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GENE THERAPY. Agustina Setiawati , M.Sc., Apt. DEFINITION. Gene therapy is a technique for correcting “defective” genes responsible for disease development. CYSTIC FIBRIOSIS. Cystic fibrosis (CF) is one of the most fatal heredity
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GENE THERAPY AgustinaSetiawati, M.Sc., Apt
DEFINITION • Gene therapy is a technique for correcting “defective” genes responsible for disease development.
CYSTIC FIBRIOSIS • Cystic fibrosis (CF) is one of the most fatal heredity • It is caused by more than 500 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene • A thick mucus which is a results of: • Alignate produced by bacteria • DNA from lysed cells • Leucocytes which accumulate due to the infection
“ When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders can result”
Several approaches of Gene Therapy • A normal gene may be inserted into a nonspecific location • An abnormal gene could be swapped • The abnormal gene could be repaired through selective reverse mutation
What is reverse mutation? Mutate - to + Mutant, inactive + + - + WT, active Mutate + to - + - - + WT, active Same site revertant WT ? active different site revertant
How Does Gene Therapy Work? Gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. The most common vector is a virus that has been genetically altered to carry normal human DNA
Advantages of Gene Therapy Enabling people to have children where natural conception is impossible (a more effective treatment of infertility). The potential for discovering cures for terrible diseases - leading to less pain and suffering. Sex selection to prevent genetic diseases. Increased availability of organs for transplant. Increased procreative autonomy (choice over some of the genetic characteristics that one's future child will possess).
Disadvantages Gene Therapy • Safety concerns - the risk of detrimental genetic effects (bad biological consequences) • Dangers associated with 'genetic reductionism' - reducing human difficulties to genetic causes and ignoring their social context (EG, disability) • Other potential harms - e.g., the perpetuation of present inequalities/ stereotypes/ power relations, both within countries and between countries, could result,
Type of Human Gene Therapy • Somatic Gene Therapy In vivo & Ex vivo • Germline Gene Therapy
SOMATIC GENE THERAPY GERMLINE GENE THERAPY “ Involve genetic modification of germ cells” “ Transfer material genetic to the body cells except germline”
Ex vivo manipulation techniques • Electroporation • Liposomes • Gold bullets (fired within helium pressurized gun) • Retrotransposons (jumping genes – early days)
In vivo techniques usually utilize viral vectors • Virus = carrier of desired gene • Virus is usually “crippled” to disable its ability to cause disease • Viral methods have proved to be the most efficient to date • Many viral vectors can stable integrate the desired gene into the target cell’s genome
Problem: Replication defective viruses adversely affect the virus’ normal ability to spread genes in the body • Reliant on diffusion and spread • Hampered by small intercellular spaces for transport • Restricted by viral-binding ligands on cell surface therefore cannot advance far.
VECTOR • An adequate carrying capacity • To be undetectable by immune system • Safe to patient • High efficiency
VIRUS as VECTOR • Retrovirus • Adenovirus • Adeno-associated virus • Herpes simplex virus
RETROVIRUS • RNA virus, so it need reverse transcription and integration when enter to the cell • Only divide in rapid proliferation cell (hair, intestine ) • Insert their material genetic in the middle of important gene • Response is reduced by immune response
group-specific antigen (gag) codes for core and structural proteins of the virus polymerase (pol) codes for reverse transcriptase, protease and integrase envelope (env) codes for the retroviral coat proteins.
ADENOVIRUS • DNA virus • Can infect slow proliferating cell, ie: blood, lung, skin • Material genetic can not be integrated to host DNA • Response is reduced by immune response
ADENO-ASSOCIATE VIRUS (AAV) • Occur naturally in human body • DNA virus • Harmless, non pathogenic, do not cause immune response • Insert their material genetic into chromosome 19 (spesific site) • Only 2 genes can be inserted to these vector • Trial to treat muscle and eye disease
Non VIRAL VECTOR • Artificial liposome • Human artificial chromosomes—introducing 47thchromosome to the cell • Chemical linking therapeutic DNA that can bind to specific receptor of cell • Cationic Liposome • Naked DNA (electroporation, gene gun, DNA injection)
Cationik liposome forms complex with DNA (negative charge) • Net charge of the complexes is positive so it can be interact to cell membrane
Human Artificial Chromosome • 47th chromosome • Microchromosome, has only 6- 10 Mb size • Developed after bacterial and yeast artificial chromosome • Grown in HT1080 cell
DNA Transcription Antisense Oligonucleotides mRNA AAAAA Translation Protein
