Gene therapy

Gene therapy , Nucleic acids , GENES , ADA-SCID(Severe combined immunodeficiency) , APPROACHES IN GENE THERAPY : Types of gene therapy , somatic gene therapy , germ line gene therapy , Gene modification , gene replacement , gene correction , gene augmentation , Gene transfer methods , viral gene transfer (biological) , non viral gene transfer : physical method ,chemical method , retrovirus vector system , adenovirus vector system , Adeno associated virus vector , Herpex simplex virus vector , Electroporation , Microinjection , Gene Gun , Calcium phosphate mediated DNA transfer , Liposome mediated gene transfer , Potential target diseases for Gene Therapy , GENE THERAPY USED TO TREAT TYPE I DIABETES , GENE THERAPY FOR CANCER TREATMENT , PARKINSON’S DISEASE , GENE THERAPY TREATMENT FOR ADA-SCID , GENE THERAPY FOR CYSTIC FIBROSI S, HEMOPHILIA , BLINDNESS
Gene therapy , Nucleic acids , GENES , ADA-SCID(Severe combined immunodeficiency) , APPROACHES IN GENE THERAPY : Types of gene therapy , somatic gene therapy , germ line gene therapy , Gene modification , gene replacement , gene correction , gene augmentation , Gene transfer methods , viral gene transfer (biological) , non viral gene transfer : physical method ,chemical method , retrovirus vector system , adenovirus vector system , Adeno associated virus vector , Herpex simplex virus vector , Electroporation , Microinjection , Gene Gun , Calcium phosphate mediated DNA transfer , Liposome mediated gene transfer , Potential target diseases for Gene Therapy , GENE THERAPY USED TO TREAT TYPE I DIABETES , GENE THERAPY FOR CANCER TREATMENT , PARKINSON’S DISEASE , GENE THERAPY TREATMENT FOR ADA-SCID , GENE THERAPY FOR CYSTIC FIBROSI S, HEMOPHILIA , BLINDNESS

1. Gene therapy Presented by P.Pavazhaviji M.Pharm I Year (II Sem ) Dept. of Pharmaceutics MTPG & RIHS Puducherry

2. NUCLEIC ACID Nucleic acids  are large molecules where genetic information is stored. There are two types of  nucleic acids : deoxyribonucleic  acid( DNA) and ribonucleic   acid( RNA). 2

3. GENES Are the basic unit of heredity carried on a chromosome. Gene is a segment of DNA that contains the instruction for making a particular protein . Mutation of Genes Results in- Failure to synthesize a particular protein. Synthesis of a defective Protein. These results in abnormalities Recognized as genetic disorders. G enetic disorders can be treated by Gene Therapy 3

4. GENE THERAPY Gene therapy can be defined as an experimental technique for correcting defective genes Inserting a normal gene to replace an abnormal gene 4

5. GENE THERAPY The first attempt, an unsuccessful one, gene therapy was performed by Martin Cline on 10 July 1980 for treating beta-thalassemia. The first approved gene therapy clinical research in the US took place on 14 September 1990, at the National Institutes of Health (NIH), under the direction of  William French Anderson . Four-year-old  Ashanti   DeSilva received treatment for a  genetic defect  that left her with ADA-SCID(Severe combined immunodeficiency). 5

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7. APPROACHES IN GENE THERAPY A. T ypes of gene therapy somatic gene therapy germ line gene therapy B . Gene modification gene replacement gene correction gene augmentation C . Gene transfer methods viral gene transfer (biological) non viral gene transfer : physical method chemical method 7

8. A) TYPES OF GENE THERAPY 8

9. TYPES OF SOMATIC GENE THERAPY 9

10. IN VIVO GENE THERAPY 10

11. EX VIVO GENE THERAPY 11

12. B) GENE MODIFICATION 1 ) GENE REPLACEMENT: Removal of a Mutant Gene sequence from the Host Genome and its replacement with a normal Functional Gene . 2) GENE CORRECTION: Involves only defective portion of a mutant gene which is altered to provide the Functional Gene . 3) GENE AUGMENTATION: D efective gene is modified by introducing a normal genetic sequence into Host Genome without altering the defective one. 12

13. C) Gene transfer methods To transfer the desired gene into a target cell, a carrier is required Such vehicles of gene delivery are known as vectors Two main classes Viral vectors Non viral vectors 13

14. 14 1) VIRAL GENE TRANSFER (BIOLOGICAL)

15. a) retrovirus vector system The First used ones Retroviruses are diploid, single-stranded, circular enveloped RNA viruses Retroviruses cause diseases such as AIDS, leukemia, and cancer Retroviruses are viruses that integrate with host genome to produce viral proteins that are extracted during gene delivery. USE- In ex-vivo therapy 15

16. b ) adenovirus vector system: Adenoviral vectors have a wide range of action and are able to deliver nucleic acids to both dividing and non-dividing cells . Adenoviruses are often responsible for respiratory, gastrointestinal and eye infection that affect humans. 16

17. c ) Adeno associated virus vector: It is a single stranded, non pathogenic small DNA virus. AAV enters host cell, becomes double stranded and gets integrated into chromosome . d ) Herpex simplex virus vector: Viruses which have natural tendency to infect a particular type of cell . Double stranded DNA virus that attack neurons (neurotropic) Helpful for gene delivery in CNS conditions 17

18. 2) NON VIRAL VECTORS PHYSICAL METHODS: Electroporation: T he cells are placed in a solution containing DNA and subjected to electrical pulse to cause holes in the membrane The foreign DNA fragments enter through holes into the cytoplasm and then to nucleus . Advantage -Simple and technically easy. Disadvantage- High risk of contamination. 18

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20. Microinjection The microinjection is the process of transferring the desirable DNA into the living cell , through the use of glass micropipette. Glass micropipette is usually of 0.5 to 5 micrometer, easily penetrates into the cell membrane and nuclear envelope . Advantage -High efficient Disadvantage -Only limited no of cell can be transformed at a time. 20

21. Gene Gun In this method DNA is coated with gold particles and loaded into a device , which is similar to gun and it generates force by which it can penetrate into cells. 21

22. b. CHEMICAL METHOD Calcium phosphate mediated DNA transfer The process of transfection involves the admixture of isolated DNA (10-100ug) with solution of calcium chloride and potassium phosphate so precipitate of calcium phosphate to be formed . Cells are then incubated with precipitated DNA either in solution or in tissue culture dish. A fraction of cells will take up the calcium phosphate DNA precipitate by endocytosis. 22

23. Liposome mediated gene transfer Liposomes are spheres of lipids which can be used to transport molecules into the cells. These are artificial vesicles that can act as delivery agents for exogenous materials including transgenes. Promote transport after fusing with the cell membrane. Cationic lipids due to their positive charge, used to condense negatively charged DNA so as to fascilitate encapsulation of DNA into liposomes endocytosis of liposomes followed by lysis releases DNA into cytoplasm 23

24. Potential target diseases for Gene Therapy 24

25. Type I diabetes is caused by the destruction of insulin-producing pancreatic β cells by an inappropriate autoimmune response . Scientists used a recombinant adeno -associated virus ( rAAV ) to insert a INS gene that results in the expression of a single-chain insulin analogue (SIA) into streptozotocin -induced diabetic rats. GENE THERAPY USED TO TREAT TYPE I DIABETES 25

26. GENE THERAPY USED TO TREAT TYPE I DIABETES First, the gene was cloned under the L-type Pyruvate Kinase (LPK) promoter, which regulates the expression of SIA in response to glucose levels. The LPK- gene was then attached to a recombinant adeno -associated virus and integrated into the host chromosomal DNA. After insertion of the rAV -LPK-gene , the rats displayed a drop in glucose levels that reached a range of normoglycaemia within one week of treatment. This form of gene therapy may provide a cure for type I diabetes for humans in the future . 26

27. The adenovirus containing the p53 tumor suppressor gene binds to the receptor in the cell membrane of the cancer cell. The vesicle breaks down , releasing the adenovirus near the cell nucleus. The adenovirus injects its gene , which contain the p53 tumor suppressor gene , into the cell nucleus. The cancer ell then makes p53 protein The p53 protein causes the cancer cell to self destruct without affecting surrounding normal cells . GENE THERAPY FOR CANCER TREATMENT: 27

28. PARKINSON’S DISEASE Deliver the GAD gene into patient’s subthalamic nucleus. The gene instructs cells to begin making GABA neurotransmitters to re-establish the normal chemical balance that becomes dysfunctional as the disease progresses. 28

29. GENE THERAPY TREATMENT FOR ADA-SCID : ADA-SCID( Severe Combined Immune Deficiency) is also known as the bubble boy disease . Affected children are born without an effective immune system and will succumb to infections outside of the bubble without bone marrow transplantation from matched donors. The therapeutic gene called ADA was introduced into the bone marrow cells of such patients in the laboratory, followed by transplantation of the genetically corrected cells back to the same patients. 29

30. GENE THERAPY FOR CYSTIC FIBROSIS In patients with cystic fibrosis, a protein called cystic fibrosis transmembrane regulator (CFTR) is absent due to a gene defect. In the absence of CFTR chloride ions concentrate within the cells. This leads to the accumulation of sticky mucous in respiratory tract and lungs. Main symptom--- Difficulty in breathing . Treated by in vivo replacement of defective gene by adenovirus vector. 30

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33. HEMOPHILIA Autosomal recessive disorder due to absence of factor VIII leading to bleeding tendencies Patients born with Hemophilia are not able to induce blood clots and suffer from external and internal bleeding that can be life threatening. In a clinical trial conducted in the United States , the therapeutic gene was introduced into the liver of patients, who then acquired the ability to have normal blood clotting . 33

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35. BLINDNESS Leber's Congenital Amaurosis (LCA) is a rare inherited eye disease that appears at birth or in the first few months of life Researchers at Moorfields Eye Hospital and University College London in London conducted the first gene therapy clinical trial for patients with RPE65 LCA. 35

36. ADVANTAGES Gene therapy has the potential to eliminate and prevent hereditary disease such as cystic fibrosis, ADA-SCID etc . It is possible to cure heart disease, AIDS and cancer . It gives someone born with a genetic disease a chance to life . It can be used to eradicate diseases from the future generations. 36

37. DISADVANTAGES Immune response to the transferred gene stimulates a potential risk to gene therapy . Viruses used as vectors for gene transfer may cause toxicity, immune responses, and inflammatory reactions in the host . Disorders caused by defects in multiple genes cannot be treated effectively using gene therapy. 37

38. REFERENCES: Nejm Gene therapy and novel drug delivery page 1-36 Intechopem Gene therapy and viral and nonviral vectors 387-402 Japi.org Human gene therapy 1-17 Anderson Germ line therapy spring 2013 1-25 Nptel.ac.in gene therapy page 1-24 38

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