1. Gene Therapy Cystic Fibrosis

2. Key ideas Understand that cystic fibrosis is a genetic disease that results in the build-up of salt inside of cells. Understand that the adenovirus is the vector for gene transfer for the gene therapy of cystic fibrosis. Explain the problem with using adenoviruses for GE Describe the alternative method (liposomes) for gene transfer.

3. What is Cystic Fibrosis (CF)? Cystic fibrosis is a genetic disease that results in the build-up of salt inside of cells to produce abnormal amounts of excessively thick and sticky mucus within the lungs, airways and the digestive system.

4. What is Cystic Fibrosis (CF)? People with CF develop an abnormal amount of excessively thick and sticky mucus within the lungs, airways and the digestive system. The mucus causes impairment of the digestive functions of the pancreas and traps bacteria in the lungs resulting in recurrent infections which lead to irreversible damage. Lung failure is the major cause of death for someone with CF. Today, life expectancy for many people with CF has increased to 38 years.

5. Symptoms of CF People with CF may have the following symptoms: persistent cough, particularly with physical effort some difficulty in breathing or wheezing with effort tiredness, lethargy or an impaired exercise ability frequent visits to the toilet salt loss in hot weather which may produce weakness poor appetite

6. WHO CAN GET CF? CF is ‘autosomal recessive’ meaning that it occurs equally in males and females. The CF gene must be inherited from both parents and it can ‘skip’ generations. In Australia, one in 2,500 babies are born with CF, that’s one every four days. On average one in 25 people carry the CF gene - most of whom are unaware that they are carriers. Because carriers of CF are unaffected (and therefore show no symptoms) it is hard for them to appreciate that CF may be a real risk. Any of us could be a carrier and we wouldn’t know – think about it, that’s about 1 million unaware carriers – it could be you!

7. WHO CAN GET CF? Genetic diagram showing the gamete combinations from cystic fibrosis gene carrying parents r = recessive allele that causes cystic fibrosis

8. What Causes CF? The Cystic Fibrosis Transfer (CFTR) Gene creates a (CFTR) protein, a channel protein that controls the flow of H 2 O and Cl − ions in and out of cells inside the lungs. Disease-causing mutations in the CFTR gene alter the production, structure, or stability of the chloride channel. All of these changes prevent the channel from functioning properly, which impairs the transport of chloride ions and the movement of water into and out of cells. As a result, cells that line the passageways of the lungs, pancreas, and other organs produce mucus that is abnormally thick and sticky. The abnormal mucus obstructs the airways and glands, leading to the characteristic signs and symptoms of CF.

9. The CFTR protein is a channel protein that controls the flow of H 2 O and Cl − ions in and out of cells inside the lungs. CF is characterized by the buildup of thick mucus in the lungs. 1. CFTR protein is working correctly Ions freely flow in and out of the cells 2. CFTR protein is malfunctioning Ions cannot flow out of the cell due to a blocked channel

10. The CFTR Protein The CFTR protein functions as a channel across the membrane of cells that produce mucus, sweat, saliva, tears, and digestive enzymes. The channel transports negatively charged chloride ions into and out of cells. The transport of chloride ions helps control the movement of water in tissues, which is necessary for the production of thin, freely flowing mucus.

11. The CFTR protein functions as a pump for Cl - ions.

12. The CFTR Protein Those with cystic fibrosis have an imbalance of salt in their bodies caused by the defective CFTR protein. Because there is too little salt and water on the outside of the cells, the thin layer of mucus that helps keep the lungs free of bacteria becomes very thick and difficult to expel by coughing. This thick mucus can clog the airways and lead to dangerous infections.

13. GENE THERAPY FOR CYSTIC FIBROSIS There is currently no cure. From birth, a person with CF undergoes constant medical treatments and physiotherapy. Approximately half of those who suffer from cystic fibrosis in Australia will die from lung disease by their late thirties. Airway gene therapy is a potential treatment for lung disease caused by cystic fibrosis, and hopefully provide an effective and safe cure for this devastating illness. Treatment generally involves: Intensive daily physiotherapy to clear the lungs Enzyme replacement capsules with food to aid digestion Antibiotic therapy to treat lung infections Aerosol mist inhalations via a nebuliser to help open the airways Salt and vitamin supplements A nutritious, high calorie, high salt, high fat diet Exercise – important to help clear the airways and build core strength.

14. Gene Therapy The genetic engineering of human cells to treat genetic diseases is called Gene Therapy. This involves introducing a good copy of a gene to compensate for the bad copy that is present due to a genetic disorder. Gene therapy involves inserting healthy and correctly functioning cystic fibrosis genes into cells that line the airways. Once established, it is hoped this new genetic information will remedy the basic cellular defect that causes cystic fibrosis and lung disease

15. Gene Therapy The current problem is to find a way to successfully ‘deliver’ the working version of the gene. To begin with, the affected cells are taken from the person’s body and the working version of the gene is either ‘spliced’ or injected into these cells. They are left to grow in the laboratory and then replaced into the person.

16. Gene Therapy A vector is needed to introduce the corrected gene in to cells of the lung 1.Viruses as Vectors Harmless virus- adenovirus/adeno associated virus 2. Liposomes as Vectors

17. 1. Viruses as Vectors for Gene Therapy One promising technique is to put the working gene inside a harmless virus, which has had most of its own genes removed – it has been ‘deactivated’. A virus that causes disease (such as the common cold) works by slipping into a cell, taking over its DNA and forcing it to produce more viruses. Similarly, a deactivated virus can enter the specific cell and deliver the working gene.

18. 1. Viruses as Vectors for Gene Therapy 1.Modify adenovirus’ genes involved in replication. This makes them harmless so they can not replicate and cause illness. 2.Grow adenoviruses in epithelial cells in a lab. Add recombinant plasmids that contain functional CFTR gene. 3.Recombinant plasmid taken up by adenovirus. Gene becomes part of virus DNA. 4.Viruses isolated from epithelial cells and purified. 5.Virus sprayed into the nostrils of patients via aerosol 6.DNA (including normal CFTR gene) injected into epithelial cells of patients lungs.

19. 1. Viruses as Vectors for Gene Therapy Genetic diseases such as cystic fibrosis are being treated using genetically engineered adenoviruses as vectors.

20. 1. Viruses as Vectors for Gene Therapy A modified adenovirus is used as the vector for transferring the healthy cystic fibrosis transfer gene (CFTR gene) into the lung cells of a CF patient. Adenovirus is used because it can specifically infect lung cells. Once inside the cell, the healthy CFTR gene makes the normal protein that permits the cell membrane to allow salt to leave the cells to restore the correct water levels inside the lung cells.

21. 1. Viruses as Vectors - concerns Adenoviruses may cause infection Adenoviruses may triggering immune response or patients may develop an immunity to them making them useless in future treatments Patients have to undergo multiple rounds of gene therapy as effects short lived Problems associated with using adenoviruses to transfer the genes have seen the use of liposomes as an alternative method.

22. 2. Liposomes as Vectors Liposomes are synthetic fat droplets into which the genes can be hidden. Cells will accept the liposomes as food and consequently take in the hidden genes.

23. 2. Liposomes as Vectors 1.Normal CFTR genes isolated from human cells & inserted into plasmids 2.Recombinant plasmids are placed back into bacteria. Gene markers pick up the cells that contain the plasmid with the CFTR gene. 3.Bacteria cloned. 4.Plasmids extracted & wrapped in lipid molecules (forming a liposome) 5.Liposomes sprayed into patients airways via a nasal aerosol. 6.Liposomes pass easily through the plasma membrane of cells and nucleus.

24. Liposomes

26. Gene Therapy for CF Summary

27. Stem Cells? Other techniques involve using stem cells. These are immature cells that have the potential to develop into cells with different functions. In this technique, stem cells are manipulated in the laboratory to accept new genes that can then change their behaviour. For example, a gene might be inserted into a stem cell that could make it better able to survive chemotherapy. This would be of assistance to those patients who could benefit from further chemotherapy following stem cell transplantation