GENE THERAPY^J CLINICAL APPLICATION ^0RECENT ADVANCES.pptx

recent gene therapy and its clinical application in various diseases..

1. GENE THERAPY, CLINICAL APPLICATION & RECENT ADVANCES PRESENTED BY- INDRAJIT SAMANTA M.PHARM ( PHARMACEUTICAL CHEMISTRY), 1 ST SEM, 1 ST YEAR DEPARTMENT OF PHARMACEUTICAL CHEMISTRY, SPER, JAMIA HAMDARD, NEW DELHI​

2. Agenda GENE THERAPY 2 Introduction Study purpose of gene therapy TARGET SITE FOR GENE THERAPY GENE THERAPY STRATEGIES TYPE & method OF GENE THERAPY VECTORS INVOLVED IN GENE THERAPY GENE EDTING TOOLS application Gene therapy products in the pharmaceutical market LIMITATIONS OF GENE THERAPY Summary

3. Introduction GENE THERAPY 3 Gene therapy is the use of genetic material(DNA) as a pharmaceutical agent to prevent or cure a disease. Is the insertion of genes into an individual cell and tissue to treat a disease. Gene therapy typically aims to supplement a defective/ mutant allele with a functional one. A functioning copy of gene is packaged into a vector. Modified virus acts as a transport vehicle for functioning gene. The first approved gene therapy experiment occurred on 14 th sept. 1990 in US, when Ashanti Desilva was treated for ADA-CID.

4. Study purpose of gene therapy This is treatment for many disease. For example: Cancers Blood disease Central nervous system disease Immune system disease Cardiovascular disease Muscular dystrophy Huntington’ disease 4 GENE THERAPY

5. HOW IT WORKS? 5 GENE THERAPY

6. TARGET SITE FOR GENE THERAPY 6 GENE THERAPY

7. GENE THERAPY STRATEGIES GENE AUGMENTATION THERAPY (GAT) TARGETED KILLING OF SPECIFIC CELLS TARGETED INHIBITION OF GENE EXPRESSION.

8. GENE THERAPY 8 GENE AUGMENTATION THERAPY (GAT)

9. GENE THERAPY 9 TARGETED KILLING OF SPECIFIC CELLS

10. GENE THERAPY 10 TARGETED INHIBITION OF GENE EXPRESSION

11. ÀPPROCHES FOR GENE THERAPY Different approaches have been tried for effective gene therapy. They are: 1. Gene modification- (A) gene addition, (B) gene correction, (C) gene silencing, and (D) cell elimination techniques. 2. Gene transfer- a) Physical (microinjection, electroporation, etc.) b) Chemical (Liposome, polymer, etc.) c) Biological (Viral and non-viral vector) 3. Gene transfer in specific cell lines- a) Somatic gene therapy b) Germline gene therapy 4. Eugenic approach (gene insertion) 11 GENE THERAPY

12. TYPE OF GENE THERAPY GENE THERAPY 12 SOMATIC CELL GENE THERAPY GERM LINE GENE THERAPY Therapeutic genes transferred into the somatic cells. Therapeutic genes transferred into the germ cells. Eg. Introduction of genes into bone marrow cells, blood cells, skin cells etc. . Eg. Genes introduced into eggs and sperms. Will not be inherited later generations. It is heritable and passed on to later generations At present all researches. directed to correct genetic defects in somatic cells. For safety, ethical and technical reasons, it is not being attempted at present. No ethical issues attached Ethical problems to be answered & precludes its use.

13. METHOD OF GENE THERAPY 13 GENE THERAPY

14. Barriers of gene delivery 14 GENE THERAPY Extracellular Epithelial Barriers: Circulation and The components such as RBC, serum proteins, Enzymes, etc Reticuloendothelial cleaning system, etc. Require inert surface and targeting molecules for specific transportation of complex in the blood Intracellular 1 Cell membrane 2. Endosomal Membrane 3. Nuclear Membrane 4. DNA releasing at Right Site and Right Time. 5. Require cationic groups for successful gene delivery

15. VECTORS INVOLVED IN GENE THERAPY : To transfer the desired gene into a targeted cell a carrier is required. such vehicle of gene delivery are known as vectors. 15 GENE THERAPY b. Adeno viruses c. Adeno associated viruses b. Physicals methods c. Chemical methods

16. viral vectors RNA viruses (Retroviruses ) 1. Murine leukaemia virus ( MuLV ) 2.Human immunodeficiency virus (HIV) 3. Human T-cell lymphotropic viruses (HTLV) DNA viruses 1. Adenoviruses 2. Adeno-associated viruses (AAV) 3. Herpes simplex virus (HSV) 4. Pox viruses 5. Foamy viruses 16 GENE THERAPY

17. Non-viral vectors NON VIRAL VECTOR SYSTEM: Physical methods- 1. Electroporation 2. Microinjection 3. Gene gun 4. Magnetofection Chemical methods- 1. DNA transfer by calcium phosphate method 2.Liposome medicated transfer 17 GENE THERAPY

18. Retrovirus vector system Retrovirus vector system They uses the enzyme reverse transcriptase to convert the RNA into DNA which is integrated into genome further enters the host cell. It have the ability to infect a wide variety of cell type with high efficiency. HERPES SIMPLEX VIRUSES : Herpes viruses includes herpes simplex viruses that rarely can cause encephalitis and infect the non dividing cells so it have the ability to transduce neurons. Its advantage is being able to infect non dividing cells that help in treating neurological disorders. 18 GENE THERAPY

19. Adenovirus vector system The AAV genome comprises of inverted terminal repeats (ITR) as both of the DNA stand and open reading frames.(ORFs). This type of viruses is being uses, because it is non-pathogenic and do not build an immune response. AVV vectors are used to deliver genes to the brain, this is possible because AAV viruses can infect non-dividing cells such as neurons. Advantages of viral vectors- They're very good at targeting and entering cells. Some target specific types of cells. They can be modified so that they can't replicate and destroy cells 19 GENE THERAPY

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21. 21 GENE THERAPY

22. Electroporation Electroporation is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane allowing chemicals, drugs, or DNA to be introduced into the cell. Short pulses of high voltage carry DNA across the cell membrane . This cause temporary formation of pores and thus allow DNA molecules to pass. 22 GENE THERAPY

23. 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 micro-meter. It easily get penetrates into the cell membrane and nuclear envelope. The desired gene is then injected into the sub cellular compartment and needle is removed. Limitation of microinjection costly skilled person required more useful for animal cells. 23 GENE THERAPY

24. Gene Gun Or A Biollistic Particle Delivery System 1. Biollistics or particle bombardment also known as gene gun technique is a physical method that uses accelerated micro projectiles to deliver DNA or a other molecule into intact tissue and cells. 2. The gene gun is a device that literally fit DNA into target cells. 3. The DNA to be transformed into the cell which are coated onto microscopic beads made of either gold or tungsten. 4.The coated beads are then attached to the end of plastic bullets and loaded into the firing chamber of the gene gun. 5. An explosive force fires the bullet with DNA coated beads towards the target cells that lie just beyond the end of the barrel. 6. Some of the beads pass through the cell wall into the cytoplasm of the target cells. 24 GENE THERAPY

25. Chemical Method DNA transfer by calcium phosphate method- The process of transfection involves the mixture of isolate DNA with solution of calcium chloride and potassium phosphate. Cell are then incubated with precipitated DNA eitherin solution or in tissue culture dish. A fraction of cells will take up the calcium phosphate DNA precipitate by endocytosis. 25 GENE THERAPY

26. Chemical Method Liposome medicated transfer- Liposome are spheres of lipids which can be used to transport molecules into cells. These are artificial vesicles that can act as delivery agent for exogenous materials including trans genes. Promote transport after fusing with the cell membrane. Cationic lipids are those having a positive charge are used for the transfer of nucleic acid. 26 GENE THERAPY

27. GENE EDTING TOOLS There are three families of engineered nucleases being used: A. Zinc finger nucleases (ZFNs) B. Transcription Activator-Like Effector-based Nucleases (TALENs) C. CRISPR-Cas system 27 GENE THERAPY

28. Zinc finger nucleases (ZFNs ) 28 GENE THERAPY

29. Transcription Activator-Like Effector-based Nucleases (TALENs) 29 GENE THERAPY

30. CRISPR-Cas system 30 GENE THERAPY

31. 31 GENE THERAPY

32. application GENE THERAPY 32 Retinal degeneration cancers Autoimmune disorder Cardiac disorders Infectious disorders Neurological disorders Designer babies Thalassemia & sickle cell anaemia

33. Severe Combined Immune Deficiency (ADA-SCID ) Severe Combined Immune Deficiency (ADA-SCID) Affected children are born without an effective immune system and will succumb to infections. The disease is caused by a mutation in gene on chromosome 20. The gene codes for the enzyme adenosine deaminase (ADA). 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. The immune system was reconstituted 33 GENE THERAPY

34. Eye diseases Choroideremia is a rare inherited cause of blindness that affects around 1 in 50,000 people. There is currently no cure. It is caused by defects in the CHM gene on the X chromosome. Without the protein produced by the CHM gene, pigment cells in the retina of the eye slowly stop working, then die off. Subretinal injection of adeno-associated virus containing a L-opsin gene. The study implicates the future potential of gene therapy in the treatment of adult colour blindness or even other adult vision disorders. 34 GENE THERAPY

35. gene THERAPY FOR CYSTIC FIBROSIS Cystic fibrosis (CF) is an inherited autosomal recessive disorder. CF affects the epithelial cells lining air passage to the lungs CF affects the epithelial cells lining air passages to the lungs. CF causes a build up of thick mucus in the airways 35 GENE THERAPY

36. LESCH-NYHAN SYNDROME It is an X-linked recessive disorder. It mostly affects male as they contain a single copy of X-chromosome. In females, a mutation must usually be present in both copies of the gene to cause the disorder. There is deficiency of the enzyme Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) or Kelly Seegmiller syndrome that affects the levels of uric acid in the body. Gene therapy involves retroviral transfer of HGPRT gene in lymphocytes. 36 GENE THERAPY

37. SICKEL CELL ANAEMIA The shape of the haemoglobin molecule is controlled by two alleles Normal Haemoglobin allele Sickle Cell Haemoglobin allele 37 GENE THERAPY

38. THALASSEMIA Thalassemia is an inherited autosomal recessive blood disease. In thalassemia, the genetic defect which could be either mutations or deletion results in reduced rate of synthesis or no synthesis of one of the globin a or ẞ- chains that make up haemoglobin. Gene transfer of a regulated ẞ-globin gene in Hemopoietic stem cells (HSCs) would reduce the imbalance between a or B-globin chains in erythroid cells. Transplantation of autologous, genetically corrected HSCs would represent an alternative therapy for thalassaemic patients lacking a suitable bone marrow donor. 38 GENE THERAPY

39. Parkinson’s disease and Alzheimer’s disease Gene therapy for Parkinson’s disease is among the most successful gene therapy studies for neurologic diseases. The first approach attempts to ameliorate Parkinson’s disease by transmitting the gene for glutamic acid decarboxylase into the subthalamic nucleus. Gene therapy for Alzheimer’s disease is attempted by delivery of the Nerve Growth Factor (NGF) gene into human central nervous system. recent in vivo trials utilizing AAV and vector are still in progress. Possibilities of treating Alzheimer’s disease using other factors or other vectors, such as lentiviral vectors, are also under investigation. 39 GENE THERAPY

40. Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) In gene therapy for HIV/AIDS, certain transgenes are transferred into haematopoietic stem cells or into T-cells. in order to confer specific protection against HIV infection to these cells. The transgene could function by inactivating. HIV-1 protein, or simply creating an environment unsuitable for HIV-1 replication. Both clinical T cell and haematopoietic stem cell gene transfer trials have demonstrated promising results 40 GENE THERAPY

41. Gene Therapy For Cancer Gene therapy for treatment of cancer is a rapidly growing field. Oncolytic viruses are employed in the first strategy. In this strategy, researchers attempt to induce the death of malignant cells by introducing specific genes into the malignant cells by oncolytic viruses. Gendicine , a recombinant human 5-adenovirus carrying a human wild-type p53 expression cassette, as the first approved anticancer drug based on gene therapy principle. Suicide-gene therapy, or prodrug activation therapy, is another strategy to treat cancer by means of gene therapy. Currently, a “dendritic cell vaccine” for recurrent prostate cancer has been recommended for approval by US FDA 41 GENE THERAPY

42. Gene Therapy in Treatment of Coronary and peripheral artery diseases The most recent gene therapy study for treatment of coronary artery disease and peripheral artery disease are aimed at patients who were unable to receive conventional therapies. In most of the studies, stimulation of angiogenesis was attempted by injection of angiogenic stimulating gene such as vascular endothelial growth factor (VEGF) gene or fibroblast growth factor (FGF). 42 GENE THERAPY

43. Other genetic diseases including certain muscular dystrophies cystic fibrosis alpha-1-antitrypsin deficiencies Huntington’s disease lysosomal storage diseases chronic granulomatous disease junctional epidermolysis 43 GENE THERAPY

44. Gene therapy products in the pharmaceutical market 44 GENE THERAPY

45. Zalmoxis August 2016 (EU) Graft-versus-host disease Thymidine kinase stimulant; DNA directed DNA polymerase inhibitor invossa July 2017 Knee osteoarthritis Overexpression of transforming growth factor beta-1 Yescarta October 2017 (USA) Relapsed or refractory diffuse large B-cell Lymphoma (DLBCL); non-Hodgkin’s lymphoma; Follicular lymphoma CD19 antagonist Luxturna December 2017 (USA) Leber’s congenital amaurosis; retinitis pigmentosa RPE65 stimulant Onpattro August 2018 (USA) Polyneuropathy of Hereditary Transthyretinmediated amyloidosis in adults Double stranded siRNA that causes degradation of mutant and wild-type TTR mRNA through RNA interference Zynteglo May 2019 (EU) Beta thalassemia Lentiviral vector that inserts a Functioning version of the HBB gene into a patient's blood Givlaari ( givosiran ) November 2019 (USA) Acute hepatic porphyria (AHP) in adults A double stranded siRNA that causes degradation of Aminolevulinate synthase 1 (ALAS1) mRNA in hepatocytes through RNA interference 45 GENE THERAPY Gene therapy drug Authorization date Indication Mechanism of action

46. PROBLEMS WITH GENE THERAPY Efficient gene transfer into target cells (Insertional mutagenesis) Adequate level of transgene expression Persistence of gene expression Regulation of gene expression Tolerance to transgene product Safety 46 GENE THERAPY

47. GENE THERAPY REGULATIONS 47 GENE THERAPY

48. LIMITATIONS OF GENE THERAPY 48 GENE THERAPY

49. LIMITATIONS OF GENE THERAPY 49 GENE THERAPY

50. LIMITATIONS OF GENE THERAPY 50 GENE THERAPY

51. Future of gene therapy Advanced technologies yielding life-changing gene therapies have come with a high price tag, especially in the context of the rare diseases on which they are often focused. The expansion of the cellular gene therapy pipeline in recent years is a primary reason for growing optimism regarding the expectations of industry and patients that some of the most difficult cancers may be treatable in some patients. Gene therapy may gain momentum as the scope stretches beyond genetic disorders. gene therapy may ultimately prove viable in stem cells that could be used to repair tissue function or in sustaining immunity to kill infected cells directly. Gene therapy is theoretically capable of addressing the significant unmet needs of both uncommon inherited diseases and more common infectious or degenerative diseases 51 GENE THERAPY

52. Summary GENE THERAPY 52 The introduction and implementation of gene therapy has opened new doors in the field of Health sciences . But implementing a safe and effective gene therapy has not been so easy due to high cost, finessing appropriate viral system, immune rejection and ethical issues. Safety is always a concern when live vector systems are used for human gene therapy the ideal expression vector for use in humans has yet to be identified. For patients to get assess to these treatments, cost effectiveness should be included in the futuristic therapies.

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54. Thank you