1. 16.5 Gene therapy 10.1 Coordination.

2. Learning outcomes By the end of this lesson I will know – The use of gene therapy is to supplement defective genes. That cystic fibrosis (CF) is caused by a recessive defective gene, resulting from a deletion mutation How to evaluate the effectiveness of gene therapy. Success criteria By the end of this lesson I can Describe the differences in amino acids resulting from mutations. Complete exam questions on cystic fibrosis List the stages of gene therapy Evaluate the effectiveness of gene therapy Complete gene therapy application

3. Starter Normal DNA – TAC GGG AAT TCG mRNA - AUG CCC UUA AGC Amino acid - Met – Pro – Leu – Ser What kind of mutations are these examples of? 1. Normal DNA – TACGGGAAATCG 2. Normal DNA – TACGGGTAATTCG 1.Substitution 2.Addition Describe the differences in amino acids, which has more effect and why?

4. Gene therapy replaces defective genes with healthy ones to treat genetically inherited diseases. The cause of the disease is tackled not just the symptoms. Cystic fibrosis is an inherited disease that can be treated by gene therapy. Gene therapy has been used in the treatment of cancer and recently, in 2008 to treat genetic blindness. What is gene therapy?

5.  Cystic fibrosis is a recessive genetic disease which affects 1 in 2000 people in the UK.  Symptoms include: ◦ Production of thick sticky mucus from the cells that line respiratory system resulting in congestion of the lungs, breathing difficulties and higher risk of infection. ◦ Blockage of the pancreatic duct with mucus which can lead to a reduced release of pancreatic juice and hence inadequate digestion. Also prevents pancreatic enzymes from reaching the duodenum and leading to the formation of fibrous cysts. ◦ The reproductive system is also blocked, preventing sufferers having children.

6.  The carrier protein cystic fibrosis transmembrane- conductance regulator (CFTR) is used to carry chloride ions (Cl - ) across epithelial cells and water naturally follows by osmosis, so epithelial cells are kept moist.  CF is caused by a mutation in the gene that codes for this protein (CFTR).  Cystic fibrosis (CF) is caused by a recessive defective gene, resulting from the deletion of a base triplet (adenine-adenine-adenine)  The deletion of the base triplet means that an amino acid is omitted from the coded protein.  Why would this affect the function of the protein?  This affects the 3D shape of the protein so that chloride ions can’t move out of the cell.

7. CFTR channel proteins Water Chloride ions Sodium ions Water Cell surface membrane Epithelial cell cytoplasm CFTR channel proteins Mucus layer Airway Epithelial cell in a healthy person

8. Cell surface membrane Epithelial cell cytoplasm Thick and sticky mucus layer Airway Epithelial cell in a person with cystic fibrosis CFTR channel proteins Chloride ions channel blocked Sodium ions Non- functional CFTR channel proteins Water does not pass into mucus

9.  CF sufferers retain chloride ions in their cells, causing them to retain water in their cells, which would normally leak out of the cells into the thin layer of mucus lining the airways.  With less water the mucus layer becomes stickier and thicker and cannot be moved out of the lungs by the beating cilia lining the airways.  Answer exam questions  Peer mark answers

10. There are 2 ways in which gene therapy can be used to treat cystic fibrosis 1) Gene replacement – defective genes are replaced by healthy ones 2) Gene supplementation – one or more copies of the healthy gene are added alongside the defective gene. The added genes are dominant so they mask the effects of the recessive alleles.

11.  1) Germ-line therapy – replacing/supplementing the defective gene in the fertilised egg. All cells will develop normally and the cells of the offspring  2)Somatic-cell therapy – targets the affected tissues, such as the lungs, so it is not passed onto future generations. Long term is to target undifferentiated stem cells so treatment is effective for the life span of the individual.  Name at least one issue with each type of therapy 1) Moral and ethical issues 2) Treatment needs to be repeated – limited success

12. It involves inserting cloned functional CFTR genes into epithelial cells of the lungs to replace defective genes. 2 techniques are being tested: (1) Wrapping CFTR genes in lipids that can be absorbed through the cell surface membranes. (2) Inserting CFTR genes into harmless viruses called adenoviruses that are then allowed to ‘infect’ the cells.

13. Adenoviruses are used as they are well adapted for infecting the cells of the airways in the lungs, normally causing colds and respiratory diseases. 1) The adenoviruses must be modified so the virus doesn’t replicate. 2)The viruses are cultured in the lab in epithelial cells. They are exposed to plasmids containing the normal CFTR gene, which is then incorporated into the viral DNA. 3)The viruses are extracted from the cells in the lab, purified and sprayed into the lungs, where they infect epithelial cells, taking the CFTR gene into the cells. 4)The CFTR channel protein is made as normal, but as the viruses do not replicate, no damage is done to the cell.

14.  Infection could occur if the genes that allow the virus to replicate have not been completely inactivated.  Viruses have a very high mutation rate so can become virulent again.  Patients treated repeatedly may develop antibodies to destroy the viruses making them resistant to the treatment.

15. Recombinant DNA technology is used to put healthy CFTR genes into plasmids which are wrapped in liposomes. Liposomes are tiny lipid droplets that fuse with the cell membrane, they can easily pass through the phospholipid bilayer (1)CFTR gene isolated from healthy human tissue, cloned and inserted into plasmids (2)Gene is inserted into liposome (fluid filled pouches made by mixing lipids with water) (3)Aerosol spray into lungs (4)Liposomes membrane contain sugars recognised by cell membrane receptors (5)Liposomes enter cell by endocytosis - genes are transported across the cells Use the diagram on page 164 to list the stages of wrapping the gene in lipid molecules

16. 2 problems must be overcome before we can effectively use liposome vectors for cystic fibrosis: 1) The aerosol spray must be fine enough to get the liposomes through the very narrow bronchioles of the lungs and into the alveoli. 2) Only a small proportion of the genes that are absorbed are expressed.

17.  SCID is a rare inherited disorder, individuals can’t show a cell-mediated immune response and are unable to produce antibodies  SCID can be caused by mutations in a gene that encodes for an enzyme called adenosine deaminase (ADA). ADA is essential for the metabolic function of a variety of body cells, but especially T- cells. The absence of this enzyme leads to an accumulation of toxic metabolic by-products within lymphocytes that cause the cells to die. ADA deficiency is the second most common cause of SCID, accounting for 15% of cases.  http://news.bbc.co.uk/1/hi/sci/tech/728512.stm http://news.bbc.co.uk/1/hi/sci/tech/728512.stm Use page 264 write down the process of gene therapy And highlight the problems with this type of therapy

18.  In pairs read through the application of gene therapy and answer the questions  Discuss