Gene Therapy

Question 1

what is gene therapy?

Answer 1

the use of nucleic acid polymers as a drug to treat diseases by therapeutic delivery into cells of patients.

Question 2

what is a gene?

Answer 2

a molecular unit of heredity of a living organism

Question 3

what is therapy?

Answer 3

attempted treatment of a health problem usually following diagnosis

Question 4

what process takes place converting DNA to RNA?

Answer 4

transcription

Question 5

what process takes place converting RNA to a protein?

Answer 5

translation

Question 6

what process takes place converting RNA to DNA?

Answer 6

reverse transcription

Question 7

what required before gene therapy can be given?

Answer 7

1. the condition in question must be understood
2. identify where the faulty gene is
3. availability of the working copy of the gene
4. cells that need to be treated should be identified and accessible
5. delivery of the working gene copies should be possible
6. link between disease and gene should be understood

Question 8

what are the 4 possible gene therapy approaches?

Answer 8

1. insert normal gene into genome hence replacing non-functional gene
2. swap abnormal gene for normal gene (correcting the mutation - so swap incorrect base for correct one)
3. selective reverse mutation
4. blocking the expression of the pathogenic gene

Question 9

which of the two is more effective for counteracting genetic diseases: germ-line therapy or somatic cell therapy?

Answer 9

germ-line therapy as the correction is inherited

Question 10

what characteristics are required of an ideal vector system?

Answer 10

1. adequate carrying capacity
2. undetectable by the immune system
3. non-inflammatory
4. safe to patients
5. efficient enough to correct the phenotype
6. long duration of expression and able to re-administer

Question 11

what is the non-viral gene therapy approach?

Answer 11

• The insertion of a normal gene into the genome to replace the abnormal disease-causing gene.
• the gene is inserted directly into the target cells

Question 12

what are some pros with using non-viral gene therapy?

Answer 12

• possible to work with large pieces of DNA
• non-toxic
• no immune response

Question 13

what are some cons with using non-viral gene therapy?

Answer 13

• inefficient

- limited to ex vivo gene transfer

Question 14

give an example of a non-vial vector?

Answer 14

liposomes

Question 15

what is a liposome and how does it work?

Answer 15

• an artificial lipid sphere with an aqueous core carrying the therapeutic DNA.
• fuses with the target cell membrane and delivers the gene to the nucleus

Question 16

how does receptor mediated endocytosis work in the non-viral gene therapy?

Answer 16

• the vector binds to the receptor on the target cell
• the plasma membrane of the target cell forms a membrane around the vector forming an endosome vesicle inside the cell
• the vesicle is disrupted releasing the vector again
• vector migrates into the nucleus.

Question 17

Explain how gene therapy works in the viral approach?

Answer 17

the vector, usually viruses that are genetically altered to carry normal human DNA, is used to deliver the therapeutic gene to the target cells.

Question 18

what are the requirements of the viral vector?

Answer 18

1. cell-specific
2. can host the therapeutic gene (size)
3. immune evasion
4. non-immunogenic or allergic
5. safe for patients and the environment
6. express gene for as long as required
7. purified in large quantities at high concentrations

Question 19

how do retroviral vectors work?

Answer 19

• DNA is made via reverse transcriptase

- DNA integrates into host chromosome via integrase

Question 20

what are advantages of using retroviral vectors?

Answer 20

• long-term expression
• low toxicity
• high capacity
• low immunity allowing repeat administration

Question 21

what are disadvantages of using retroviral vectors?

Answer 21

• lack of cell specificity

- only infects dividing cells

Question 22

how do adenoviral vectors work in gene therapy?

Answer 22

• vector binds to membrane of target cell
• vector packaged in vesicle
• vesicle breaks down releasing the vector
• vector injects new gene into nucleus
• vector has a dsDNA genome which doesn’t integrate into the chromosomal DNA of the host and remains as a separate DNA strand in the nucleus which is transcribed from there.

Question 23

what are advantages of using adenoviral vectors?

Answer 23

• Exhibit high transfection efficiencies, ex vivo and in vivo.
• Infects both dividing and non-dividing cells.
• Replicate to high titres in vitro: large quantities possible

Question 24

what are disadvantages of using adenoviral vectors?

Answer 24

• More likely to be attacked by host immune system

- Potential to trigger inflammatory responses, thus no repeat administration.

Question 25

what are adeno-associated vectors?

Answer 25

vectors that contain single stranded DNA and integrates the DNA into the host genome at a single site on chromosome 19.

Question 26

what are advantages to using adeno-associated vectors?

Answer 26

• can infect dividing and non-dividing cells

- they are nontoxic so don’t trigger an immune response, hence no inflammation

Question 27

what are disadvantages to using adeno-associated vectors?

Answer 27

• Small so can only carry two genes

- Difficult to make in large quantities

Question 28

what is the herpes simplex viral vector mostly used for?

Answer 28

gene transfer in the nervous system

Question 29

what are advantages to using the herpes simplex viral vector?

Answer 29

• Large genome so can accommodate larger genes
• Can infect wide range of tissues (incl. muscle, liver, and nerve/lung)
• Complications due to HSV-1 infections are rare

Question 30

what are disadvantages to using the herpes simplex viral vector?

Answer 30

• Relatively untested

- Stigma due to “Herpes”

Question 31

what are some disadvantages to gene therapy?

Answer 31

• Short-lived nature as rapidly dividing cells cause DNA to be diluted
• cause an immune response, therefore reduced effectiveness
• viral vectors could be toxic, they can cause an immune/inflammatory response or they may have targeting issues
• difficulty treating multi-gene disorders
• insertional mutagenesis (virus attacking wrong cells, or DNA integrated into wrong position, which can cause other diseases)

Question 32

why is cystic fibrosis an ideal candidate for gene therapy?

Answer 32

• it is a single gene defect;
• it is a recessive condition, with heterozygotes being phenotypically normal (suggesting gene dosage effects are not critical);
• the main pathology is in the lung, which is accessible for treatment;
• it is a progressive disease with a virtually normal phenotype at birth, offering a therapeutic window.

Question 33

why can’t gene therapy be delivered by the instillation of vector-containing fluids into the lungs for treating cystic fibrosis?

Answer 33

• potential of aspiration

- enhanced alveolar exposure as a result of bulk flow of fluids into the lungs causing adverse reactions

Question 34

why can’t gene therapy be delivered by oral inhalation of aerosolised vector-containing fluids into the lungs for treating cystic fibrosis?

Answer 34

-small particles target the peripheral airways and the alveolar region of the lungs which would be ineffective for treating cystic fibrosis as there’s the possibility of inducing adverse effects.

Question 35

why can’t viral vectors help with the treatment of cystic fibrosis?

Answer 35

because of inefficient transduction from luminal surfaces and immune responses which the restrict efficacy of repeated application.