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Flashcards in Gene Therapy Deck (35)
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1
Q

what is gene therapy?

A

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

2
Q

what is a gene?

A

a molecular unit of heredity of a living organism

3
Q

what is therapy?

A

attempted treatment of a health problem usually following diagnosis

4
Q

what process takes place converting DNA to RNA?

A

transcription

5
Q

what process takes place converting RNA to a protein?

A

translation

6
Q

what process takes place converting RNA to DNA?

A

reverse transcription

7
Q

what required before gene therapy can be given?

A
  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
8
Q

what are the 4 possible gene therapy approaches?

A
  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
9
Q

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

A

germ-line therapy as the correction is inherited

10
Q

what characteristics are required of an ideal vector system?

A
  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
11
Q

what is the non-viral gene therapy approach?

A
  • 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
12
Q

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

A
  • possible to work with large pieces of DNA
  • non-toxic
  • no immune response
13
Q

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

A
  • inefficient

- limited to ex vivo gene transfer

14
Q

give an example of a non-vial vector?

A

liposomes

15
Q

what is a liposome and how does it work?

A
  • 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
16
Q

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

A
  • 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.
17
Q

Explain how gene therapy works in the viral approach?

A

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

18
Q

what are the requirements of the viral vector?

A
  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
19
Q

how do retroviral vectors work?

A
  • DNA is made via reverse transcriptase

- DNA integrates into host chromosome via integrase

20
Q

what are advantages of using retroviral vectors?

A
  • long-term expression
  • low toxicity
  • high capacity
  • low immunity allowing repeat administration
21
Q

what are disadvantages of using retroviral vectors?

A
  • lack of cell specificity

- only infects dividing cells

22
Q

how do adenoviral vectors work in gene therapy?

A
  • 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.
23
Q

what are advantages of using adenoviral vectors?

A
  • 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
24
Q

what are disadvantages of using adenoviral vectors?

A
  • More likely to be attacked by host immune system

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

25
Q

what are adeno-associated vectors?

A

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

26
Q

what are advantages to using adeno-associated vectors?

A
  • can infect dividing and non-dividing cells

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

27
Q

what are disadvantages to using adeno-associated vectors?

A
  • Small so can only carry two genes

- Difficult to make in large quantities

28
Q

what is the herpes simplex viral vector mostly used for?

A

gene transfer in the nervous system

29
Q

what are advantages to using the herpes simplex viral vector?

A
  • 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
30
Q

what are disadvantages to using the herpes simplex viral vector?

A
  • Relatively untested

- Stigma due to “Herpes”

31
Q

what are some disadvantages to gene therapy?

A
  • 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)
32
Q

why is cystic fibrosis an ideal candidate for gene therapy?

A
  • 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.
33
Q

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

A
  • potential of aspiration

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

34
Q

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

A

-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.

35
Q

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

A

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

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