypothetically possible in humans. Unfertilized ova will need to berecovered from superovulated woman or donor. The nucleus of the cell was removeand replace with nucleus of donor cell and embryo grow in the cuture which contain modified human genome this step isalso expensive and difficult.10.
Reimplantationinto the mother:98%of the embryo lost roughly this is the limiting factor in human in vitrofertilizations. Following natural abortion is normal and only 15% in vitrofertilization have chances of successful pregnancy. To increase the chance of success manyembrtos are reimplanted at a time and would may result in twins or triplets.Techniquesfor gene transfer:Manytechniques are used for gene transfer some are elaborate here;1. Electroporation:cellsare mixed with DNA solution and connected with electric current.
Current pulsescreates 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 anyother technique. Electroporation allow the integration of gene into thechromosome of host cell.2. DNA Microinjection:It is use to deliver the foreign gene into themouse embryo at the early stage of development DNA is mostly injected in themale pronucleus of fertilized mouse egg. Embryo is then implanted into the fertilized foster mother. The foreigngene is inserted into the every cell’s chromosome and present in every cell ofthe 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 Cell fusion:It is a technique in which two cells are fuse which have some common traits. itis basically use for the production of monoclonal antibody. These monoclonalantibody cell lines are formed by fusion with animal myeloma cells which couldnot produce antibodies.
10 The antibodies obtained from this culture was pureand use in agricultural and medicinal purposes. Diagnostic kits and humanvaccines are based on monoclonal antibodies.