Gene Therapy

In this slide, You will get to learn abut Gene Therapy and different types of gene therapy. Various method of Gene Therapy and Advantage & Disadvantage and Recent advances in Gene Therapy.

1. GENE THERAPY Presented by:- Shaikh Nisar Ali ( M . pharm ) Dept. of Pharmacology Guided by:- Mrs . Arati Malpani . (Ph.D) (Associate Professor) 29 January 2021

2. Advances in Biochemistry and Molecular biology have helped to understand the genetic basis of inherited disease. It was a dream of the researchers to replace the defective genes with new one and cure genetic disorders. DEFINATION:- Gene therapy can be broadly defined as the transfer of genetic material to cure the disease or at least to improve the clinical status of the patient. The gene used in gene therapy are called GENE DRUG.

3. Principle :- Gene therapy is the process of inserting genes into cells to treat diseases. The newly introduced genes will encode proteins and correct deficiencies that occour in genetic disease. It involves genetic manipulation in animals or humans to correct disease and keep organisms in good health. Initial experiment is carried out first on animals than on Humans.

4. Approaches for Gene therapy Somatic Cell Gene Therapy Germ Cell Gene Therapy

5. Somatic Cell Gene therapy:- The Non-Reproductive cells ( Non-sex cells ) of an organism are referred to as Somatic Cells. These are cells of organs other than sperm or egg cells. Example- Bone marrow cells , Blood cells , skin cells, intestinal cells. Somatic cell gene therapy involves insertion of an expressible gene into Somatic cells and at present all research on gene therapy is directed to correct genetic defects in Somatic Cells. Here Gene therapy is given to somatic cells that’s why Its called Somatic Cells Gene therapy.

6. Germ Cell Gene Therapy :- The Reproductive cells ( Sex cells ) of an organism are referred to as Germ Cells. Here Gene Therapy is given to germ cells that’s why its is called Germ cell gene therapy. Gene therapy involving the introduction of DNA into germ cells is passed to Successive Generation. For safety , Ethical and technical reason , germ cell gene therapy is not being attempted at present.

7. There are 2 Types of gene therapy:- EX-VIVO IN-VIVO

8. EX-VIVO Here cells are modified outside the body and then transfer back again. It involves patient own cells for culture and genetic correction and then return back to patient. Called as EX-Vivo because cells are treated outside the body. IN-VIVO Invivo Gene therapy involves direct delivery of therapeutic gene (DNA) in to target cells of a particular tissue of the patient. Genes are changed in cell when the cells are still in the body. Called IN-Vivo because the gene is transferred into the cell inside patient’s body.

9. Steps involved in Ex Vivo gene therapy :- First patients own cells are isolated with genetic defects from patient. Grow cells in culture. Introduce the therapeutic gene to correct gene defects. Select the genetically corrected cells(stable Transformants ) and grow. Transplant the modified cells in to the patient.

10. Ex-Vivo Gene Therapy

11. Vectors in gene Therapy :- VECTORS :- The carrier particles or molecules that are used to deliver genes to Somatic Cells are called Vectors. Types of vectors:- 1) Viruses. Human artificial Chromosomes. Bone Marrow Cells.

12. Viruses:- Viruses such as Ret e rovirus , adenovirus, Murine leukemia Virus etc. are used as vectors. The vectors frequently used in gene therapy are virus particularly reterovirus . RNA is a genetic material in reterovirus . As RNA enters host cells , it synthesize DNA by reverse transcription. The formed viral DNA (Provirus) gets incorporated into DNA of host cell. IT is normally harmless , but some reterovirus convert Normal cells to cancer cell.

13. Human Artificial Chromosomes(HAC) The Human Artificial Chromosomes are the synthetic chromosomes that can replicate with other chromosomes , besides encoding human protein. As the use of reterovirus in gene therapy is associated with heavy risk such as cancer , This problem can be overcome if human artificial chromosomes is used. HAC was developed in 1997 and it possesses the characteristics of Human Chromosomes.

14. Bone Marrow cells:- Bone marrow cells contain totipotent embryonic stems cells. These cells are capable of dividing and differentiating into various cell types. Ex- RBC , Platelets , B & T Lymphocytes. Bone Marrow cells are most widely used in gene therapy for disease like SCID , Sickle Cell Anaemia .

15. Stratagies OF Gene Therapy:- 1 ) Gene Augmentation therapy :- Used to treat disease caused by mutation that stops a gene from producing a functioning product such as protein. Thus therapy Adds DNA containing a functional version of lost gene back in to the cells. The new gene produce a functioning product at sufficient levels to replace the protein that was originally missing.

16. 2) Gene Inhibition Therapy:- It is suitable for treatment of infectious disease , cancer , and inherited disease caused bt inappropriate gene activity. This therapy inhibits or eliminate the activity of gene that encourages the groeth of Disease related cells. Aim is to introduce a gene whose product either- Inhibits the expression of another gene. Interferes with the activity of product of another gene. EX- Cancer is sometimes the result of over activation of an ONCOGENE ( Gene which stimulate cell growth). So by inhibiting or eliminating the activity of that Oncogenethrough gene inhibitiob therapy it is possible to prevent further cell growth and stops cancer in its track.

17. 3) Killing of specific cells :- Its is suitable for disease such as cancer that can be treated by destroying certain group of cells. Aim is to insert DNA into disease cell that causes thet cell to die. Achived by one of 2 ways :- Insert DNA that contain ”Suicide Gene” that produces a highly toxic product which kills the diseases cells. Insert DNA that causes expression of a protein that marks the cell so that the diseases cells are attacked by body’s natural Immune System. It is essential with this method that the inserted DNA is targeted appropriately to avoid the death of cells hat are functioning Normally.

18. Various types of Gene Transfer Technique Natural Methods- Congugation Agrobacterium Mediated Transfer Reteroviral Transduction Physical Methods- Microinjection Biolistics Method

19. Chemical Methods- DNA transfer by Polyethylene glycol (PEG) Liposome Mediated transport Electrical Methods- Electroporation Electrofusion

20. Electroporation :- It is an efficient process to transfer DNA into cells. Microscopic pores are induced in biological membrane by applicaation of electric field. These pores are known as electropores which allows the molecule , ions , water to pass from one side of membrane to another. Or in simple terms , here electric field is applied on the cells – because of this there is a disturbance on the phospholipid bilayer of the cells – because of this polar molecules enters in to the cells.

21. Electroporation

22. Electroporation

23. Advantages :- Fast Method Less Costly Simultaneously , large number of cells can be treated. Disadvantages:- Cell damages because of disturbances.

24. Microinjection :- Here DNA is directly injected into cells . Specifically in to nucleus or cytoplasm. Fine tipped ( 0.5 – 1.0 Micrometer ) diameterglass needle or pipette is used. Microinjection can be done by only skilled person. Computerised control of holding pipette needle , Microscopic stage and video technology has improved the efficiency of technique.

25. Micro Injection

26. Limitation :- Coslty Skilled personal Required More useful for Animal cells

27. Biolistics :- Simplest methodof direct introduction of therapeutic DNA into target cells. Looks like a pistol but works more like a Shotgun Golden Pellets. The Gene gun fires DNA into the cells The DNA to be transformed into the cells is coated on to microscopic beada of either gold or tungsten. The coated beads are then attachedto the end of plastic bullets.

28. An explosive force fires the bullet towards the target cells that lie just beyond the end of the barrel. After the beads pass through the cell wall into cytoplasm of the target cells. Then beads and DNA dissociate and cells become transformed.

29. Gene Gun

30. Gene Gun

31. Liposome Mediated Gene Transfer:- Liposomes are spheres of lipids which can be used to transport genes or molecules in to the cell. These are artificial vesicles that can act as a delivery agent for exogenous materials. They are considered as spheres of lipid bilayer surrounding the molecule to be transported and promote transport after fusing with cell membrane.

32. Liposome mediated gene transfer

33. Liposome mediated gene transfer

34. Clinical Application :- Severe Combined Immuno Defficiency ( SCID) Hemophilia Parkinson’s disease Cancer HIV Retinal Disease Colour Blindness Diabetes mallitus

35. First Gene therapy:- The first gene therapy was performed to treat the defect in the gene which synthesizes Adenosine Deaminase(ADA).

36. Bone marrow cells with the help of ADA enzyme produce B&T Lymphocyte which are responsible for Immune response. Gene undergoes transcription and translation process to form ADA enzyme which is a protein. Suppose If that gene is faulty then no B&T lymphocyte are formed which weakens the Immune response. This disease is called SCID ( Severe Combine Immuno Deficiency ). The first gene therapy was performed on 4 year girl in 1990 who was suffeeing from SCID. What scientists did was.....

37. They Isolate ADA synthesizing gene . This gene was introduced in to lymphocyte from that girl using reterovirus. Because of this temporarily B&T cells starts synthesize. The immune system becomes normal. But life of lympocyte is short thatswhy regular interval of therapy is required. If given at early stage this correction can be permanent. ADA synthesizing gene Using Reterovirus Lymphocyte Bone Marrow Cells Transformed lymphocyte is introduced in bone marrow of girl.

38. Advantages of Gene therapy:- Gives a chance to live a normal life to a patient born with genetic disease. Gives a hope of healthy life to Cancer patient , Alopecia patient etc. Disadvantages of Gene therapy :- Costly Taking frequent gene therapy is dificult as it is very costly. New things introduced in body leads to immune response. As not much study done and not FDA approved , It is not not safe for humans.

39. Current status... Till Right now USFDA has not approved any human gene therapy for sale. Reasons..? In 1999, A 18 year old Jesse Gelsingur died from multiple organ failire afyer treatment for Ornithine Transcarbaylase (OTC) . DEATH was triggered by severe immune response to Adenovirus. Like this there are many reasons ... One problem with gene therapy is that one does not have control over where the gene will be inserted into genome.

40. Recent Advances :- Genes get into brain using Liposomes coated in polymer called polyethylene glycol found potential for treating Parkinson’s disease. Sickle cell anaemia is successfully treated in Mice. Severe Combined Immuno Difficiency (SCID) treatment is found successful by Gene therapy. Curing hemophilia B in mice is found Successful. Research is going on Animal level for treating Amyotropic Lateral Sclerosis.

41. Stem cell therapy gives hope to prevent Inherited neurological disease where scientists from University of Manchester have used stem cell gene therapy to treat a fatal genetic brain disease. A very large variety of therapeutic genes are under investigation such as Tumour supressor , Suicide antiangiogenesis , Micro-RNA genes. Cancer gene therapy is not yet indicated in clinical practice however basic and clinical advanceshave been reported and gene therapy is promising. New therapeutic approach for treatment of gastrointestinal tumours.

42. Gene therapy holds promise for treating wide range of disease such as Cancer , Cystic fibrosis , Diabetes , Hemophilia , Alopecia , AIDS etc. Researchers are still atudying how and when to use gene therapy. Currently in US gene therapy is available only as a part of clinical trail.

43. THANK YOU 29 January 2021