Gene therapy

Question 1

what is gene therapy?

Answer 1

Treatment method based on the delivery of nucleic acids to cells (transfection) to treat a medical condition

Question 2

what is transfection?

Answer 2

a procedure that introduces foreign nucleic acids into cells to produce genetically modified cells

Question 3

how can gene therapy be used?

Answer 3

▫ Introduce a new gene OR Replace a defected/mutated gene
= using a Plasmid DNA
▫ Silence a gene that is not working properly ( STOP protein expression)
= Small interfering RNA (siRNA) OR Short-Hairpin RNA (shRNA)

Question 4

when are gene therapy used to treat patients?

Answer 4

1. single gene defect diseases (genetic/ hereditary)
2. poly-genetic or non-hereditary disease

Question 5

what are some examples of single gene defect diseases (genetic/ hereditary) used in gene therapy?

Answer 5

haemophilia

Question 6

what are some examples of poly-genetic or non-hereditary disease used in gene therapy?

Answer 6

• cancer
• cvd
• hepatitis

Question 7

what is poly-genetic or non-hereditary disease?

Answer 7

more than one gene/ environmental factors involved, harder to treat

Question 8

how does SARS-COV-2 vaccines prevent disease?

Answer 8

using gene therapy to provide antibodies to target the spike proteins

providing an immune response

Question 9

what are the different techniques of gene therapy transfection methods?

Answer 9

in-vivo therapy (increase risk)
ex-vivo therapy

Question 10

how do you transfer genetic material?

Answer 10

non-viral delivery vectors (synthetic)
-liposomes
-CNTs
viral delivery vectors

Question 11

how do viruses work?

Answer 11

two viral cycles - lytic and lysogenic

Question 12

explain the two viral cycles

Answer 12

Question 13

what are lytic viruses?

Answer 13

virus enters host,
replicates, and lyses
(burst open) occurs
causing death of host cell
immediately

Question 14

what are lysogenic/latent viruses?

Answer 14

Some viruses have the ability to become dormant inside the cell
-They are called latent viruses
-They may remain inactive for
long periods of time (years)
Later, they activate to produce
new viruses in response to some
external signal

Question 14

what are some examples of lysogenic/latent viruses?

Answer 14

• HIV and Herpes viruses

Question 15

what is an example of a retrovirus?

Answer 15

HIV

Question 16

what is a retrovirus ?

Answer 16

A retrovirus is a type of virus that contains RNA as its genetic material and uses the enzyme reverse transcriptase to convert its RNA into DNA after infecting a host cell.

This DNA is then integrated into the host’s genome, where it can be transcribed and translated to produce new viral particles.

Question 17

what are some viral drug delivery systems?

Answer 17

• retroviral/retroviruses
• adenoviral
  -adeno-associated viruses (AAV)

Question 18

what are retroviral/retroviruses? and an example

Answer 18

100nm
contain RNA not DNA
use reverse transcriptase
When a retrovirus infects a cell, it injects its RNA and reverse transcriptase enzyme into the cytoplasm of that cell

HIV

Question 19

what are adenoviruses? and an example

Answer 19

90-100nm
double stranded DNA
cause respiratory/ intestinal/ eye infections in humans

COMMON COLD

Question 20

what are adeno-associated viruses (AAV)?

Answer 20

small DNA viruses
can’t replicate individually therefore require a HELPER VIRUS (e.g adenovirus/ herpes)
not known to cause disease

Question 21

what are the differences between the viral vectors/drug delivery systems?

Answer 21

Question 22

what was within the AZ/Oxford COVID-19 vaccine?

Answer 22

adenovirus vaccine vector from a chimpanzee
- harmless/ weakened adenovirus =stronger immune response

Question 23

what are non-viral delivery vectors?

Answer 23

liposomes - lipoplexes
polymers - polyplexes
CNTs- carboplexes

Question 24

what do non-viral delivery vector require?

Answer 24

cationic drug delivery systems required (positively charged) to increase cell uptake as cell membranes tend to be negatively charged OR drug is negatively charged therefore increased affinity to the drug to the DDS

Question 25

what is an advantage of non-viral delivery vectors?

Answer 25

no immune response = re-administer no limitation on size of therapeutic gene delivery compared to viral vectors

Question 26

what is a disadvantage of non-viral delivery vectors?

Answer 26

less efficient than viral vectors

Question 27

What are the advantages of using AAV vs adenoviruses for gene therapy?

Answer 27

low gene expression and low levels of immunity

Question 28

What are the main issues with in vivo gene therapy ?

Answer 28

the possibility that the introduction and expression of virus proteins may activate endogenous pathogenic viruses Possible immune reaction from the vectors used

Question 29

what vectors can cause an immune reaction in vivo

Answer 29

Viruses, bacterial plasmids, nanoparticles

Question 30

how does ex-vivo gene therapy work?

Answer 30

1. Cells are modified outside the body and then transplanted back in again 2. Cells from the patient’s blood or bone marrow are removed and grown in the lab 3. The cells are exposed to the gene therapy vector that is carrying the desired gene ▫ E.g. the virus enters the cells and inserts the desired gene into the cells’ DNA 4. The cells grow in the laboratory and are then returned to the patient by injection into a vein

Question 31

what are the limitations of ex-vivo gene therapy?

Answer 31

❖Complex procedure ❖Maintenance of cells in vitro ❖Indirectly introducing the desired-containing cells into an organism may trigger immune responses ❖The “modified/transfected” cells also may : ▫ May be rejected by the patient ▫ Malfunction ▫ Trigger an immune response or not entirely work at all once transplanted

Question 32

mRNA-based therapies compared to DNA

Answer 32

mRNA delivery is safer than whole virus or DNA delivery = not infectious as it cannot integrate into the host genome * mRNA is processed directly in the cytosol whereas DNA needs to reach the nucleus to be decoded

Question 33

what are the disadvantages of mRNA-based therapies? GENERALLY

Answer 33

* mRNA has a short half-life * Not Immunogenic * mRNA needs a DDS

Question 34

what are the Advantage of mRNA-based therapies? IN VIVO

Answer 34

* Safe & efficient transportation in vivo ▫ Without degradation in the circulation ▫ Crossing the cellular plasma membrane ▫ Reaching cytosol

Question 35

What do you think the issues, hurdles that face gene therapy ?

Answer 35

* Immune response reduces the effectiveness to repeated doses * Issues with viral vectors - Risks of toxicity, inflammatory responses * Problems with integrating therapeutic DNA into the genome - Rapid dividing cells and Short term effect * Polygenic disorders - CVD, Cancer, Diabetes, Alzheimer’s disease * Insertional mutagenesis - Integration of DNA in a sensitive place in genome e.g. Tumour suppressor gene ➔cancer * Expensive treatment