Gene therapy

This will provide you information about gene therapy, its types, methods of gene delivery and some potential target diseases for gene therapy.

1. PRESENTATION ON GENE THERAPY Presented to: Himanshi Ma'am Presented By: Vikas Dagar R oll N o : 1 9 01212200 10 M. P harma (P’C eutics ) 2 ND S emester DEPARTMENT OF PHARMACEUTICAL SCIENCES, GURU JAMBHESHWAR UNIVERSITY OF SCIENCES & TECHNOLOGY, HISAR

2. CONTENTS INTRODUCTION GENE THERAPY What are Genetic Disorders? Several approaches to gene therapy Why gene therapy? HISTORY OF GENE THERAPY TYPES OF GENE THERAPY IN VIVO GENE THERAPY EX VIV O GENE THERAPY VECTORS IN GENE THERAPY METHOD OF GENE DELIVERY IN PHYSICAL METHOD CHEMICAL METHOD POTENTIAL TARGET DISEASES ADVANTAGES DISADVANTAGES

3. INTRODUCTION Gene therapy is the introduction of genes into existing cells to prevent or cure a wide range of diseases. It is a technique for correcting defective genes responsible for disease development . The first approved gene therapy experiment occurred on september 14,1990 in US, when Ashanti DeSilva was treated for ADA-SCID.

4. GENE THERAPY Gene therapy is experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells inserted of using drugs or surgery . Genes are carried on chromosomes and the basic physical and functional units of heredity. Genes are specific sequences of nucleotides that encode instruction on how to make proteins.

5. What are Genetic Disorders? Genetic Disorder is a disease caused by a “variation” or “mutation” of a gene. Genetic Disorder can be passed on to family members who inherit the genetic abnormality. A small number of rare disorder are caused by a mistake in a single gene. Most disorder involving genetic factors, such as heart disease and most cancers, arises from a interplay of multiple genetic changes and environmental factors.

6. Several approaches to gene therapy : Inserting a normal gene to replace abnormal gene. Inactivating, or “knocking out ,” a mutated gene that is functioning improperly. Introducing a new gene into the body to help fight a disease.

7. Why gene therapy? Gene therapy can be used for a number of diseases, such as severe combined immune- deficiencies , hemophillia , Parkinson’s disease, cancer and even HIV through a number of different approaches. This technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells inserted of using drugs or surgery.

8. Vector and its ideal properties TARGET the right cells. INTIGRATE the gene in the cells. ACTIVATE the gene. AVOID harmful effects. No universal vector exists.

9. HISTORY OF GENE THERAPY 1953: scientists Francis Crick & James Watson determined double helical structure of DNA. 1973: American doctor Stanfeild Rogers tried to treat sisters with Hyperargininemia using human pappiloma virus. 1980: Dr. Martin Cline – first attempted at human gene therapy in university of California , L.A. 1984: the human gene therapy working group (HGTG) created.

10. 1999: Death of Jesse Gelsinger , the first casuality in gene therapy.

11. TYPES OF GE NE THERAPY SOMATIC CELL GENE THERAPY Therapeutic genes transferred into somatic cells. Eg. Introduction of genes into bone marrow cells, blood cells, skin cells etc. GERM LINE GENE THERAPY Therapeutic gene transferred into the germ cells. Eg. Genes introduced into eggs & sperms.

12. SOMATIC CELL GENE THERAPY Will not be inherited later generation. At present all researches directed to correct genetic defects in somatic cells. GERM LINE GENE THERAPY It is heritable & passed on to later generations. For safety, ethical & technical reasons, it is not being attempted at present.

13. IN VIVO GENE THERAPY Direct delivery of therapeutic gene into target cell into patients body. Carried out by viral or non viral vector systems. It can be the only possible option in patients where individual cells cannot be cultured in vitro in sufficient number(e.g. brain cells). In vivo gene transfer is necessary when cultured cells cannot be re-implanted in patients effectively.

14. Example of IN VIVO gene therapy In patient 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 and it draws water from surrounding. This lead to the accumulation of sticky mucous in respiratory tract and lungs. Treated by in vivo replacement of defective gene by adenovirus vector.

15. EX VIVO GENE THERAPY Isolate cells with genetic defect from a patient Grow the cells in culture Introduce the therapeutic genes. Select genetically corrected cells and grow. Transplant the modified cells to the patient.

16. Example of EX VIVO gene therapy 1 st gene therapy – to correct deficiency of enzyme, Adenosine deaminase (ADA) . Performed on a 4yr old girl Ashanthi DeSilva. Was suffering from SCID- severe combined Immunodeficiency. Caused due to the defect in gene coding for ADA. Deoxy adenosine accumulate and destroys T lymphocytes.

17. VECTORS IN GENE THERAPY To transfer the desired gene into a target cell, a carrie r is required. Such vehicles of gene delivery are known as vectors . 2 main classes - Viral vectors - Non viral vectors

18. VIRAL VECTORS 1 ) RETROVIRUS VECTO R S SYSTEM The recombinant retroviruses have the ability to integrate into the host genome in a stable fashion. Can carry a DNA of size- less than 3.4kb Replication defective virus particles Target cell dividing

19. 2) ADENO VIRUS VECTOR SYSTEM Adeno virus with a DNA genome -good vectors. Target- non dividing human cell. Eg . Common cold adenovirus

20. 3) ADENO ASSOCIATED VIRUS VECTOR It is human virus that can integrate into chromosome 19. It is a single st r and e d, non pathogenic small DNA virus. AAV enter host cell , becomes double st r and e d and gets integrated into chromosomes .

21. 4) HERPE S SIMPLEX VIRUS VECTOR Viruses which have natural tendency to infect a particular type of cell. They infect and persist in nervous cells.

22. NON VIRAL VECTOR SYSTEM 1) PURE DNA CONSTRUCT Direct introduction of pure DNA construct into target tissue. Efficiency of DNA uptake by the cells and expression rather low. Consequently, large quantities of DNA have to be injected periodically.

23. 2) LIPOPLEXES Lipid DNA complexes; DNA construct surrounding by artificial lipid layer. Most of it gets degraded by lysosomes. 3) DNA MOLECULAR CONJUGATES Commonly used synthetic conjugate is poly-L- lysin bound to specific target cell receptor.

24. Therapeutic DNA is then made to combine with the conjugate to form a complex. It avoids lysosomal breakdown of DNA. 4) HUMAN ARTIFICIAL CHROMOSOME Can carry a large DNA , with one or more therapeutic genes with regulatory elements.

25. METHODS OF GENE DELIVERY PHYSICAL METHOD GENE GUN Employs a high pressure delivery system to shoot tissue with gold or tungsten particles that are coated with DNA

26. MICROINJECTION Process of using a glass micropipette to insert microscopic substances into a single living cell. Normally performed under a specialized optical microscope setup called a micromanipulator.

27. SONOPORATION: We use ultrasonic frequency to deliver DNA into cells . Which can disrupt the cells membrane and allow DNA to move into cells.

28. ELECTROPORATION : In this method we use short pulses of high voltage to carry DNA across the cell membrane which makes a shock to cause temporary formation of pores and thus allow DNA molecules to pass .

29. Some of the drawbacks of electroporation can be using of high- voltage plasma discharge DNA was efficiently delivered following very short pulses.

30. CHEMICAL METHODS USING DETERGENT MIXTURE - Certain charged chemical compounds like Calcium phosphates are mixed with functioning cDNA of desired function. - The mixture is introduced near the vicinity of recipient cells. - The chemicals disturbs the cell membrane, widens the pore size and allows cDNA to pass through the cell.

31. LIPOFECTION - It is a technique used to inject genetic materials into a cell by means of liposomes. - Liposomes are artificial phospholipid vehicles used to deliver a variety of molecules including DNA into the cells. INORGANIC NANOPARTICALS: It is one of the non-viral gene delivery system where we use gold-silica and iron oxide and calcium phosphate some of the benefits are storage stability , low manufacturing cost and often time , low immunogenecity .

32. POTENTIAL TARGET DISEASES Gene therapy can be used for a number of diseases, such as Parkinson’s disease, cancer Alzheimer's disease and cystic fibrosis through a number of different approaches.

33. Gene Therapy Used in Cancer Cancer is basically a disease of cells characterized by the loss of- -Normal cellular growth -Maturation -Multiplication and thus Homeostasis is disturbed . Gene therapy for cancer is currently focused in multiple areas, including : Genetically engineered viruses that directly kill cancer cells, Gene transfer to alter the abnormal functioning of cancer cells, and Immunotherapy (which includes CAR T-cell therapy), which helps the immune system better find and kill tumour cells.

34. Genetically Engineered Viruses This approach uses specially modified viruses (called oncolytic viruses) that target and destroy cancer cells while leaving normal cells unharmed. The viruses, engineered to contain certain genes, are designed to infect cancer cells and, once inside, to produce proteins that cause the cells to die.

35. Gene Transfer In gene transfer, researchers introduce a foreign gene directly into cancer cells or into surrounding tissue. The goal is that the newly inserted gene will cause the cancer cells to die or prevent cancer cells and surrounding tissue from funnelling blood to tumours, depriving them of nutrients they need for survival. While this approach has a great deal of promise, it presents scientists with several obstacles as well, including “gene silencing,” in which the implanted genes fail to switch on.

36. Immunotherapy CAR T-cell therapy, which seeks to enhance the natural cancer-fighting ability of patient’s own T cells, is one type of immunotherapy. A sample of a patient’s T cells is collected and mixed with viruses carrying several specific genes.The viruses deliver these genes to the T cell’s nuclei, where they are incorporated into the cell’s DNA. The genes cause the T cells to express a special protein called a chimeric antigen receptor , or CAR, on their surface. The CAR directs the T cell to the tumour cell using a specific “address,” and the CAR T cell is then equipped to rapidly destroy the cancer cell. When the cells, now called CAR T cells, are infused into the patient, they seek out tumour cells and then proliferate to generate many more cancer-killing cells.

37. Gene Therapy Used in Cystic Fibrosis Cystic fibrosis is a genetic disease that causes mucus to build up in a patient’s lungs. As a result, patients suffer from blocked airways and bacterial infections . A set of industry collaborations could help bring a gene therapy developed by the UK Cystic Fibrosis Gene Therapy Consortium into clinical testing. The treatment uses a type of virus called a lentivirus to deliver a healthy copy of a gene called CFTR (Cystic Fibrosis Transmembrane Regulator) , which causes cystic fibrosis when it carries a mutation. The gene therapy will be given by inhalation to better target the right cells.

38. Different mutations have been identified in the CFTR gene that can cause cystic fibrosis, and gene therapy may be the most effective way to combat all of them.   UK Cystic Fibrosis Gene Therapy Consortium was the first to show that repeated doses of a gene therapy delivered in fat droplets could be effective in  treating cystic fibrosis  in a Phase IIb trial, but the therapy was not equally effective in all patients . Dutch biotech ProQR is developing a drug that  binds to mutated RNA  of the CFTR gene to restore the production of a healthy CFTR protein.

39. London biotech Verona Pharma recently obtained positive Phase II results for a drug that simultaneously  inhibits two enzymes  in order to reduce inflammation, clear mucus membranes and dilate the lungs.

40. Gene therapy Used in Parkinson's disease Parkinson's disease (PD) is a progressive neurological condition that is the result of the death of the cell that contains and produces dopamine in substantia nigra. People with PD may develop disturbance in their motor activities. Gene therapy in Parkinson's disease  consists of the creation of new cells that produce a specific neurotransmitter (dopamine), protect the neural system, or the modification of genes that are related to the disease. Then these cells are transplanted to a patient with the disease .

41. Gene Therapy Used in Alzheimer Disease Alzheimer is a neurodegenerative disorder in which there is loss of cholinergic neurons at the basal forebrain. Treatment of this disease involves the delivery of Nerve Growth Factor (NGF) which prevents the neuronal loss in addition to ameliorating deficits in learning and memory associated cells.

42. The intracerebral transplantation of genetically modified fibroblasts has been applied to an Animal Model wherein a sparingly small amount of the Cholinergic neurons has been observed to produce NGF . Current pharmacological intervention consist on the administration of L-dopa, a dopamine precursor. The L-dopa therapy increases dopamine production of the remaining nigral neurons . These treatments try to reduce the symptoms of the patient focusing on increasing the production of dopamine but they do not cure the disease .

43. The new treatments for PD are in clinical trials and most of them are centred on  gene therapy . Researchers expect to compensate the loss of dopamine or to protect the dopamine neurons from degeneration. The pharmacological and surgical therapies for PD focus on compensating the ganglia dysfunction caused by the degeneration of the dopaminergic neuron from substantia nigra

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

45. DISADVANTAGES Long testing therapy is not achieved by gene therapy due to rapid dividing of cells benefits of gene therapy is short lived . Immune response due the transferred gene stimulate a potential risk to gene therapy. Disorders caused by defect in multiple genes cannot be treated effectively using gene therapy. Viruses used as vectors for gene transfer may cause toxicity , immune responses , and inflammatory reactions in the host.