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GENE THERAPY

GENE THERAPY. Methods for treatment of human diseases Current methods: 1 Medicine tablets by oral 2 Medicine by injection. 3.Medicine by nasal spray 4 Medicine by applying to skin. Methods in the future: 5 Cell therapy 6 Gene therapy. GENE THERAPY.

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GENE THERAPY

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  1. GENE THERAPY

  2. Methods for treatment of human diseases Current methods: 1 Medicine tablets by oral 2 Medicine by injection. 3.Medicine by nasal spray 4 Medicine by applying to skin. Methods in the future: 5 Cell therapy 6 Gene therapy

  3. GENE THERAPY

  4. Gene therapy:Transfer of therapeutic gene into the diseased tissue Hemophiliac dogs with coagulation factor IX deficiency Normal dog: blood clots in about 8 to 10 minutes Diseased dog: blood clots in about 50 to 60 minutes 1-hour procedure of GT infusion, 15 month of expression, 20 min for clotting Dr. Kenneth Brinkhous (North Carolina Univ)

  5. Diseases for applying gene therapy Disease Defect Target cell Severe combined Adenosine deaminase 4 Bone marrow cells or immunodeficiency T-lymphocytes Hemophilia Factor VIII, Factor IX deficiency Liver, muscle, fibrob. Cystic fibrosis Loss of CFTR gene Airspaces in the lung Hemoglobulinpathies or  globulin gene Bone-marrow cells 1-antitrypsin deficiency 1-antitrypsin Lung or liver cells Cancer Many causes Many cell types Neurological diseases Parkinson’s, Alzheimers Direct injection into the brain Cardiovascular Restenosis, arteriosclerosis Vascular endothelium Infectious diseases AIDS, hepatitis B T cells, macrophages, Liver cirrhosis Fibrogenesis Hepatocyte growth factor Autoimmune disease Lupus, diabetes MHC, 2-microglobulin

  6. In humans Cancer 69%

  7. The Food and Drug Administration (FDA) has not yet approved any human gene therapy product for sale. • 2) Immune response. It reduces gene therapy effectiveness and • makes repetitive rounds of gene therapy useless • 3) Problems with viral vectors . Toxicity, immune and inflammatory responses, also fears that viral vector may recover disease-causing ability 4) Multigene disorders. Most commonly occurring disorders, such as heart disease, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. • 1) Short-lived nature of gene therapy. Very hard to achieve any long-term benefits without integration and even with it. General concerns Four major problems with gene therapy:

  8. Gene therapy could be very different for different diseases • Gene transplantation • (to patient with gene deletion) • Gene correction • (To revert specific mutation in the gene of interest) • Gene augmentation • (to enhance expression of gene of interest) • Targeted killing of specific cells by introducing killer gene • Gene ablation – targeted inhibition of gene expression

  9. Gene therapy In vivo Ex vivo

  10. EX VIVO IN VIVO in vivo and ex vivo schemes http://laxmi.nuc.ucla.edu:8237/M288/SChow_4_10/sld005.htm

  11. In vivo gene therapy 1. The genetic material is transferred directly into the body of the patient 2. More or less random process; small ability to control; less manipulations 3. Only available option for tissues that can not be grown in vitro; or if grown cells can not be transferred back

  12. Ex vivo gene therapy • The genetic material is first transferred • into the cells grown in vitro 2. Controlled process; transfected cells are selected and expanded; more manipulations 3. Cells are usually autologous; they are then returned back to the patient

  13. Transgenes Integrated Not integrated - stable expression; may provide a cure for episomes (plasmids), Random mutagenesis Is not an issue - Random insertions in heterochomatin Gene can be inactivated; In euchromatin -- Can disrupt important host genes; Long-term consequences are unknown - Expression is transient; Repeated treatments nesessary

  14. How episomes and integrated trasgenes behave in dividing cells Integral transgene Episome Loss of plasmid

  15. Methods of gene delivery (therapeutic constructs) 1 Injection of naked DNA into tumor by simple needle and syringe -- DNA coated on the surface of gold pellets which are air-propelled into the epidermis (gene-gun),mainly non applicable to cancer 2 DNA transfer by liposomes (delivered by the intravascular, intratracheal, intraperitoneal or intracolonic routes) 3 Biological vehicles (vectors) such as viruses and bacteria. Viruses are genetically engineered so as not to replicate once inside the host. They are currently the most efficient means of gene transfer.

  16. 5. Transgene should be responsible for its regulatory elements (on/off system) 6. Vector should be able to target specific cell types Desirable characteristics of gene delivery vector 1. High titer or concentrations (>108 particles/ml) 2. Easy and reproducible method of production 3. Precise and stable introduction of transgene 4. Vector should not elicit immune response in the host

  17. Injections of naked DNA

  18. Covalently closed circular form is more stable that open plasmid • Intravascular delivery •  liver and muscle • Intramuscular delivery Naked DNA gene therapy -- Results in a prolonged low level expression in vivo -- Very cheap

  19. Passive Active to increase the pre-existing immune response to the cancer initiates an immune response against an unrecognised or poorly antigenic tumor DNA vaccines Cancer immunotherapy Antiviral and antibacterial (But, traditional vaccines are better when available)

  20. www.malaria-vaccines.org.uk Current attempts with naked DNA vaccination in infectious diseases Tuberculosis, Lyme disease Helicobacter pylori Malaria HIV hepatitis B and C Influenza Papilloma Cytomegalovirus T cells recognise liver cell with malarial parasite inside Produce IFN-gamma IFN-gamma stimulate antigen presentation

  21. several peptide epitopes that we know are recognised by T-cells (a so-called multi-epitope string) DNA vaccines (and other vaccines too) prime immune system with properly presented antigen immunologically important components of the malaria pathogen whole protein called thrombospondin related adhesion protein (TRAP). DNA vaccine encoding an immuno recognisable insert

  22. Ballistic DNA Injection, particle bombardment, microprojectile gene transfer (gene guns) Invented for DNA transfer to plant cells Fully applicable to mamalian cells plasmid DNA is precipitated onto 1-3 micron sized gold or tungsten particles. Discharge: helium pressure, or high-voltage electronic Light micrograph of DNA-coated gold beads in the skin after gene-gun vaccination

  23. Duchenne muscular dystrophy (DMD) X-linked recessive disorder; 1/3500 boys worldwide About 30% of cases represent new mutations. Absence of dystrophin, a cell membrane protein (approximately 0.01 % of skeletal muscle protein). All muscles involved 1. Generalized weakness and muscle wasting affecting limb and trunk muscles first. Wheels at 12 y.o. Life threatening dysrhythmia or heart failure develops in about 10 %. Death at 10th-20th after pulmonary problems (breathing)

  24. Why muscles are enlarged in DM patients? Increased fibrous connective tissue revealed by this trichrome stain. There are larger overly contracted muscle fibers with scattered small degenerating or regenerating fibers Degenerated muscles contain lots of fibrous and adipose tissue

  25. Normal muscles and DM muscle muscles stained for dystrophin with monoclonal antibodies dystrophin is not evident myofibers are circumscribed by the darkly-staining dystrophin wider variation in myofiber diameters increased connective tissue

  26. Dystrophin Provide links between the intracellular cytoskeleton and the actin filaments with the extracellular matrix Whole complex stabilizes the membrane. Laminin2α2: congenital MD chr 6 Sarcoglicans: Limb Girdle MDs (4 types) Duchenne and Becker MDs

  27. DM is good model disease as ballistic GT is available for muscles Problem: Native gene is 2,4 Mb in size (quite unusual) mRNA is 14 kb in size (also too big for any vector) IDEA: Dystrophin can retain significant function even when missing large portions of its sequence (Becker’s phenotype) Becker’s phenotype is anyway better than complete Duchenne ! Patient: exon 17–48 removed (48% of the coding region),

  28. Deletion variants of dystrophin for GT ABD= actin-binding domains most, but not all, of the spectrin-like repeats are dispensable for the function of dystrophin. Scott Harper et al., 2002

  29. GT with dystrophin minigene in mice with DM phenotype MDX mice with premature stop codon in exon 23; no dystrophin GT treated Non-treated

  30. Jun. 05, 2003 French Muscular Dystrophy Association (AFM) and Transgene announced that the results of their Phase I trial on gene transfer for Duchenne/Becker's Muscular Dystrophy Nine patients in three groups: Muscle segment were taken out for examination a single injection of low dose of plasmid with dystrophin a single injection of high dose of plasmid with dystrophin Two injections of high dose each of plasmid with dystrophin Expression of dystrophin is found in 1 to 10 percent of muscle fibers for group 1 and 2 ; for all 3 patients in group 3 No immune reactions; no side effects !!!!

  31. Liposomes Next level idea – why naked DNA? Lets’ wrap it in something safe to increase transfection rate Lipids – is an obvious idea ! Therapeutic drugs

  32. Liposomes are formed by the self-assembly of phospholipid molecules in an aqueous environment. Anionic liposome www.emc.maricopa.edu/faculty/ farabee/BIOBK/

  33. Cationic liposomes Positively charged lipid heads • positively charged lipid droplets • can interact with negatively charged DNA • to wrap it up and deliver to cells Inside liposomes DNA is resistant to degradation Lipofectin, lipofectamine, lipofectase….

  34. Lab procedure for liposome preparation

  35. Liposome disadvantages Liposomes are rapidly cleared from the circulation and largely taken up by the liver macrophages How to overcome it? liposome surface ligands decrease degradation (monosialoganglioside or polyoxyethyle)

  36. Modified liposomes (stealth liposomes) hydrophilic polyoxyethylene lipids incorporated into liposome Increased half-life is due to a reduced coating (opsonisation) of these liposomes by plasma proteins cholesterol, polyvinyl-pyrrolidone polyacrylamide lipids, glucoronic acid lipids are working the same…. So liver cells not able to uptake them

  37. Complex multilayer liposomes (Piedmont) Able to transport medication through the epidermal and dermal layers of the skin via the lipid-rich intercellular channels. The medication can be directed specifically to the targeted area

  38. Cochleates – multilayer lipid rolls 1. Storageable without any problems – could be lyophilized (at least one year as a lyophilized powder at room temperature)! 2. Durable – survive multiple membrane fusion event (fuse-release drug-disengage-fuse-release..) 3. Can survive in GI tract Cochleates have been shown to be an effective oral delivery system.

  39. Immunoliposomes for active targeting Antibodies to intracellular myosin target liposomes to infarcted areas of heart Antibody against tumor specific molecules will target them to tumors

  40. Liposomes could serve as tumor specific vehicles (even without special targeting) Liposomes better penetrate into tissues with disrupted endothelial lining

  41. delivery of genes by liposomes Cheaper than viruses No immune response Especially good for in-lung delivery (cystic fibrosis) 100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector

  42. Cystic fibrosis most common lethal genetic disorder in Caucasian populations (1 in 2000 live births.) . Among African and Asian is really rare a defect in the CFTR gene (cystic fibrosis transmembrane conductance regulator ) irregular chloride and sodium ion conductance in epithelial cells of many organs (increased uptake of sodium ions). Sweat glands (too much salty secretion) Lungs create thick mucus secretion (prone to infections, constant cough, leading cause of death) Pancreas is damaged (leads to diabetes) Digestive tract (constipation)

  43. Lungs in cystic fibrosis CF lungs Normal lung Normal alveolar appearance dilated crypts filled with mucus and bacteria. lung did not collapse when it was removed postmortem CF lungs filled with mucus

  44. Cystic fibrosis lungs are prone to infections Pseudomonas aeruginosa easily colonise mucus in the dilated lungs Neutrophiles are activated, then overactivated The battle between neutrophils and bacteria leads ultimately to lung fibrosis and damage "Hyperinflammation" as recruited neutrophils unable to eradicate bugs, instead damage lung tissue. Mucus protects bugs and promotes hypermutation

  45. Pancreas in cystic fibrosis Normal pancreas Distended CF cripts filled with mucus Pancreatic enzymes not able to leave the gland; they damage the gland Less insulin produced Impaired glucose tolerance and diabetes Survivors to 25 years old: 1/3 with impaired glucose tolerance and with 1/3 diabetes

  46. Treatment of CF 1. Many different antibiotics are required to clear infections. Staphylococcus aureus, Haemophilius influenzae, Aspergillus fumigatus - the same picture…

  47. CFTR gene (chromosome 7) 27 exons, 1480 aa ATP binding domain

  48. CFTR product Delta F508 mutation 50% of all patients are homozygotes with this mutation 30% are heterozygotes, delta F508/X an incompletely folded, protease-sensitive form; Rapidly degrades before entering Golgi complex

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