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Gene therapy - Types, Advantages and genetic manifestations in molecular medicine
- 1. GENE THERAPY Dr. Reenaz Shaik MD Pathology
- 2. CONTENTS • Introduction • Types and Approaches • Genetic Manifestations of Molecular Medicine • Gene Therapy and Molecular Medicine • Gene Therapy in various diseases • Ethical Issues • National Guidelines 2
- 5. ◉ Gene therapy can be defined as the use of genetic manipulation for treatment of disease. ◉ Gene therapy offers the potential of a one-time cure for devastating inherited disorders. It has application to many diseases for which current therapeutic approaches are ineffective. 5
- 6. ◉ Mechanism: • Replacing a disease-causing gene with a healthy copy of the gene • Inactivating a disease-causing gene that is not functioning properly • Introducing a new or modified gene into the body to help treat a disease. ◉ Gene therapy products are being studied to treat diseases including cancer, genetic diseases and infectious diseases. 6
- 7. HISTORY 7
- 8. ◉ The first attempt at modifying human DNA was performed in 1980, by Martin Cline. ◉ The first successful nuclear gene transfer in humans, approved by the National Institutes of Health, was performed in May 1989 8
- 10. 10 Somatic Gene Therapy Germ line Therapy Therapeutic genes are transferred into somatic cells. Therapeutic cells are transferred into germ cells Ex: Blood cells, skin cells, bone marrow cells Examples: Eggs and sperms These will not be inherited Heritable and passed on to later generations Ethically approved For safety, ethical and technical reasons , its not attempted
- 11. 11
- 12. ◉ In-vivo Gene Therapy: - Direct delivery of therapeutic gene to the patient with the help of some vector. - Used when individual cells cannot be cultures in vitro. - Example: CFTR gene therapy with Adenovirus. 12
- 13. ◉ Ex-vivo Gene Therapy: Example: ADA enzyme gene therapy in SCID. 13 Isolate cells with genetic defect Grow cells in culture Introduce the therapeutic gene Isolate the corrected cells Transplant the cells I the patient
- 14. CORRECTION METHODS • Introduction of exogenous gene, chimeric, or modifier deoxyribonucleic acid (DNA) sequences which might replace or add to the function of defective gene • Expression of microribonucleic acid (RNA)-adapted short-hairpin RNA and small interfering RNA • Gene editing through homologous recombination or by using clustered regularly interspaced short palindromic repeats (CRISPR)-guided Cas9 (CRISPR-associated protein 9) or by other gene-modifying techniques. 14
- 15. VECTORS 15
- 16. 16
- 17. 17
- 18. IDEAL VECTOR ◉ Allow the introduction of the gene of interest and its appropriate expression in target tissue without unwanted side effects. ◉ Be specifically targeted to the cell/tissue of interest. ◉ Be expressed in a regulated manner. ◉ If a viral vector is used, it should be readily and reproducibly grown to high titre. 18
- 20. ◉ Physical Methods: - Gene Gun: High pressure delivery where DNA is coated with gold and tungsten and injected. - Microinjection: It uses micro pipette and micromanipulation to introduce the modified gene into the living cell. ◉ Chemical Methods - Detergent Mixtures: Calcium phosphate mixed with cDNA introduced near the cell, will disrupt cell membrane and the gene is taken inside the cell. - Lipofection: Liposomes are used which are artificial phospholipid vesicle. 20
- 22. ADVANTAGES ◉ Can potentially cure a disease ◉ Only has to be given one time. ◉ Long-lasting effects. ◉ Positive effects passed down through generations. ◉ Rapidly-changing technology. 22
- 23. DISADVANTAGES Expensive. Zolgensma, a gene therapy to treat spinal muscular atrophy, is the most expensive medication in the U.S. It costs $2.1 million for a course of treatment. Experimental Potentially dangerous Ethical issues May cause infection 23
- 25. ◉ Current recombinant deoxyribonucleic acid (DNA) technologies allow for the rapid identification of genes and manipulation of genetic material. ◉ This enables medical researchers to examine cellular physiology at a molecular level. ◉ Most diseases are complex and multifactorial. They result from a complex series of events involving changes in the level of expression of many genes and/or environmental factors and behaviour. ◉ Monogenetic ◉ Multigenetic ◉ Multifactorial 25
- 26. APPLICATIONS 26
- 27. CANCER ◉ Insertion of a gene (such as a tumor suppressor gene) would alter the phenotype of a malignant cell. ◉ Gene transfer of cytokines or other immune mediators to - Augment host immune responses - The genetic modification of neoplastic cells to promote immunogenicity - Treatment of localized cancers with genes encoding viral or bacterial enzymes that convert prodrugs into toxic metabolites - Transfer of genes that provide enhanced resistance to conventional chemotherapy 27
- 28. INFECTIOUS DISESASES ◉ Gene therapy strategies for diseases caused by rapidly proliferating infectious pathogens include intracellular immunization and polynucleotide vaccines. ◉ Gene-therapy-induced vaccination for these pathogens may represent an effective strategy by acting classically to “prime” innate immunity prior to exposure to the pathogen. ◉ Intracellular immunization seeks to transform cells into cells that are refractory to infection. 28
- 29. GENETIC VACCINATION ◉ DNA vaccines consist of a bacterial plasmid with a strong viral promoter, the gene of interest, and a polyadenylation/transcription termination sequence. ◉ The plasmid is grown in bacteria (Escherichia coli), purified and injected or blasted into target tissues of the recipient. ◉ Rapid progress is being made toward the development of a DNA vaccine for HBV. 29
- 30. ORGAN TRANSPLANTATION ◉ Adenovirus vectors is used for efficient gene transfer to the lung and heart in a post transplantation setting. ◉ The efficacy of such procedures show the feasibility of genetic modification of the graft to reduce post transplantation rejection, such as chronic graft vascular disease in cardiac allograft rejection. ◉ The graft rejection process could be modified by inserting specific genes of immunosuppressive molecules or by transfecting genes of antisense molecules to block expression of an important mediator of graft rejection 30
- 31. CELLULAR TRANSPLANTATION ◉ Unrelenting shortage of donor organs for whole organ transplantation has resulted in the use of every known method to promote successful transplantation outcomes ◉ Hepatocyte cell transplantation has become an experimental treatment for individuals rejected as candidates for organ (liver) transplantation. 31
- 32. AGING ◉ Muscle atrophy occurs as part of the normal aging process. Muscle strength decreases up to one-third in humans between the ages of 30 and 80. ◉ Recent studies using an adeno-associated virus vector and the gene for insulin like growth factor 1 showed that injection of aged mice with this construct totally prevented the decrease in muscle mass seen in aging 32
- 33. CRISPR 33
- 34. CRISPR ◉ Genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. ◉ It is based on a simplified version of the bacterial CRISPR- Cas9 antiviral defence system. ◉ By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo. 34
- 36. • How can “good” and “bad” uses of these technologies be distinguished? • Who decides which traits are normal and which constitute a disability or disorder? • Will the high costs of gene therapy make it available only to the wealthy? • Could the widespread use of gene therapy make society less accepting of people who are different? • Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability? 36
- 38. ◉ National Guidelines for Gene Therapy Product (GTP) Development and Clinical Trials” prepared by the Indian Council of Medical Research and Department of Biotechnology in 2019. ◉ Based on the data reported from tertiary care hospitals genetic diseases such as - Haemophilia, thalassemia, sickle-cell anaemia, certain forms of muscular dystrophies, retinitis pigmentosa, primary immunodeficiency in children, lysosomal storage disorder and cystic fibrosis. 38
- 39. ◉ Gene therapy clinical trial approval and evaluation process. ◉ Composition of committee reviewing gene therapy clinical trial applications ◉ Duration of record keeping: 5 years for documents and 15 years for patient records ◉ Follow-up of patients: 5- 10 years ◉ Germ cell gene therapy is prohibited. 39
- 40. REFERENCES ◉ An introduction to gene therapy and molecular medicine by Thomas F Kesrina ◉ Both G, Alexander I, Fletcher S, Nicolson TJ, Rasko JE, Wilton SD, Symonds G. Gene therapy: therapeutic applications and relevance to pathology. Pathology. 2011 Oct;43(6):642-56 ◉ Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan RA, Moen R, et al. (August 1990). "Gene transfer into humans – immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction". The New England Journal of Medicine. 323 (9): 570–578 40
- 41. THANK YOU 41