Transcript 10. Coordinatrion

16.5 Gene therapy
10.1 Coordination.
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
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.
2.
Substitution
Addition
Describe the differences in amino acids, which has more
effect and why?
What is gene therapy?
• 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.


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.
The carrier protein cystic fibrosis transmembraneconductance 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.

Epithelial cell in a healthy person
Water
Chloride ions
CFTR
CFTR
channel
channel
proteins
proteins
Airway
Sodium ions
Water
Mucus
layer
Cell surface
membrane
Epithelial cell
cytoplasm
Epithelial cell in a person with cystic fibrosis
NonCFTR
functional
channel
CFTR channel
proteins
proteins
Chloride ions
channel blocked
Airway
Sodium ions
Water does not pass
into mucus
Thick and sticky
mucus layer
Cell surface
membrane
Epithelial cell
cytoplasm




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
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.



1)
2)
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
Moral and ethical issues
Treatment needs to be repeated – limited success
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.
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.



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.
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
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.
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 Tcells. 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
Use page 264 write down the process of gene therapy
And highlight the problems with this type of therapy



In pairs read through the application of gene
therapy and answer the questions
Discuss