Gene therapy Flashcards
what is gene therapy?
Treatment method based on the delivery of nucleic acids to cells (transfection) to treat a medical condition
what is transfection?
a procedure that introduces foreign nucleic acids into cells to produce genetically modified cells
how can gene therapy be used?
▫ 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)
when are gene therapy used to treat patients?
- single gene defect diseases (genetic/ hereditary)
- poly-genetic or non-hereditary disease
what are some examples of single gene defect diseases (genetic/ hereditary) used in gene therapy?
haemophilia
what are some examples of poly-genetic or non-hereditary disease used in gene therapy?
- cancer
- cvd
- hepatitis
what is poly-genetic or non-hereditary disease?
more than one gene/ environmental factors involved, harder to treat
how does SARS-COV-2 vaccines prevent disease?
using gene therapy to provide antibodies to target the spike proteins
providing an immune response
what are the different techniques of gene therapy transfection methods?
in-vivo therapy (increase risk)
ex-vivo therapy
how do you transfer genetic material?
non-viral delivery vectors (synthetic)
-liposomes
-CNTs
viral delivery vectors
how do viruses work?
two viral cycles - lytic and lysogenic
explain the two viral cycles
what are lytic viruses?
virus enters host,
replicates, and lyses
(burst open) occurs
causing death of host cell
immediately
what are lysogenic/latent viruses?
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
what are some examples of lysogenic/latent viruses?
- HIV and Herpes viruses
what is an example of a retrovirus?
HIV
what is a retrovirus ?
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.
what are some viral drug delivery systems?
- retroviral/retroviruses
- adenoviral
-adeno-associated viruses (AAV)
what are retroviral/retroviruses? and an example
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
what are adenoviruses? and an example
90-100nm
double stranded DNA
cause respiratory/ intestinal/ eye infections in humans
COMMON COLD
what are adeno-associated viruses (AAV)?
small DNA viruses
can’t replicate individually therefore require a HELPER VIRUS (e.g adenovirus/ herpes)
not known to cause disease
what are the differences between the viral vectors/drug delivery systems?
what was within the AZ/Oxford COVID-19 vaccine?
adenovirus vaccine vector from a chimpanzee
- harmless/ weakened adenovirus =stronger immune response
what are non-viral delivery vectors?
liposomes - lipoplexes
polymers - polyplexes
CNTs- carboplexes
what do non-viral delivery vector require?
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
what is an advantage of non-viral delivery vectors?
no immune response = re-administer
no limitation on size of therapeutic gene delivery compared to viral vectors
what is a disadvantage of non-viral delivery vectors?
less efficient than viral vectors
What are the advantages of using
AAV vs adenoviruses for gene
therapy?
low gene expression and low levels of immunity
What are the main issues with in vivo
gene therapy ?
the possibility that the introduction and expression of virus proteins may activate endogenous pathogenic viruses
Possible immune reaction from the vectors used
what vectors can cause an immune reaction in vivo
Viruses, bacterial plasmids, nanoparticles
how does ex-vivo gene therapy work?
- Cells are modified outside the body and then
transplanted back in again - Cells from the patient’s blood or bone marrow are removed and grown in the lab
- 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 - The cells grow in the laboratory and are then returned to the patient by injection into a vein
what are the limitations of ex-vivo gene therapy?
❖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
mRNA-based therapies compared to DNA
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
what are the disadvantages of mRNA-based therapies? GENERALLY
- mRNA has a short half-life
- Not Immunogenic
- mRNA needs a DDS
what are the Advantage of mRNA-based therapies? IN VIVO
- Safe & efficient transportation in vivo
▫ Without degradation in the circulation
▫ Crossing the cellular plasma membrane
▫ Reaching cytosol
What do you think the issues, hurdles
that face gene therapy ?
- 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
