Learning Intentions What causes cystic fibrosis? How to treat cf using Gene Therapy Differences between Germ-Line and Somatic Cell Gene Therapy

Cystic Fibrosis Cystic fibrosis is caused by a recessive allele. 3 bases (A-A-A) are missing – a deletion mutation. Causes 1 amino acid to be missing = a faulty protein. (The CFTR chloride ion channel protein) Prevents Cl ion channel from opening. Less water enters the cell membrane. The membranes stay dry and the mucous is sticky.

To Treat Cystic Fibrosis: Gene replacement – replace the faulty gene Gene supplementation – put a dominant gene next to (not replacing) the faulty one

How to replace/supplement the faulty gene Germ-Line Gene Therapy. Replace/supplement the faulty gene in a blastocyst, sperm or egg. All genes will then develop normally. Faulty gene not passed onto next generation. Currently against the law.

Somatic-Cell Gene Therapy Targets tissues affected – eg lungs. Faulty gene still passed onto future generations. Has to be repeated.

Getting the cloned, healthy gene into the patient. Using a harmless virus – (eg an adenovirus which causes respiratory diseases). The correct gene is introduced, using the virus as a vector, with a nostril spray (USA) or Using a lipid molecule to carry the gene in a plasmid = a liposome. Also via a nostril spray (UK)

Harmless virus Grown with plasmid with normal CFTR gene Viruses that take up gene are purified Into patient Adenovirus injects DNA into epithelial cells of lungs or Normal CFTR gene into bacterial plasmid Cloned Plasmids taken out Wrapped in lipids = liposome Into patient Liposome passes across cell membrane of lung epithelial cells

The technique of somatic gene therapy involves inserting a normal gene into the appropriate cells of an individual affected with a genetic disease, thereby permanently correcting the disorder. Figure 1.2 outlines the simplest methods of getting genes into the person's cells using either viruses (which carry the human gene, in place of one of their own genes, into a cell) or liposomes (small fat-like molecules which can carry DNA into a cell). In some cells, the gene or genes become inserted into a chromosome in the nucleus. The target cells might be bone marrow cells, which are easily isolated and re-implanted. Bone marrow cells continue to divide for a person's whole life to produce blood cells, so this approach is useful only if the gene you want to deliver has a biological role in the blood. Delivery of a gene that has a biological role in, say, the lungs, muscle, or liver would have to occur within those target organs. In many cases, accessing the appropriate tissue or, if the gene is required in multiple tissues (e.g. muscles throughout the body) ensuring it can be delivered where it is needed, is a major problem

Severe Combined Immunodeficiency (SCID) SCID is inherited. Sufferers cannot produce antibodies. They do not have the gene to produce the enzyme ADA, that destroys pathogen toxins.

Gene therapy for SCID : Isolate/cut out healthy ASA gene using restriction endonuclease Insert into virus Grow in lab (in vitro) Mix with patient’s white blood cells (T Cells) or bone marrow stem cells (more recent) Inject into patient

In Vitro In vitro