GENE THERAPY Presented by- Urvashi Shakarwal M Pharm Pharmacology Amity Institute of pharmacy ‘Amity University Lucknow U.P. M Pharm PharmacologyIntroduction Gene therapy is to transfer a gene from one DNA molecule to another DNA molecule. It is the insertion of genes into the individual’s cell and tissues to treat a disease. The directed desirable gene transfer from one organism to another and the subsequent stable integration and expression of foreign gene into the genome is referred as genetic transformation. Unstable transformation occur when DNA is not integrated into host genome. Stable transformation occur when DNA is integrated into host genome and is inherited in subsequent generations. The transferred gene is known as transgene and the organism that develop after a successful gene transfer is known as transgenic organism.Steps involved in gene therapy Identification of the defective gene Cloning of normal healthy gene Identification of target cell / tissue / organ Insertion of the normal functional gene te Urvashi Shakarwal M Pharm Pharmacology .Approaches involved in gene therapy Germ line gene therapy: > where germ cells (sperm or egg) are modified by the introduction of functional genes, which are integrated into their genome. >» Therefore changes due to therapy would be heritable and would be passed on to later generation. > Theoretically, this approach should be highly effective in counteracting genetic disease and hereditary disorders. Urvashi Shakarwal M Pharm PhatinacologySomatic gene therapy: > where therapeutic genes are transferred into the somatic cells of a patient. » Any modifications and effects will be restricted to the individual patient only and will not be inherited by the patients offspring or any later generation. Urvashi Shakarwal M Pharm PharmacologySomatic Therapy vs Germline Therapy Somatic Therapy ‘Gerinline Therapy 13 ave introduced into somatic cells, [Genes ure introduced into germ line ceil and wil co Is are confined to the recipient, [Changes will be passed to the future generations. ie are issue specific in most instancestinfigh frequency of insertional mutations an ir expression} although not locaiion specificbbserved in this process and cause ierdiogsnis many. In many instances, it may not ences, ible to reconstitute normal level and tissue stribution, fecinical expertise for soomatic ane ate still many technical difficulties it wanipulations J viiro introduction of gene offntoduetion of genes into germ cells. snd replanting somatic eells in body’ to ake them functional is developed,Vectors involved in gene therapy To transfer the desired gene into a target cell a carrier is required. Such vehicle of gene delivery are known as vectors. ‘Types of Vectors, 1. Viral Vectors 2. Non-Viral Vectors | | b) Adeno Vira ©) Adeno Asso b) Naturally Occurring co Enhance delivery by physical methods 1 Viruses, dl) Herpes Simplex Virus ©) Enhance delivery by chemical methods Urvashi Shakarwal M Pharm Pharmacology 7Viral vectors Viral vectors are mostly derivatives of viruses that infects animals. + Viruses have evolved a way of encapsulating and delivering their genes to human cells to remove disease-causing genes and insert therapeutic ones > Virus bind to their hosts and introduce their genetic material into the host cell |.Free replicating viruses- that multiply within the cell, but do not integrate into the genome of the host. 2.Integrateing viruses-that can integrates into the host genome. They enters the cell, copy their RNA genome into DNA. Urvashi Shakarwal M Pharm PharmacologyTypes of viral vectors Retrovirus vector system They uses the enzyme reverse transcriptase to convert the RNA into DNA which is integrated into gemone further enters the host cell. It have the ability to infect a wide variety of cell type with high efficiency. Urvashi Shakarwal M Pharm PharmacologyAdenovirus vector system The AAV genome comprises of inverted terminal repeats (ITR) as both ends of the DNA stand and open reading frames.(ORFs). This type of viruses is being used, because it is non pathogenic and do not build an immune response, AVY vectors are used to deliver genes to the brain, this is possible because AAV viruses can infect non-dividing cells such as neurons. 40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. Urvashi Shakarwal M Pharm PharmacologyAdvantages of viral vectors Urvashi Shakarwal M Pharm Pharmacology.Non viral vector system Physical methods- 1.Electroporation 2.Microinjection 3.Gene gun or a biolistic particle delivery system 4. Magnetofection Chemical methods- 1.DNA transfer by calcium phosphate method 2.Liposome medicated transfer Urvashi Shakarwal M Pharm Pharmacology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 14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 micrometer. 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. Limitations of microinjection -Costly -Skilled person required -More useful for animal cells. | me Urvashi Shakarwal M Pharm PhatinacologyGene gun or a biolistic particle delivery system |.Biollistics or particle bombardment also known as gene gun technique is a physical method that uses accelerated micro projectiles to deliver DNA or other molecule into intact tissues 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. Urvashi Shakarwal M Pharm Pharmacology ®4.The coated beads are then attached to the end of the 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.Magnetofection Magnetofection is a simple and highly efficient transfection method that uses magnetic fields to concentrate particles containing nucleic acid into the target cells. Iie Urvashi Shakarwal M Pharm PharmacologyChemical methods 1.DNA transfer by calcium phosphate method The process of transfection involves the mixture of isolated DNA with solution of calcium chloride and potassium phosphate. Cell are then incubated with precipitated DNA either in solution or in tissue culture dish. A fraction of cells will take up the calcium phosphate DNA precipitate by endocytosis. Urvashi Shakarwal M Pharm Pharmacology2.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 agents for exogenous materials including transgenes. Promote transport after fusing with the cell membrane. Cationic lipids are those having a positive charge are used for the transfer of nucleic acid. Urvashi Shakarwal M Pharm PharinacologyVarious strategies for gene therapy 1. Gene augmentation therapy 2. Inhibition of gene expression 3. Gene therapy to achieve pharmacological effects/ killing specific cells Urvashi Shakarwal M Pharr PharmacologyApplications of Gene Therapy Gene Therapy for Genetic Disorders 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 ADA deaminase (ADA). Chromosome 20 Urvashi Shakarwal M Pharr Pharmacology» 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 Urvashi Shakarwal M Pharm PharmacologyChronic Granulomatus Disorder (CGD) CGD is a genetic disease in the immune system that leads to the patients’ inability to fight off bacterial and fungal infections that can be fatal. investigators in Germany treated two patients with this disease some of the blood-making cells are taken from the patient The normal gene is placed into the cells using special viruses called retroviruses. The cells are then able to produce the normal protein. Urvashi Shakarwal M Pharm Pharmacology 3.Hemophilia » Patients are not able to induce blood clots and suffer from external and internal bleeding that can be life threatening. » Inaclinical trial conducted in the United States , the therapeutic gene was introduced into the liver of patients, who then acquired the ability to have normal blood clotting time. > The therapeutic effect was transient because the genetically corrected liver cells were recognized as foreign and rejected by the healthy immune system in the patients and curative outcome by gene therapy Urvashi Shakarwal M Pharm Phatinacology EyGene therapy trial for inherited blindness 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. Urvashi Shakarwal M Pharr Pharmacology aeGene therapy for acquired diseases Cancer » Multiple gene therapy strategies have been developed to treat a wide variety of cancers, including suicide gene therapy, oncolytic and therapeutic gene vaccines. » Two-thirds of all gene therapy trials are for cancer and many a ere are entering the advanced stage, including a Phase rial » Additionally, numerous Phase | and Phase II clinical trials for cancers in the brain, skin, liver, colon, breast and kidney among others, are being conducted in academic medical centers and biotechnology companies, using novel technologies and therapeutics developed on-site. Urvashi Shakarwal M Pharm PharmacologyOther acquired diseases » The same gene therapeutic techniques have been applied to treat other acquired disorders such as viral infections (e.g. influenza, HIV, hepatitis), heart disease and diabetes, among others. » Some of these have entered, or will soon be entering, into early phase clinical trials. Urvashi Shakarwal M Pharm Pharmacology 3Uses of gene therapy Clinical gene transfer applications Vaccine development Production of transgenic animals Treatment of cancer and AIDS Gene discovery Gene therapy Enhancing the resistance of plant Genetically modified organism Urvashi Shakarwal M Pharm PharmacologyTHANK YOU