Therapeutic potential of regulatory RNAs Flashcards
What can siRNAs do to a mutated genes mRNA?
“knock-down” the mRNA
Which body cells possess the machinery which constitutes a functional siRNA/miRNA pathway?
All body cells
What takes up siRNAs?
RISC complex (RNA Induced Silencing Complex)
What does the RISC complex use siRNA for?
A scanning target for mRNA
What happens to mRNA with sequence complementarity to siRNA?
It is bound by the siRNA
What happens to mRNA once bound to siRNA?
It is degraded by enzymatic components of the RISC complex
What kind of organisms use siRNA/RISC to combat viral infection?
Plants and other lower organisms
What do plants and other lower organisms use RISC for?
A means of combating viral infection
How is siRNA manipulated by scientists?
an siRNA that has sequence complementarity to any mRNA of interest is designed and this can be used to knock down mRNA
What is an example of a type of gene that siRNA can be used to target?
Oncogenes
What is the philadelphia chromosome?
A fusion of chromosome 9 and 21
What is the issue with the philadelphia chromosome?
Where the chromosomes fuse creates a BCR-ABL oncogene which is cancerous
What is caused by the BCR-ABL oncogene?
Leukaemia
How can siRNA help therapeutically with leukaemia?
siRNA against fusion point between BCR and ABL gene is designed,
Why is the siRNA against leukaemia on the philadelphia chromosome targeted specifically against the fusion point of the BCR and ABL gene?
So it doesn’t target any normal BCR or ABL genes that aren’t fused
Where is the BCR gene found?
Chromosome 22
Where is the ABL gene found?
Chromosome 9
Why is siRNA a useful way of targeting viral infections?
siRNA can be quickly redesigned to keep up with mutations of the virus
Why can siRNAs not be given orally?
The acid contents in the stomach degrades siRNAs
How must siRNAs be adminitered?
Intravenously
What is an issue with administering siRNAs intravenously?
There are exonucleases in the blood that will recognise the siRNAs as foreign and start to degrade them, as well as immune cells that do the same thing
Issue with siRNA size?
They are rapidly filtered by the kidneys and so cleared
Issue with siRNAs and plasma membrane?
They have an overall -ve charge (RNA phosphate backbone) so they can not cross cell membranes
How is the immune response to siRNAs combated?
You can change the RNA structure that repels the immune system and exonucleases
What edits are done to siRNA to prevent exonucleases and the immune system from attacking?
Add a 2’ O-methyl group instead of a hydroxyl group, phosphorothionate linkages
In what direction do most exonucleases act?
5’ to 3’
How does adding a 2’ O-methyl group to siRNA prevent exonuclease degradation?
Recognition from the exonuclease is prevented
What do phosphorothioate linkages replace?
Phosphodiester bonds
Difference between phosphodiester bonds and phosphorothioate bond?
Oxygen atom is changed to a sulfur
What are locked nucleic acids?
Altered structures of DNA or RNA, not strictly DNA or RNA–> they are synthetic nucleic acids
Features of locked nucleic acids?
Oxygen present at the 2’ carbon which is crosslinked to carbon at position 4
Why are locked nucleic acids used?
Exonucleases and the immune system wont recognise them
Benefit of locked nucleic acids other than not being recognised?
They bind more potently to their targets than siRNAs do
How are siRNAs helped to cross the cell membrane?
They are encased in lipid nanoparticles
What types of lipids make up lipid nanoparticles?
Neutral lipids and cationic lipids
How is the -ve charge of siRNAs neutralised?
Cationic lipids are +vely charged
How is the molecular weight of siRNAs increased?
Polyethylene groups
Which two things to lipid nanoparticles do that helps siRNAs?
Remove the -ve charge and increase their molecular weight
When are lipid nanoparticles used?
When a systematic delivery is wanted
What type of delivery is used when you want to deliver siRNAs to a specific organ or cell type?
Targeted delivery
What encases siRNAs in targeted delivery?
Targeted cationic polymers
What make up targeted cationic polymers?
polycations, polyethylene groups, targeting ligand
What is the targeting ligand specific to in targeted delivery?
A receptor on the surface of the cell/organ you are aiming for
How is the polymer nanoparticle taken up in targeted delivery?
Receptor mediated endocytosis
Issues with receptor mediated endocytosis?
The molecules are not readily released from the endosome
What happens if the contents of an endosome is not readily released?
They get transferred to a lysosome where they are degraded
What is endosomal escape?
The release of something from an endosome
What is used to aid endosomal escape?
Using proton sponge groups
Where are proton sponge groups added?
siRNA complex
What do proton sponge groups do?
Cause protons to be pumped into the endosome, eventually causing it to burst
Examples of successful in vivo trials?
Haemophilia, ebola
What causes hemophilia?
mutations in clotting factors, so they are not produced–> persistent bleeding
Where are clotting factors produced and what with?
In the liver, with anticoagulants
Method of targeting hemophilia with siRNAs?
Targeting anti clotting factors, restoring the balance between clotting factors and bleeding factors
Successful siRNA of hemophilia?
Attaching GalNAc groups (ligands) to antithrombin siRNAs
What do GalNAc groups do?
Bind to proteins found on the surface of liver cells
Which ligand does GalNAc attach to?
ASGPR receptor
Result of GalNAc siRNA?
Reduced levels of antithrombin being produced
How was siRNA used against ebola?
siRNA designed against murkona strain of ebola
What kind of delivery was used for the ebola siRNA?
LMP (systematic delivery)
Which animals were used to test the ebola siRNA and did it work?
Monkeys, and yes
