Gene Therapy

1. Gene Therapy Outcomes: -describe and explain the causes and symptoms of c.f. -compare germline and somatic gene therapy and describe the techniques used for each. -construct a flow chart/diagram showing the stages in gene therapy for cystic fibrosis

2. Cystic fibrosis • Common; 1 in 20,000 white people have CF • Caused by mutant recessive allele, in which three DNA bases (AAA) are missing • Normal CFTR gene produces a protein with 1480 amino acids • The mutation results in a single amino acid being omitted from the protein • This is enough to prevent the protein transporting chloride ions across epithelial cells • What kind of mutation causes CF…? • A deletion mutation

3. Symptoms of CF… • CFTR is a chloride ion channel protein that transports chloride ions out of epithelial cells • Water follows by osmosis • Therefore epithelial membranes are normally kept moist. • In CF, however, the membranes remain dry and any mucus they make is viscous & sticky. • What are the symptoms of CF? • Mucus congestion in the lungs; infection; poor gas exchange; blocked pancreatic ducts; poor digestion; mucus in sperm ducts; infertility

4. Gene therapy – various kinds • Gene replacement • Gene supplementation – healthy dominant genes added alongside the defective recessive gene. • Why does gene supplementation work? • The effects of the recessive allele are hidden by the dominant allele.

5. Gene therapy – various kinds • Germ-line therapy – replacing or supplementing defective genes in a fertilized egg. • Why is this a more permanent solution? • All the cells which develop from the egg will contain the supplementary genes as will the offspring / future generations • Germ-line gene therapy is currently banned – why? • The moral / ethical implications of long term genetic change; “designer babies”

6. Gene therapy – various kinds • Somatic-cell gene therapy • This targets just the affected tissue e.g. lungs. • Added gene not present in the gametes, not passed to future generations • Why does somatic-cell gene therapy need to be repeated? • E.g. Lung cells continually die / are replaced • If the defective genes are not replaced, why does this kind of gene therapy work? • E.g. lung cells produce both non-functional AND functional protein

7. CF and somatic-cell gene therapy… • Do you remember from GCSE…? • How can cloned normal CFTR genes be delivered into lung cells? • Using a harmless virus… • Wrapping the gene in lipid molecules…

8. Using a virus to deliver genes… • Adenovirus made harmless • Adenovirus grown in epithelial cells, along with plasmids containing the normal CFTR gene • CFTR gene becomes incorporated into the DNA of the adenovirus • Viruses isolated and purified • Modified virus given to the patient • Adenovirus inject their DNA, containing the normal CFTR gene, into the lung epithelial cells. • How do you think the modified virus is administered to the patient? • Nasal spray

9. Using lipids to deliver genes… • Normal CFTR genes isolated from healthy human tissues • CFTR genes inserted into bacterial plasmid vectors, along with a gene marker • Modified plasmid re-introduced back into their bacterial host • Bacteria cultured / in vivo cloning of the plasmid & CFTR gene • Plasmids extracted and wrapped in lipid to make a liposome • Administered to the patient (aerosol / inhaled) • Why do the liposomes pass into the lung epithelial cells? • Because lipid molecules can easily pass through the phospholipid portion of the cell surface membrane

10. Drawbacks… • Adenoviruses may cause infections • Patient’s immune system may attack modified virus • Liposome aerosol not fine enough to enter bronchioles • Once CFTR genes are delivered into the target cells, few are expressed • What does “CFTR” stand for? • Cystic fibrosis trans-membrane-conductance regulator • What else has gene therapy been used to cure? • SCID page 265

11. Gene therapy can be used to treat SCID

12. Effectiveness of gene therapy… • Effect is short-lived / repeat treatment needed (with somatic-cell gene therapy) • Can cause immune response / rejection of the gene itself, or of the vector or liposome. This is made worse by the secondary response of antibodies. • Viruses may cause toxic, inflammatory or immune responses. Also disabled viruses may recover their pathogenicity • Only a few of the genes reaching the target cells are actually expressed • Gene therapy is most effective on diseases cause by a single gene mutation: why is this a drawback? • Many diseases such as arthritis / CHD / diabetes are caused by variations in a number of genes

13. Ethics… • What is normal and what is a disability? Red hair? Black skin? Who decides? • Should we cure or prevent disability? Isn’t it just part of normal species variation? • Gene therapy research / treatment is very expensive… Shouldn’t we spend money on proven treatments..? • Germ-line therapy would be very effective, but what could be the long term effect of introducing inheritable genes into a population? Could we select one race in preference to another? (eugenics)

14. The spec…

15. Questions… • Explain how two parents without CF can have a child with CF • Why does somatic-cell gene therapy fail to provide a permanent cure for CF? • Describe two ways that a normal CFTR gene can be delivered to the lungs of a patient with CF • Why does the secondary immune response provide a particular problem for patients undergoing gene therapy? • Which drawbacks of somatic-cell gene therapy do not apply to germ-line gene therapy?