Gene Therapy

Question 1

What is gene therapy?

Answer 1

Gene therapy is the treatment of a disease by genetic manipulation of a patient’s cells.
This is a form of drug delivery in which altered cells produce the needed proteins continuously at a therapeutic level.

Question 2

What can gene therapy used for?

Answer 2

• Correct an inborn error of metabolism.
• Repair an acquired genetic abnormality.
• To provide a new function to a cell.

Question 3

Where diseases are caused by a deficiency in a protein that should be produced by the body, there are two treatment options. What are they?

Answer 3

• Admission of the deficient proteins to alleviate the symptoms (traditional therapy).
• Restoration of gene function that has been lost (gene therapy).

Question 4

What are the two approaches for gene therapy?

Answer 4

Replacement and addition.

Question 5

Explain gene replacement.

Answer 5

• Remove defective gene.
• Incorporate functioning allele.
• Requires recombination event (technically difficult).

Question 6

Explain gene addition.

Answer 6

• A functional gene is integrated into the genome.
OR
• It exists independently.

Question 7

What are the two main targets for gene therapy?

Answer 7

Germ cells and somatic cells.

Question 8

Describe the targeting of germ cells in gene therapy.

Answer 8

This is the modification of egg or sperm cells, or a fertilized egg. This change is inheritable and is passed on to the next generation.

Question 9

Describe the targeting of somatic cells in gene therapy.

Answer 9

This modification affects only the cells of the body, the change us not passed onto the next generation. This may be in-vivo or ex-vivo.

Question 10

What routs of administration of gene therapy may be used?

Answer 10

• Inhalation.
• Oral administration.
• Intramuscular injection.
• Intravenous injection.
• Ex-vivo administration.

Question 11

What characteristics should an ideal delivery vehicle for gene therapy have?

Answer 11

• Efficient delivery.
• Relatively specific for target cells.
• Limited immune response – the immune system may detect the vehicle as an invader.
• Allow transgene expression.
• Carry large pieces of DNA.

Question 12

Give some examples of delivery vehicles used in gene therapy.

Answer 12

• Direct injection of naked DNA (used in germline therapy).
• Viruses (infective but replication-defective) – e.g. retroviruses or adenoviruses.
• Non-viral vehicles – e.g. liposomes.

Question 13

What physical methods can be used to introduce new genes in gene therapy?

Answer 13

Electroporation, gene gun, sonoporation, magnetofecation.

Question 14

Describe the technique of electroporation used for gene delivery.

Answer 14

This method uses short pulses of high voltage to carry DNA across the cell membrane. This shock causes temporary formation of pores in the cell membrane, allowing DNA molecules to pass through.

Question 15

Describe the technique of using a gene gun for gene delivery.

Answer 15

DNA is coated with gold particles and loaded into a device which generates a force to achieve penetration of DNA/gold into the cells.

Question 16

Describe the technique of sonoporation used for gene delivery.

Answer 16

This technique uses ultrasonic frequencies to deliver DNA into cells. The
process of acoustic cavitation is thought to disrupt the cell membrane and allow DNA to enter into cells.

Question 17

Describe the technique of magnetofection used in gene delivery.

Answer 17

DNA is complexed to magnetic particles, and a magnet is placed underneath the tissue culture dish to bring DNA complexes into contact with a cell monolayer.

Question 18

Describe retroviruses.

Answer 18

These are single stranded RNA viruses; this RNA is then converted to double-strand DNA by reverse transcriptase.
The viral DNA is then incorporated into the host cell chromosome where it becomes stable and permanent.
These viruses infect only dividing cells, making them suitable for ex-vivo gene therapy.

Question 19

Explain how retroviral vectors for gene delivery are created.

Answer 19

1) The therapeutic DNA is first cloned into the retroviral vector.
2) A packaging cell line expressing retroviral genes necessary for replication and viral packaging then is transfected with the retroviral vector DNA.
3) The transfected DNA is transported to the nucleus of the transfected cell and the retroviral vector DNA integrates into the genome of the transfected cell.
4) The infective recombinant retroviral particles are released from transfected cells.
5) Purify and characterise the released recombinant retroviral particles to ensure they contain the therapeutic DNA and that they remain replication-deficient.
6) Once characterised, the therapeutic retroviral vector must be amplified to produce stocks of sufficient titre for therapeutic administration.

Question 20

How do retroviral vectors deliver genes in gene therapy?

Answer 20

1. The retroviral vector encoding the therapeutic gene binds to specific cell surface receptors and enters the cell by endocytosis.
2. The viral vector then is released from the endosome and its RNA genome is converted to DNA before being transported into the nucleus of the cell.
3. Once inside the nucleus, retroviral DNA is integrated randomly into the genome of the infected cell.
4. The integrated retroviral and/or therapeutic DNA then is transcribed and translated into a functional protein using the transcriptional and translational machinery of the host cell.

Question 21

What are the disadvantages of using retroviral vectors for gene therapy?

Answer 21

•	Uncontrolled integration which could result in:
o	Splitting important gene.
o	Activating oncogene.
o	Integrating into inert region (methylated region).
•	Not suitable for non-dividing cells.
o	Cystic fibrosis, muscular dystrophies.
•	Provoke an immune response.
•	Not specific (infect other cells).

Question 22

What are adenoviruses?

Answer 22

These are double-stranded DNA viruses which infect non dividing cells. They are replication-defective and the DNA they contain isn’t integrated to the chromosome of the cell. This means that the expression of the genes is transient, not permanent.

Question 23

How are adenoviral vectors for gene delivery constructed?

Answer 23

1. First the therapeutic DNA is cloned into an adenoviral shuttle vector.
2. The adenoviral shuttle vector and replication-deficient adenoviral DNA are then co-transfected into a packaging cell line.
3. Once inside the cell, the process of homologous recombination allows DNA to be swapped between the adenoviral DNA and the shuttle vector, thus generating recombinant adenoviruses that encode the therapeutic gene.
4. Generated adenoviruses are translocated to the nucleus, where they are replicated and packaged and then released from the infected cells.
5. The recombinant adenoviruses are purified and characterised to ensure that they have remained replication-deficient and that they express the appropriate therapeutic DNA.
6. The characterised adenovirus is amplified to generate high-titre stocks required for in vivo administration.

Question 24

How do adenoviral vectors deliver genes in gene therapy?

Answer 24

1. The recombinant adenoviral vector enters the cell by receptor- mediated endocytosis.
2. In the endosome the viral particle is released from this internal cellular compartment and the adenoviral capsid is translocated into the nucleus.
3. Once inside the nucleus, the therapeutic DNA is transcribed from the episomal recombinant adenoviral genome and, if necessary, the therapeutic RNA is translated.

Question 25

How does liposome mediated gene delivery work?

Answer 25

1. Negatively charged DNA is first condensed and then mixed with the cationic lipid mixture.
2. The complexes of DNA and liposomes that form are then administered to the target tissue or cells, where the complexes are taken up by endocytosis.
3. Once inside the cell, the complexes are released from the endosomes and enter the target cell nucleus, where the therapeutic DNA is transcribed and translated to express the therapeutic gene product.

Question 26

What causes SCID?

Answer 26

Due to adenosine deaminase (ADA) gene defect in lymphocytes. A build-up of adenosine is specifically toxic to T and B lymphocytes.

Question 27

What treatments are available for SCID?

Answer 27

• Bone marrow transplants.
• Infusion of PEG-ADA (polyethylene glycol-modified ADA).
• Gene therapy (ex-vivo gene therapy).

Question 28

Explain how gene therapy works for the treatment of SCID.

Answer 28

Gene therapy for SCID begins with the removal of bone marrow cells from the patient. These cells are infected with a modified retrovirus carrying the ADA transgene.
Then the modified cells are injected into the body. Immunity is often restored, with 20-30% of lymphocytes expressing the normal ADA gene.

Question 29

What is cystic fibrosis?

Answer 29

Cystic fibrosis is a monogenic, recessive disorder (hereditary disease). It is caused by a mutation in a gene for a sodium transporter called CFTR.
This mutation is associated with the production of a thick, sticky mucus that clogs the lungs, making breathing difficult. This also creates an environment that us susceptible to bacterial infection.

Question 30

What two types of gene therapy are available for the treatment of cyctic fibrosis?

Answer 30

In vivo and ex vivo gene therapy.

Question 31

Describe the in vivo gene therapy used for the treatment of cystic fibrosis.

Answer 31

Transgenes are inserted into liposomes or lipoplexes, or viral vector systems. They are then delivered to the site of action (e.g. lung) by aerosol spray.

Question 32

Describe the ex vivo gene therapy used for the treatment of cystic fibrosis.

Answer 32

Cells are removed from the patients bone marrow, they are grown in the lab and certain cells are modified with a transgene. These modified cells are then reintroduced to the patient.