GENE THERAPY

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1 GENE THERAPY Presented By AMRUTHA FELIX

DEFINITION:

DEFINITION GENE THERAPY:- “The process of inserting genes into cells to treat diseases” “An introduction of a normal functional gene into cells,which contain the defective allele of concerned gene with the objective of correcting a disorder or an acquired disorder” “The treatment of genetic disease by introducing proper genes into cells of target organ” “Deliberate alteration of human genome for alleviation of disease” 2

GENE THERAPY AND REMEDIAL GENE:

GENE THERAPY AND REMEDIAL GENE Gene used to treat a genetic disease is called REMEDIAL GENE Germ line gene therapy :- Remedial gene introduced into germ cells.(egg,sperm,zygote) Somatic gene therapy :- Remedial gene introduced into somatic cells.(liver cells,skin cells..) Gene replacement therapy :- Remedial gene may replace function of defective gene Gene inhibition therapy :- Remedial gene may block the activity of the defective gene Gene augmentation therapy :-Remedial gene may add the copy of a mutated gene Suicide gene :- Remedial gene may kill specific cells 3

GUIDELINES:

GUIDELINES As per National Institutes of Health (NIH,USA) Pure form Effective method for delivering gene into derived tissue or cell Risk to the patient evaluated and minimised Disease untreatable by other strategies Data from preliminary experiments with animal models or human models should be available 4

MODELS:

MODELS Two types :- Identifying suitable target cells in an affected patient,removing them,carrying out the gene transfer invitro & returning the genetically & phenotypically corrected cells to the patient. Introduction of a vector directly into a patient that is targeted to the appropriate tissue or organ in the patient’s body. 5

STRATEGIES:

STRATEGIES 6 b) GENE INHIBITION THERAPY a)GENE AUGMENTATION THERAPY

APPROACHES:

APPROACHES 7

EMBRYO THERAPY :

EMBRYO THERAPY Treatment of genetic disease by introducing a proper remedial gene into cells of 2-10 days old embryo. Cystic Fibrosis :- an   autosomal recessive genetic disorder  that affects most critically the  lungs, and also the  pancreas,liver, and intestine. It is characterized by abnormal transport of  chloride   and sodium across an  epithelium, leading to thick, viscous secretions. 8

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EX VIVO GENE THERAPY (TRANSPLANTATION/TISSUE GRAFTING):

EX VIVO GENE THERAPY ( TRANSPLANTATION/TISSUE GRAFTING ) Applied to selected tissues whose cells can be cultured in laboratory. STEPS Isolate cells with genetic defect from a patient Grow cells in culture Introduce the therapeutic gene to correct gene defect Select the genetically corrected cells and grow Transplant the modified cells to the patient Eg:- liver transplantation, bone marrow transplantation, kidney transplantation 10

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IN VIVO GENE THERAPY:

IN VIVO GENE THERAPY Direct delivery of a remedial gene construct into proper organ of a patient to correct the genetic defect Liver,muscle,skin,spleen,lungs,brain & blood cells are the potential candidates of this approach Less time consuming Easy Alzheimer’s disease,brain tumor etc.; can be treated Vectors – adenovirus, herpes simplex virus 12

ANTISENSE GENE THERAPY or GENE INHIBITION THERAPY:

ANTISENSE GENE THERAPY or GENE INHIBITION THERAPY The treatment of genetic diseases by introducing a remedial gene that prevents the expression of the specific defective gene is called antisense gene therapy Mainly used in the treatment of cancer 13

STEPS:

STEPS The defective gene in the patient’s cells produces mRNA by transcription The mRNA gets translated into a defective protein. Accumulation of this protein results in a genetic disease A single stranded oligonucleotide complementary to mRNA binds with the mRNA & forms an RNA-DNA hybrid The DNA strand blocks the translation initiation signals in the mRNA so the defective protein has not been produced The oligonucleotide strand blocking the translation of mRNA of defective gene is called antisense DNA. 14

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VECTORS IN GENE THERAPY:

VECTORS IN GENE THERAPY Vectors are the carrier particles or molecules used to deliver genes to somatic cells They include :- Viruses Human Artificial Chromosome (HAC) Bone marrow cells 16

VIRUSES:

VIRUSES Retrovirus* Oncoretrovirus Adenovirus Adenoassociated virus Herpes virus RETROVIRUS Frequently used Genetic material –RNA Synthesizes DNA(viral DNA –Provirus) from RNA as it enters host cell Provirus gets incorporated into the DNA of the host cell Proviruses are usually harmless.*some retroviruses convert normal cells into cancerous ones. 17

HUMAN ARTIFICIAL CHROMOSOME(HAC):

HUMAN ARTIFICIAL CHROMOSOME(HAC) Synthetically produced vector DNA Possess the charachteristics of human chromosome Size ranges from 1/10 th to 1/5 th of a human chromosome Carry human genes that are too long Risk in using retroviruses can be overcome by HAC 18

BONE MARROW CELLS:

BONE MARROW CELLS Bone marrow contains ‘ totipotent embryonic stem (ES) cells’-capable of dividing and differentiating into various cell types ( eg .; RBC,platelets,macrophages etc.;) Widely used method for genetic diseases. Sickle cell anemia,thalassemia,osteoporosis etc.; are likely to be cured. 19

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20 GENE DELIVERY PROCESS OF INTRODUCING FOREIGN DNA INTO HOST CELLS NECESSARY FOR GENE THERAPY & THE GENETIC MODIFICATION OF CROPS

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NON VIRAL METHODS:

NON VIRAL METHODS MICROINJECTION the direct-pressure injection of a solution into a cell through a glass capillary—is an effective and reproducible method for introducing exogenous material into cells in culture. MATERIALS Cells - The best cells for microinjecting will be large, easily adherent, with a pronounced nucleus. In theory, any mammalian cell can be injected, although some types provide more challenges than others. Micropipette - Glass capillary tubing for fabricating micropipette, Micropipette puller for preparing the glass micropipettes. DNA - Any plasmid containing a cytomegalovirus (CMV) promoter-driven reporter gene that may be assayed in individual cells may be used for monitoring the efficiency of microinjection. Microinjection apparatus – Microscope, Micromanipulator, Microinjector 22

METHODS:

METHODS Cell preparation - Inoculate the cells onto cover slips in 35 or 60 mm tissue-culture plates. Use the appropriate growth medium for the cells buffered with 10–20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)  , pH 7.5. Inoculate the cells at a low density so that there is minimal contact between adjacent cells. Micropipette preparation - Immediately before use, fabricate a number of micropipettes from 1-mm outer diameter, thin-walled capillary glass with an inner filament (the filament aids filling the very tip of the micropipette with injection solution) using a micropipette puller according to the manufacturer’s directions. DNA preparation – Centrifuge the purified DNA solution at 10,000–15,000g for 10 min prior to loading the supernatant into the micropipette. Using a 27G needle attached to a 1-mL syringe, load a few microlitres of DNA solution through the back-end of the micropipette. 23

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Fit the micropipette to the microinjector and lower the micropipette tip into the culture medium. Too long a delay between loading the micropipette and immersing the tip in the medium may cause the liquid in the tip of the micropipette to evaporate, resulting in blockage. d. Microinjection Apparatus Set-Up Ensure that the micromanipulator is set with all three axes at the center of their movement ranges. Place the dish containing the cell-covered cover slips in the center of the microscope stage, select the lowest power objective available, and focus the microscope on the cells. Rack the microscope objective up a few millimeters so that the plane of focus of the microscope is now a little above the cells. Insert a microinjection pipette into the micropipette holder. Place the holder in the clamp of the micromanipulator and, using the pitch adjustment on the clamp, align the micropipette so that its tip projects into the optical axis of the microscope. 24

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5 . Look down the microscope while using the micromanipulator joystick to position the micropipette tip in the center of the field of view. Bring the micropipette tip into focus by moving the Z-axis of the micromanipulator. Slowly and very carefully, begin to rotate the Z-axis of the micromanipulator, lowering the micropipette tip toward the cells. As the cells come into focus, stop lowering the micropipette tip. Set the microinjector to provide a burst of injection pressure for as long as the footswitch is held down. Prior to injecting any cells, try a test injection to ensure that the DNA solution is flowing by pressing the microinjector footswitch 25

e. Injection procedure:

e. Injection procedure Identify a target cell within the field. Microinjection is a concert of three simultaneous actions: Twisting the Z-axis control of the micromanipulator joystick, lowering the micropipette tip toward the cell. Movement of the joystick so that the micropipette penetrates the cell. Depression of the footswitch of the microinjector to inject the DNA into the cell. 3. Immediately after injection, reverse the direction of movement of the joystick to withdraw the micropipette and release the footswitch. 26

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GENE GUN:

GENE GUN A device for injecting cells with genetic information. Often referred to as  bioballistics  or  biolistics . Is able to transform almost any type of cell, including plants, and is not limited to genetic material of the nucleus: it can also transform organelles, including plastids. Mostly applied for plant cells. However, there is much potential use in animals and humans as well. Gene guns have also been used to deliver DNA vaccines. 28

ELECTROPORATION:

ELECTROPORATION Electroporation designates the use of short high-voltage pulses to overcome the barrier of the cell membrane. By applying an external electric field, which just surpasses the capacitance of the cell membrane, transient and reversible breakdown of the membrane can be induced. This transient, permeabilized state can be used to load cells with a variety of different molecules, either through simple diffusion in the case of small molecules, or through electrophoretically driven processes allowing passage through the destabilized membrane – as in the case for DNA transfer. Some effort has been put into optimization of conditions for DNA electrotransfer, and the latest development in equipment opens possibilities to design various pulse combinations. There are three ways to deliver pulses for DNA transfer: Just short, high-amplitude pulses. Just long, low-amplitude pulses. Short, high-amplitude pulse followed by long, low amplitude pulses. 29

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IMPALEFECTION:

IMPALEFECTION derived from two words –impalement & infection method of gene delivery using  nanomaterials , such as carbon nanofibres , carbon nanotubes ,  nanowires . Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. Plasmid DNA containing the gene, intended for intracellular delivery, is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. Cells that are impaled by nanostructures can express the delivered gene(s). 31

PURE DNA CONSTRUCTS:

PURE DNA CONSTRUCTS The direct introduction of pure DNA construct into the target tissue. Efficiency of DNA uptake by the cells and its expression are rather low. Large quantities of DNA have to be injected periodically. The therapeutic gene produces the protein in the target cells which enter the circulation and get degraded. 32

DNA MOLECULAR CONJUGATES:

DNA MOLECULAR CONJUGATES The use of DNA molecular conjugates avoids the lysosomal breakdown of DNA. Large sized therapeutic DNA (>10 kb) can be delivered to target tissue Most commonly used synthetic conjugate is poly-L-lysine. 33

LIPOFECTION:

LIPOFECTION Technique used to inject genetic material into a cell by means of  liposomes , which are vesicles that can easily merge with the cell membrane since they are both made of a  phospholipid bilayer . Lipofection generally uses a positively charged (cationic) lipid to form an aggregate with the negatively charged (anionic) genetic material. Highly efficient, low toxicity, easy to use. 34

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SONICATION:

SONICATION The act of applying sound energy to agitate particles in a sample, for various purposes. Used to speed dissolution, by breaking intermolecular interactions. Used to disrupt cell membranes and release cellular contents – sonoporation . 36

POLYPLEXES:

POLYPLEXES Complexes of polymers with DNA are called polyplexes. protect the DNA from undesirable degradation during the transfection process. consist of cationic polymers 37

VIRAL METHODS:

VIRAL METHODS Also known as transduction Involve virus mediated gene transfer into a cell type cultured invitro Adenoviruses,Adeno -associated viruses,Baculoviruses,Herpes viruses & Retroviruses Much more efficient gene transfer rates than non viral gene delivery Criteria for the viral vectors :- Unlike their wild variant ,they have to be replication defective to prevent uncontrolled spreading invivo The virus itself should not possess undesirable properties The viral genome must be able to accommodate the therapeutic gene 38

RETROVIRUS VECTOR SYSTEM:

RETROVIRUS VECTOR SYSTEM 39

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Retrovirus is used as the vector by artificially removing the structural gene gag & pol are deleted and promoter gene is included Can carry a therapeutic DNA of maximum size 5kb Retroviral vector DNA can be used to transform the cells A plasmid in association with a retrovirus, a therapeutic gene and a promoter is referred to as plasmo virus which is capable of carrying a DNA less than 3.4 kb The target cells must be in a dividing stage Limitation – efficiency of delivery & integration of therapeutic DNA are very low 40

ADENOVIRAL VECTOR SYSTEM:

ADENOVIRAL VECTOR SYSTEM Good vector Infect most of the non dividing human cells Eg; common cold adenovirus Requires periodic administration of recombinant virus Efficiency enhanced by developing a virus that can specifically infect the target cells. 41

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42 OUTLINE OFGENE DELIVERY PROGRAM USING ADENOVIRUS

ADENO-ASSOCIATED VIRUS VECTOR SYSTEM :

ADENO-ASSOCIATED VIRUS VECTOR SYSTEM Human virus that can integrate into chromosome Single strand,non pathogenic,small DNA virus (4.7 kb) Good vector for the delivery of therapeutic genes Recombinant viruses are created by using two plasmids and an adenovirus (helper virus ) by a special technique. 43

HERPES SIMPLEX VIRUS VECTOR SYSTEM (HSV):

HERPES SIMPLEX VIRUS VECTOR SYSTEM (HSV) Have a natural tendency to infect in particular type of cells They infect and persist in non-dividing nerve cells. A human pathogen that causes cold sores & encephalitis An ideal vector for invivo gene therapy of many nervous disorders HSV has a double stranded DNA of about 152 kb length as its genome About 30 kb HSV genome can be replaced by a cloned DNA without lose of its basic charachteristics . 44

DISEASES & GENE THERAPY:

DISEASES & GENE THERAPY GENE THERAPY FOR INSULIN DEPENDENT DIABETES MELLITUS GENE THERAPY FOR CANCER GENE THERAPY FOR AIDS GENE THERAPY FOR NERVOUS SYSTEM DISORDERS ALZHEIMER’S DISEASE PARKINSON’S DISEASE GENE THERAPY FOR HEMOPHILIA B 45

GENE THERAPY FOR INSULIN DEPENDENT DIABETES MELLITUS:

GENE THERAPY FOR INSULIN DEPENDENT DIABETES MELLITUS Diabetes Mellitus – 2 types Type 1 – Insulin dependent ( auto immune disease due to destruction of insulin producing beta cells of pancreas) Type 2 – Non Insulin dependent Gene therapy is for type 1 diabete mellitus.Somatic gene therapy is attempted.Liver is the target organ. 46

GENE THERAPY FOR CANCER:

GENE THERAPY FOR CANCER Leading cause of death Uncontrolled growth and division of cells Some developments of gene therapy of cancer include:- Tumor Necrosis Factor gene therapy TNF-protein produced by human macrophages provide defense against cancer cells,brought out by enhancing the cancer fighting ability of Tumor Infiltrating Lymphocytes (TILS),a special type of immune cells TILS were transformed with a TNF gene (along with a neomycin resistant gene) – used for the treatment of malignant melanoma(cancer of melanin producing cells) some improvement observed 47

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Suicide gene therapy Suicide gene –encodes thymidine kinase enzyme TK phosphorylates nucleosides to nucleotides- used for synthesis of DNA GANCICLOVIR(GCV) has close structural resemblance to thymidine TK by mistake phosphorylates GCV to triphosphate -GCV - false unstable nucleotide Triphosphate -GCV inhibits DNA polymerase Elongation of DNA molecule stops at the point of false nucleotide Triphosphate -GCV enter and kills the neighbouring cancer cells ( bystander effect) Ganciclovir is thus used to kill the cancer cells Mainly used in brain tumors The vector used in this type of therapy is HSV (Herpes Simplex Virus) 48

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Gene Replacement Therapy The gene p53 codes for a protein with molecular weight 53 kilo dalton is considered to be a tumor suppressor gene. The protein which it encodes bind with DNA & inhibits replication Tumor development may be due to the damaged p53 gene Replacement of damaged p53 gene by a normal gene (by adenovirus vector system ) showed encouraging results. Eg:- in liver cancer 49

GENE THERAPY FOR AIDS:

GENE THERAPY FOR AIDS ACQUIRED IMMUNO DEFICIENCY SYNDROME,caused by Human Immunodeficiency Virus (HIV – RNA virus) Some developments include :- rev & env genes A mutant strain of HIV virus lacking rev & env genes are developed ( due to lack of these genes virus cannot replicate) T – lymphocytes from HIV infected patients are removed and mutant viruses are inserted into them. Modified T – lymphocytes are cultivated & injected into the patient Viruses cannot multiply as they lack essential genes but stimulate the production of CD8(Cluster Determinant antigent 8) cells which are killer lymphocytes. 50

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Gene to inactivate gp120 gp120 – glycoprotein present in the envelope of HIV Essential for binding of virus to host cell & replication F105 – gene that produce an antibody that inactivates gp120 Genes of HIV protein HIV protein synthesizing genes are attatched to DNA of mouse virus Genetically modified viruses are injected into AIDS patients HIV genes stimulate normal body cells to produce HIV proteins HIV proteins stimulate the production of anti HIV antibodies – prevent the HIV replication. 51

GENE THERAPY FOR NERVOUS SYSTEM DISORDERS:

GENE THERAPY FOR NERVOUS SYSTEM DISORDERS ALZHEIMER’S DISEASE Neuro degenerative disorder Treatment involves the delivery of nerve growth factor which prevents the neuronal in addition to the ameliorating deficits in learning & memory associated cells. PARKINSON’S DISEASE a progressive neurological condition resulting from the death of the cell that contains and produces dopamine in substantia nigra. Grafts of fetal substantia nigra dopamine neurons into the dopamine depleted brain indicated the delivery of dopamine from grafted cells could lessen the movement disturbances. 52

GENE THERAPY FOR HEMOPHILIA B:

GENE THERAPY FOR HEMOPHILIA B  Is a blood clotting disorder caused by a mutation of the Factor IX gene, leading to a deficiency of Factor IX which ultimately leads to severe bleeding. Normal treatment is replacement of factor IX but is a failure due to short half life. Using retroviral vector system,genes for the synthesis of factor IX were inserted into the liver cells of dogs The dogs no longer displayed the symptoms of hemophilia . 53

REFERENCES:

REFERENCES Pharmaceutical Biotechology by Daan J A Crommelin & Robert D Sindelar . Biotechnology by V.Kumaresan . Essentials of Biology & Biotechnology by Bir Bahadhur . Biotechnology by U.Satyanarayana . A textbook of Biotechnology by R.C Dubey . www.wikipedia.com http://www.genetherapynet.com 54

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