Niyati Savur RA1611014010097 RecombinantDNA TechnologyAssignment Gene Therapy Genetherapy is an experimental technique that uses genes to treat or preventdisease. It involves inserting a gene into a patient’s cells instead of usingdrugs or surgery. Several approaches to gene therapy that are being tested are: · Replacing a mutated, diseasecausing gene with a healthy gene. · Inactivating or ‘knocking out’ amutated gene that is functioning properly.
· Introducing a new gene into the bodyto help fight a disease. How does it work? Gene therapy is used to introduce ahealthy copy of a gene into the body to compensate for damaged or abnormalgenes. ØIf a mutated gene produces an abnormal protein, or if the protein ismissing, gene therapy can be used to introduce a normal copy of the gene torestore the function of the protein. ØA gene that is inserted directly into the cell does not function. Acarrier called a vector is genetically engineeredto carry the gene. ØThe vector can be injected or given intravenously (by IV) directly intothe specific tissue, where it is taken up by individual cells. ØAnother method is to expose the patient’s cells to the vector in alaboratory.
The cells are returned to the body. If this is successful, the newgene carried by the vector will produce a functional protein. There are two classes of vectors: 1) Recombinant Vectors: · Also called biological nanoparticles or viral vectors.
· During viral replication, viruses introduce their genetic material intothe host cell.· They then utilize the cell machinery to replicate their DNA. · Retroviruses have their DNA copied into the host cell.
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· This characteristic is exploited by substituting the viral geneticmaterial with the therapeutic DNA. · A number of viruses have been used in human gene therapy, such asretroviruses, adenoviruses, herpes samples, vaccinia and adeno-associatedvirus. 2) Non-Viral Electroporation: An electric field is applied to cells to increase permeabilityof the cell membrane, allowing DNA to enter the cell. The gene gun: Used for delivery ofexogenous DNA to cells, a method known as biolistics. Types of Gene Therapy 1)Somatic Gene Therapy and GermlineGene Therapy. · In somatic gene therapy, genedefects in the somatic cells (such as blood cells) are corrected. Thesecorrections are not passed to the next generation.
· In germline gene therapy, modificationsare made to the sperm and ova (germline cells). These changes will be passed tothe offspring. 3)In vivo and ex vivo genetherapy · One method to introduce the exogenous gene is to isolate the cells so thatthey can be modified in vitro. This way, the cells can be selected and culturedto increase their numbers.
The cell clones are then reintroduced into the body.This treatment is known as ex vivo genetherapy. · In the second method, the gene is directly introduced into the cells bythe mutation. This method is called exvivo gene therapy. · Ex vivo gene therapy is suitable for treating disorders in blood cells.Blood cells are derived from pluripotent stem cells in the bone marrow.
They canbe easily isolated, cultured in vitro and returned to the body. This method hasbeen used in the treatment of adenosine deaminase (ADA) deficiency. · Clinical trials for treatment of Gaucher’s disease, Fanconi’s anaemia andHurler’s syndrome are underway. · Ex vivo treatment is not possible for treating diseases such as cysticfibrosis and muscular dystrophy. · In the first case, epithelial lung cells are difficult to culture in vitroas they divide slowly. Also, these cells are difficult to access.
Anotherreason is that it is difficult to repopulate the lungs of affected individualswith cells cultured in vitro. For these reasons, these disorders can be treated byinjecting the exogenous gene directly into the affected cells, i.e. by in vivotreatment. Thus, muscular dystrophy can be treated by injecting the Dystrophingene into muscle cells, CF by introducing the CTFR gene into cells of theairway, etc. Approaches to Gene Therapy · In straightforward cases, gene therapy involves inserting a functionalcopy of a gene in a specific tissue. · Sometimes this approach is not enough to fix the problem, in which casedifferent methods have to be used. Certain situations thatarise may be: Dominant negative: ØSome mutations in genes lead to production of a dominant negative protein.
ØThis type of protein may block a normal protein from doing its job. ØIn this case, adding a functional copy of the gene will not help becausethe protein will still be there. Gain-of-function: This mutation makes a protein actabnormally.
A gain-of-function mutation activates a protein even when there isno signal, leading to cancer. Improper Regulation: Problems in gene regulations changewhen, where and how much protein is produced. Solutions: 1)Repairing mutations: A few techniques involvereplacing a defective copy of a gene with a healthy one. · The term SMaRT stands forSpliceosome-Mediated RNA Trans-splicing.
· Targets and repairs the Mrna transcripts copied from the mutated gene.· This technique just replaces the part of the mrna that contains themutation instead of replacing the whole gene. 2)Gene splicing: This approach “turns off” a gene sothat no protein is made from it. Gene silencing can target a gene directly orthey can target mrna transcripts from a gene. 3)Triple Helix FormingOligonucleotide Gene Therapy targets the DNA sequence of a mutated gene to prevent itstranscription. Oligonucleotides bind specifically to the groove of doublestranded DNA, preventing its transcription. 4) RNA Interference takes advantage of thecells virus killing machinery.
· A short piece of RNA is introduced. This has a sequence complementary tothe mrna transcript of a gene.· The RNA piece binds to the complementary mrna, forming a double strandedRNA molecule, which is destroyed by the cell since it mimics viral geneticmaterial. 5) Ribozymes are RNA molecules thatact as enzymes. They are designed to target and destroy mrna transcriptsencoded by the mutated gene so no protein can be made from it. 6) Genetically modifying immunecells to target specific molecules:· Immune cells can be modified to recognize a specific antigen using genetherapy. When they are returned to the patient, the modifies cells find anddestroy any cell carrying the antigen.
· Immune cells can also be modified to make certain molecules, such as adrug. When returned to the body, the cells target and destroy specific antigenas well as release the disease-fighting product. Ethical and MoralIssues of gene therapy · Gene therapy raises many ethical concerns because it involves changing thebody’s basic instructions.
· Germline gene therapy is controversial as it may lead to long term sideeffects in the foetus, or it may impact the foetus in unexpected ways. Alsosince the therapy is used to treat unborn individuals, they cannot choosewhether they want to have the treatment or not. · It also raises the question of which disorders or traits require genetherapy to be corrected. The function of many genes and the traits theydetermine are still being studied. It will lead to the question of whether genetherapy is required to treat what is considered an “undesirable” trait.
