Gene therapy

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1. Presented by: Khadga Raj Aran M.Pharm(Pharmacology) Isf college of pharmacy Moga, Punjab. HUMAN GENE THERAPY

2. contents Introduction Types of Gene Therapy Approaches in Gene Therapy Vectors used in Gene Therapy Method of Gene Delivery Advantages Disadvantages Application of Gene therapy conclusion

3. INTRODUCTION Gene therapy is an experimental technique in which introduction of normal genes into cells in place of missing or defective ones in order to correct genetic disorders. 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. TYPES OF GENE THERAPY Therapeutic genes transferred into the   somatic cells . Eg. Introduction of genes into bone marrow cells, blood cells, skin cells etc. Will not be inherited later generations. At present all researches directed to correct genetic defects in somatic cells. Therapeutic genes transferred into the  germ cells . Eg. Genes introduced into eggs and sperms. It is heritable and passed on to later generations. For safety, ethical and technical reasons, it is not being attempted at present. Somatic cell gene therapy Germ line gene therapy

5. Ex vivo gene therapy :- transfer of genes to cultured cells and reinsertion. In vivo gene therapy :- direct delivery of genes into the cells of a particular tissue in the body APPROACHES IN GENE THERAPY

6. EX VIVO GENE THERAPY

7. 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 defect in gene coding for ADA. Deoxy adenosine accumulate and destroys T lymphocytes. Disrupts immunity , suffer from infectious diseases and die at young age.

8. 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 numbers (e.g. brain cells). In vivo gene transfer is necessary when cultured cells cannot be re-implanted in patients effectively.

9. EXAMPLE OF IN VIVO GENE THERAPY In patients with cystic fibrosis, a protein call 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 leads to the accumulation of sticky mucous in respiratory tract and lungs. Treated by in vivo replacement of defective gene by adenovirus vector .

10. VECTORS IN GENE THERAPY To transfer the desired gene into a target cell , a carrier is required such vehicles of gene delivery are known as vectors. 2 main classes Viral vectors Non viral vectors Viral vectors 1) retrovirus vectors system: Can carry a DNA of size – less than 3.4kb

11. Conti…… Retroviruses used in gene therapy so that any genes that are harmful to man are removed. Corrective genes are then added to replace the removed genes, and the new, modified retrovirus is then introduced into the patient . Adeno virus vector system Gene therapy using an Adenovirus vector. A new gene is inserted into an adenovirus vector, which is used introduce the modified DNA into a human cell.If the treatment is successful,the new gene will make a functional protein. Lentivirus : It is a retrovirus it has a single stranded RNA genome .used in haemophilia A , Rheumatoid arthritis , Diabetes mellitus.

12. NON VIRAL VECTORS 1) pure DNA construct Direct introduction of pure DNA construct into target tissue . Therapeutic DNA is then made to combine with the conjugate to form a complex. It avoids lysosomal breakdown of DNA. 2) Lipoplexes Lipid DNA complexes; DNA construct surrounded by artificial lipid layer. Most of it gets degraded by lysosomes

13. Gene Gun Employs a high-pressure delivery system to shoot tissue with gold or tungsten particles that are coated with DNA 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 . METHODS OF GENE DELIVERY PHYSICAL METHODS

14. Conti… Sonaporation Using ultrasonic frequency to deliver DNA into cell. Allows disrupt cell membrane and DNA move into cell. Eletroporation Method that uses short pulses of high voltage to carry DNA across cell membrane. Thus cause temperature formation of pore in cell membrane allows gene to pass through.

15. CHEMICAL METHODS USING DETERGENT MIXTURES Certain charged chemical compounds like Calcium phosphates are mixed with functional 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. LIPOFECTION It is a technique used to inject genetic materials into a cell by means of liposomes. Liposomes are  artificial phospholipid vesicles  used to deliver a variety of molecules including  DNA  into the cells.

16. OTHER TYPES OF GENE THERAPY GENE AUGMENTATION THERAPY Most common form of gene therapy Foreign gene replaces missing or defective gene. Eg. Replacement of defective gene p53 by a normal one in liver cancer. .

17. GENE INHIBITION THERAPY Done to block the overproduction of some proteins. 2 types – Antigene and antisense therapy. Antigene – blocks transcription using antigene oligonucleotide Antisense – blocks transalation using antisense oligonucleotide

18. IS GENE THERAPY SAFE? Gene therapy is under study to determine whether it could be used to treat disease. Several studies have already shown that this approach can have very serious health risks, such as toxicity, inflammation, and cancer. some of the risks may be unpredictable; however, medical researchers, institutions, and regulatory agencies are working to ensure that gene therapy research is as safe as possible. The U.S. Food and Drug Administration (FDA) regulates all gene therapy products in the United States and oversees research in this area.

19. ADVANTAGES Gene therapy has the potential to eliminate and prevent hereditary diseases such as cystic fibrosis, ADA- SCID etc. It is a 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 diseases from the future generations.

20. DISADVATAGES Long lasting therapy is not achieved by gene therapy; Due to rapid dividing of cells benefits of gene therapy is short lived . 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.

21. APPLICATION OF GENE THERAPY Gene Therapy Reduces parkinson’s disease symptoms It significantly improved the weakness of the symptoms such as tremors motor skill problem and rigidity Main overactive brain region :the subthalamic nucleus should be introduced with GAD gene That would produce GABA Neurotransmitters. It is an inhibitor chemical then they could potentially quiet . that brain region and alleviate tremors Cystic fibrosis Cystic fibrosis affects the epithelial cells of the body, but the life threatening problems mainly affect the lungs CF is caused by a mutation in the gene cystic fibrosis transmembrane coductance regulator.CFTR gene found in chromosome 7. product f this gene is CFTR protein . The aim is to get the gene into the cells so that it can make the normal protein, i.e. CFTR

22. Contd.. In Cancer Multiple gene therapy have been developed to treat a wide variety of cancers including suicide gene therapy, anti-angiogenesis and therapeutic gene vaccines. Gene therapy against cancer include the introduction of tumor suppressor genes, gene that induces apoptosis or gene inhibitor tumor angiogenesis. Hemophilia For purpose with hemophilia therapies could be designed to deliver genes that express missing factor viii and ix meaning individual would no longer need to injected exogenous clotting factors Fat metabolism disorder In 2012 glybera became the first viral gene-therapy treatment to be approved in Europe. Treatment uses in cure lipoprotein lipase deficiency.

23. Recent advances in Gene therapy In march 2017 French scientists reported on clinical research of gene therapy to treat sickle-cell disease. In August 2017 , the FDA approved tisagenlecleucel for acute lymphoblastic leukemia. Tisagenlecleucel is an adoptive cell transfer therapy for B-cell acute lymphoblastic leukemia.  T cells from a person with cancer are removed, genetically engineered to make a specific T-cell receptor that reacts to the cancer, and are administered back to the person.

24. Conti … This is the first form of gene therapy to be approved in the United States. In October, a similar therapy called axicabtagene ciloleucel was approved for lymphoma. In a new gene therapy method developed by University of Florida researchers found treatment for a common form of blindness that strikes both youngsters and adults.

25. CONCLUSION Theoretically, gene therapy is the permanent solution for genetic diseases. But it has several complexities. At its current stage, it is not accessible to most people due to its huge cost. A breakthrough may come anytime and a day may come when almost every disease will have a gene therapy Gene therapy have the potential to revolutionize the practice of medicine.

26. REFERENCES Dubey R.C, A textbook of biotechnology , 1 st edition(2004), S Chand and company, New Delhi Gupta P.K, Elements of Biotechnology , 1 st edition(2001), Rastogi Publications, Meerut. Satyanarayana U, Biotechnology , 1 st edition, Book and allied (P) Ltd, Kolkata. http://www.medindia.net/articles/genetherapy_treatment.htm http://en.wikipedia.org/wiki/Gene_therapy

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