Gene Delivery And Therapy Flashcards
What does DNA encode?
Genetic information
Where is DNA located ?
Nucleus
What is a mutation?
A change in base pair sequence
How many coding genes do humans have?
20-25,000 coding genes
What is the function of H1 histone?
To package DNA into structural units called nucleosomes
What is chromatin?
A double-stranded helical structure of DNA
What are the main proteins in chromatin?
Histones
How do histones play a role in gene regulation?
DNA wraps around histones, therefore they play a role in gene regulation
What is a nucleosome?
Nucleosomes are the basic units of chromatin compaction
Roles of nucleosomes
Regulation of transcription
Regulation of replication
Cell reprogramming
DNA repair
Define gene therapy
Gene therapy is a technique that modifies a persons genes to treat or cure a disease.
Most common methods of gene therapy (3)
- Replacing a disease causing gene with a healthy copy of the gene
- Inactivate a disease causing gene that isn’t functioning properly
- Introducing a new gene into the body to help treat the disease
Barriers to non-viral gene delivery
Gene delivery vehicles can interact with plasma proteins or blood cells
They can aggregate and clog capillaries
Extravasation (leakage) into surrounding tissue can occur with leaky vasculature
Lysosomal degradation
List common gene delivery systems (8)
Biolistic delivery
Micro injection
Electroporation
Cationic polymers
Cationic liposomes
Cationic particles
Retroviral vectors
Adenoviral vectors
Describe how Cationic substances (polymers, liposomes, particles) deliver genes to the cells
Utilise negative charge on DNA and cell membrane
Mediate gene transfer gene transfer to membrane
Uptake by endocyctosis
(Positive carrier draws to negative cell membrane)
Describe how physical methods (electroporation, micro injection) deliver genes to the cell
Utilise penetration of cell and nuclear membrane
Electroporation & biolistic delivery penetrate cell membrane
Micro injection penetrates nuclear membrane
Describe how virus-mediated delivery, delivers genes into a cell
Gene of interest (GOI) is encapsulated into a virus particle
Delivered into cell via receptor mediated endocytosis
How does retroviral delivery differ from adenoviral delivery
For retroviral delivery, GOI enters the nucleus and integrates into the cell genome
For adenoviral delivery, GOI remains expressed in the cytoplasm
What is LPLD?
Lipoprotein lipase deficiency
A rare, inherited genetic condition where there are increased levels of fat in the blood
What conditions are associated with LPLD
Acute pancreatitis (inflammation of pancreas)
Lipemia (plasma appear milky due to high fat content)
Eruptive xanthomas (yellowish raised spots on skin which are fat deposits)
What is Glybera?
Glybera is a gene therapy used for the treatment LPLD
It’s given as one administration
What was the major downside of Glybera gene therapy?
Cost
$1.6 million per patient
Made it not commercially viable
What are the reasons why you would use non-viral delivery
More biocompatible
High nucleic acid loading capacity - not restricted
Safer
Better transfection efficiency
Describe what a lipoplexes is
Cationic liposome + DNA = lipoplexes
What characteristics to lipoplexes show in vitro?
Protected from nuclear degradation
Lipoplexes trigger cellular uptake
Facilitates release of DNA
Excellent transfection efficiency
What characteristics lipoplexes show in vivo?
Interaction with negatively charged blood components
Get trapped in mucous layers
Become embolised in capillaries
What is the purpose of stealth liposomes?
Make liposomes less susceptible to phagocytes until the lipoplexes reaches the target
Describe a method of stealth technology
Using PEG
PEG shielding until lipoplex reaches the target
PEG is removed when target is reached
How does PEG work in stealth technology?
PEG coating appears to phagocytes as harmless
Forms an aqueous cloak around the lipoplex
What is PEG?
A non-toxic, synthetic polymer used in stealth technology
It is hydrophilic and unable to interact with DNA or lipids
List 1 pro and 1 con of using PEG
Pro - Provides longer blood circulation time
Con - There is dose dependant inhibition of transfection (preventing cell uptake of GOI)
