Gene Therapy

Question 1

What is cystic fibrosis?

Answer 1

What is cystic fibrosis? A genetic disease affecting the exocrine glands. It causes the production of abnormally thick mucus, leading to the blockage of the pancreatic ducts, intestines, and bronchi and often resulting in respiratory infection.

Question 2

Why does CF cause a respiratory problems?

Answer 2

Missing triplet in the gene coding for chloride pumps
CFTR protein not formed properly
Chloride ions are not pumped to the inside surface of the lungs/out of epithelial cells
The water potential of the mucus is not lowered, so water does not enter the mucus from the cell by osmosis
Causes the mucus to be thick and sticky
Inhibits respiration (as moist surface req. on alveoli for gas ex.)

Question 3

What is the aim of gene therapy?

Answer 3

To treat a genetic disease by replacing defective genes in a patient with copies of a new DNA sequence or by supplementing defective genes with non defective ones

Question 4

State two possible methods for replacing defective genes

Answer 4

Somatic cell therapy and germ line therapy

Question 5

What are the day to day treatments of CF?

Answer 5

Antibiotics used to fight off infections
Inhaling enzymes that break down the mucus allowing it to be coughed up
Steroids
Diet high in calories and rich in fat and protein as not much absorbed
Insulin and other drugs to treat symptoms of CF like diabetes
Daily chest physiotherapy massage to keep airways open

Question 6

Define: Gene therapy

Answer 6

The deliberate ‘repair’ or replacement of damaged genes

Question 7

Describe how germ line therapy works

Answer 7

Replacement of genes in a blastocyst or eggs/sperm
Blastocyst/gametes removed and screened
Cell from blastocyst removed/gametes isolated
Cell/gametes are treated by insertion of new gene
IVF (rest of a blastocyst can be disposed of) and (re)implantation

Question 8

Describe the advantages and disadvantages of germ line therapy

Answer 8

+ Child is born completely healthy with no chance of having the disease (no symptoms)
- Ethical issues with altering blastocysts

Question 9

Describe somatic cell therapy

Answer 9

Healthy allele is added to the affected cells (eg via an asthma pump). Supplementation
This requires repeated application of the gene as the cells have limited life spans and do not pass the supplemented “healthy” allele onto the next generation

Question 10

State two delivery methods for somatic cell therapy used to treat CF in the lungs

Answer 10

Using (adeno)viruses which have had the healthy CFTR allele inserted
Using liposomes containing plasmids with the healthy CFTR allele
Both delivery methods sprayed into lungs

Question 11

What are some symptoms of CF?

Answer 11

Build up of mucus in the lungs, pancreatic duct and sperm ducts
Pancreatic duct is blocked so enzymes don’t reach duodenum and digestion is incomplete/impaired
Large apetite
Congestion and difficulty in breathing due to mucus in lungs
Increased likelihood of lung infection

Question 12

Describe how adenoviruses are used to treat CF in the lungs

Answer 12

Healthy CFTR gene removed from donor cell of a person who does not have CF
The gene is cut out using restriction enzymes
Viruses are grown in a sample of epithelial cells
Plasmids extracted and cut using same restriction enzymes to produce the matching sticky ends
DNA ligase used to splice DNA fragment to plasmid
Plasmids taken up by adenoviruses and gene becomes part of viral DNA
Viruses isolated and purified and then sprayed/inhaled into lungs
Viral DNA passed to epithelial cells’ nuclei

Question 13

What are the advantages and disadvantages of using adenoviruses to treat CF?

Answer 13

+ Provides treatment of disease/relieves symptoms without genetic screening of embryoes

• Immune system can develop resistance/create antibodies for the viruses so the virus type must be changed often for treatment
• Delivery in inefficient, unsure if the allele is delivered and not all epithelial cells’ DNA will be changed
• Non-permanent fix - supplemented gene not passed onto the next generation
• Virus may revert to a disease causing form

Question 14

Describe how liposomes with plasmid vectors are made and used to treat CF in the lungs

Answer 14

Plasmids with the healthy CFTR allele are inserted into bacteria
Bacteria multiply and produce many copies of the plasmid
Plasmids extracted and coated in a sphere of phospholipids forming a liposome
Sprayed into lungs via nasal/mouth spray/inhaler
Thses liposomes can easily pass through the cell membranes of the epithelial cells in the lungs

Question 15

What is a vector?

Answer 15

Plasmid which transfers genetic material into a cell from somewhere else (eg. a virus, another bacterium or a liposome)

Question 16

What are some advantages of gene therapy?

Answer 16

Treatment of genetic diseases/relief of
symptoms
Ability to fully cure a genetic disease

Question 17

What are some disadvantages of gene therapy?

Answer 17

Belief that screening is an invasion of privacy
Possible increase in number of abortions and ethical issues associated with this
Individuals known to be carriers/affected are put in higher risk groups for insurance making insurance difficult to get/very expensive
Regulation of therapy so that it is only used to treat diseases and not used to modify/choose characteristics in a child (“designer babies”)

Question 18

Define: Recombinant DNA

Answer 18

DNA which results from the combination of fragments from two different organisms/when foreign DNA is incorporated into the plasmid from a bacterium

Question 19

Define: Restriction endonuclease

Answer 19

Enzymes which cut DNA molecules between specific base sequences

Question 20

State some of the applications of genetic engineering

Answer 20

Transfer of genes into bacteria to produce useful enzymes or proteins like insulin
Transfer of new genes into crops and livestock so they acquire beneficial traits (eg Disease resistance)
Transfer of genes into humans to cure genetic diseases

Question 21

What are the main stages in gene transfer, as used in genetic therapy

Answer 21

Isolation of the specific DNA fragment/gene
Insertion of the foreign DNA fragment/gene into a vector
Insertion of vector into suitable hosts for replication
Identification of the cells that took up the gene by use of genetic markers
Growth of the host cells
Extraction of the vectors containing the specific gene

Question 22

State some disadvantages of using recombinant DNA technology?

Answer 22

Quantity production of complex proteins or peptides which cannot be made by other methods
Removal of the need to use extracts from mammalian organs
Uses in the production of higher yield crops with superior traits
Health benefits in treating genetic diseases

Question 23

State some disadvantages of using recombinant DNA technology?

Answer 23

Concerns over its misuse
Technically complicated and therefore very expensive on an industrial scale
Difficulties in identifying the genes of value in a large genome
Synthesis of the required protein may involve several genes each coding for a single polypeptide
Treatment of DNA with restriction enzymes produces millions of fragments which are of no use
Not all eukaryote genes will express themselves in prokaryrote cells

Question 24

What is so dangerous about bacteria being able to readily exchange genetic material?

Answer 24

This recombinant DNA may get into other organisms

Could lead to herbicide resistant weeds

Question 25

What are the advantages of treating CF with gene therapy as opposed to using non-genetic treatments?

Answer 25

Gene therapy is more effective and reduces/treats the symptoms (more/better)
Reduces the number of injections and antibiotics needed to be taken by the patient
Removes the need for dietary restrictions on patient
Less intensive monitoring of patient needed

Question 26

Describe arguments for and against the genetic screening of normal individuals for genetic disorders

Answer 26

+ Prevents disease causing alleles being passed on
+ Reduces the incidence of the gene in the population
- Increased anxiety in patients who may not develop the disease
- Which can affect lifestyle choices
- Confidentiality issues
- Insurance companies may refuse cover to individuals with disease causing alleles

Question 27

Why must the same restriction enzyme be used to cut the vector plasmid and the chromosomal DNA when forming recombinant DNA?

Answer 27

To give matching sticky ends so that the foreign DNA fragment can be joined to the vector DNA by complementary base pairing

Question 28

What is the function of DNA ligase in gene therapy?

Answer 28

Splice sticky ends together and thus join the foreign DNA fragment into the vector DNA

Question 29

What events must occur once a normal CFTR gene is inserted into a lung epithelial cell of a patient with CF for the treatment to work?

Answer 29

Gene gets into the nucleus
Gene is incorporated/inserted into chromosome
Transcription of gene and translation of resulting mRNA to give rise to the functional protein