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Gene Therapy : information of gene therapy techniques
- 1. Gene Therapy: informationof gene therapy techniques Content: 1.Gene therapy 2.History 3.Types 4.Vectors 5.Gene delivery 6.Advantages 7.Disadvantages presented by: Pardhe Ayodhya Tanaji M Pharm 1st year (Pharmacology)
- 2. What is gene: Geneis basic physical and functional unit of the heredity. This transfer the genetic information from one generation to next generation. This genes made up of DNA. Gene therapy: Gene therapy is the introduction of genes into existing cells to prevent or cure a wide range of diseases. This technique used for correcting defective genes or either modifying expressions of individual genes which responsible for disease development. Gene therapy can be achieved by using: 1. Replacement of the mutated gene which causes disease with healthy copy of gene 2. Inactivation or knocking out the mutated gene which have not properly functioning 3. Introduction of the new gene into the human body to help the fight against disease.
- 3. History First approvedgene therapy experiment occurred in 14 September 1990 AT THE National Institute of Health under the direction of William French Anderson in US . when 4 year old Ashanti Desilva receive treatment for her genetic defect with ADA- SCID a severe immune deficiency. In 1999 Dr. Claudio bordigon performed first procedure of gene therapy by using hematopoietic stem cells as vectors to deliver genes intended to correct hereditary disease. In 2003 researchers inserted genes into brain for first time. They used liposomes as a vector are small enough to cross blood brain barrier. In 2007 first gene therapy trial for inherited retinal disease. In 2011 medical community accepted it can cure HIV as in 2008 , Gero Hutter has cured a man from HIV using gene therapy. 2011-2015 research treated number of disease by using gene therapy.
- 4. Types of genetherapy 1. Somatic gene therapy 2. Germ line gene therapy Sr. no. Somatic gene therapy Germ line gene therapy 1. Therapeutic genes transfer into the somatic cells Therapeutic gene transfer into the germ cells 2. Introduction of genes into bone marrow cells, skin cells, blood cells Introduction of genes into eggs and sperm 3. Will not be inherited next generation It will be inherited and pass to the next generation 4. At present all researchers directed to correct genetic defect in somatic cells For safety ethical and technical reason it not being attempted at present
- 5. Types of somaticgene therapy 1. In vivo gene therapy: in this direct delivery of genes into cells of particular tissues in the body 2. Ex vivo gene therapy: Transfer of genes to cultured cells and reinsertion
- 6. Ex vivo genetherapy steps Isolate cells with genetic defect from patients Grow in the cell culture Introduce the therapeutic genes Select genetically corrected cells Transplant the modified cells to the patients
- 7. Examples of exvivo gene therapy 1st gene therapy- to correct deficiency of enzyme, adenosine deaminase(ADA). Performed on 4 year old girl Ashanthi desilva. She was suffering from SCID- severed combined immunodeficiency. Caused due to defect in gene coding Deoxy adenosine accumulate and destroy T lymphocytes Disrupts immunity, suffer from infectious disease and die at young age.
- 9. In vivo genetherapy In this direct delivery of Therapeutic gene into target cells into patients body. Viral and nonviral vectors are carried the gene. This gene therapy only possible when patients individual cells cannot cultured invitro sufficient amount. Eg. Brain cells This therapy required when cultured cells cannot reimplement in patients effectively.
- 10. Examples of invivo gene therapy In patients with cystic fibrosis a protein called cystic fibrosis transmembrane regulator (CFTR) is absent due to gene defect. In absence of CFTR chloride ion concentrate within cells and draws water from surrounding. This cause accumulation of sticky mucous in respiratory tract and lungs. This treated by in vivo replacement of defective gene by adenovirus vector.
- 11. Types of vectorsin gene therapy Vector: vector is the DNA molecule that has the ability to self replicate in appropriate host cells and serves as vehicle that carry DNA fragment or insert to be cloned. There are mainly two types of vectors 1)Viral vector a)Retrovirus vector b) adeno virus vector c)Adeno associate virus vector d) Herpes simplex virus vector 2) Nonviral vector a)Pure DNA construct b)Lipoplexes c)DNA molecular conjugates d)Human artificial chromosome
- 12. 1.Viral vectors a)Retrovirus vector: thisrecombinant retrovirus have the ability to integrate into host genome in stable fashion. It carry DNA size is <3.4kb Target cell is dividing b) Adenovirus vector: It does not integrate into the genome and not replicate during cell division. It is good vector when adenovirus with DNA genome. Target: nondividing human cells Eg. Common cold adenovirus
- 13. c) Adeno associatedvirus: It is human virus that can integrate into chromosome 19. It is single stranded, non pathogenic small DNA virus. This virus enter into host cells becomes double stranded and gets integrated into chromosome. d) Herpes simplex virus: Which have natural tendency to infect a particular type of cell They infect and persist in nervous cells.
- 14. 2) Nonviral vectors a)PureDNA construct: Direct insertion of pure DNA construct into target tissue Efficiency of DNA uptake by cells and expression is low b)lipoplexes: This is the lipid and DNA complex In this DNA construct surrounded by artificial lipid layer It degraded by lysosomes c)Human artificial chromosomes: It can carry large DNA that is with one or more therapeutic genes with regulatory elements.
- 15. Methods of genedelivery 1.Physical method a)Gene gun b) Microinjection 2. Chemical methods a)Using detergent mixture b)Lipofection
- 16. 1.Physical methods a)Gene gun: Thisemploys high pressure delivery system to shoot tissue with gold and tungsten particle that coated with DNA. gene gun in gene therapy b) microinjection: In this using the glass micropipette for insertion of microscopic substances into the cells. Performed under optical microscope setup called micromanipulator. microinjection in gene therapy
- 17. 2. Chemical methods a)Usingdetergent mixture: In this calcium phosphate used because it contain charge it mix with functional cDNA of desired function. This mixture is introduced vicinity of recipient cells. The chemicals disturb cell membrane, widens the pore size and allowcDNA to pass through cell. b)lipofection: This technique used to injrct genetic material into cell by liposomes. Liposomes are artificial phospholipid vesicles used to deliver variety of molecules including DNA into cells.
- 18. Advantages of genetherapy It is possible to cure heart diseases Used to eradicate diseases freom the future generation Potential to eliminate hereditary diseases such as cystic fibrosis AIDS and cancer. Improve persons quality of life Prevent the transmission of faulty gene into future generation It can fight an existing cancer and other diseases on molecular level without using therapeutic drugs, radiotherapy It can use out of body treatment action that are safer and efficient Improve body ability to fight disease
- 19. Disadvantages Viruses usedas a vectors for gene transfer may cause toxicity, immune response and inflammatory reaction in host cell. Disorder cause by defect in multiple genes cannot treated effectively by using gene therapy. Long lasting therapy not achieved by gene therapy Random insertion can disturb normal gene New gene might be inserted into wrong location in the DNA Only somatic cells therapy allow
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