Therapeutic potential of regulatory RNAs

What can siRNAs be used for?

• to knock down a harmful mRNA expressed from a particular mutated gene

• treatment of situations where the disease is caused by a harmful exogenous element

What does dicer do?

Processes siRNAs to allow them to be taken up by the RISC complex

What does RISC stand for?

RNA Induced Silencing Complex

siRNA pathway

• guiding strand of siRNA scans cytoplasm for sequence complimentarity

• when perfect match is found (base pairing between guide RNA and mRNA), enzymatic components of RISC activated + degrade the mRNA.

How long are the siRNAs and why?

22 nt so they can be recognised by the RISC complex

What is this pathway used for?

• in humans: regulates translation of mRNA

• in plants: defense

exists in all cells

What is manipulation of the pathway used to study?

genetic function

• design siRNAs that are complementary to a gene of interest

• transfect into cells

• destruction of mRNA

• study phenotypic effects.

one potential target

oncogenes

e.g. BCR-ABL fusion → Philadelphia chromosome 22

chromosomal translocation causing leukaemia.

how can siRNAs be used against BCR-ABL fusion

design siRNA against the fusion point → destruction of fused mRNA and not the normal ones → reduce potential of leukaemia

example against a virus

design siRNA against mutations that might occur in the virus

obtain virus → sequence → detect mutations → redesign siRNA to target mutated strain of virus

Issues with siRNAs

• quickly destroyed by exonucleases bloodstream, kidneys (bc small size) + stomach and by immune cells

• don’t naturally cross plasma membranes because they are negatively charged so repel

• need to target to specific cell/tissue type - need to trigger receptor-mediated endocytosis

What do exonucleases do?

Destroy foreign genetic material e.g. from viruses

Present in bloodstream

How can you protect siRNAs from exonucelase attack?

add a 2’O-methyl group to the base ring

because most exonucleases are 5’ → 3’ and this modification blocks it.

How to reduce attack of siRNAs from the immune system?

Phosphorothioate linkages - replace phosphodiester bonds with a sulphur.

What are locked nucleic acids?

Oxygen linked from the 2’ to the 4’ position

it is a synthetic nucleic acid - not DNA or RNA

Reduces attack from exonucelases and immune system.

Binds to target mRNAs more potently

How to make the siRNAs cross the plasma membrane

Encase them in lipid nanoparticles (LNP)

Mixtures of cationic and neutral lipids - form spherical structure

neutralises negative charge on siRNA

increases molecular weight to stop it being cleared by the kidneys

What does adding PEG groups do?

Polyethene groups

increases molecular weight even further

How do LNPs help siRNAs cross the cell membrane?

Made of the same thing so they fuse and siRNA released into cytoplasm.

Good for systemic delivery - to deliver to lots of cells in the body.

How to do a targeted delivery

Use polymer nanoparticles

Structure of polymer nanoparticles

A: siRNA

B: polycationic groups (neutralise negative charge)

C: PEG to increase molecular weight

D:targeting ligand - made specific to a receptor on the cell type of interest

How is it taken up in targeted delivery?

receptor-mediated endocytosis

siRNAs encased in endosomes → lysosome

need endosomal escape to stop them being transferred to lysosome.

How does endosomal escape occur?

Add proton sponge groups to lipid nanoparticle

induces pumping of hydrogen ions into endosome → osmotic swelling → endosome ruptures → siRNA released → taken up by RISC complex.

How have siRNAs been used to treat haemophilia?

clotting factors + anti-clotting factors produced in liver

haemophilia = mutation in a gene encoding a clotting factor → profuse bleeding

If you can knock down an anti-clotting factor e.g. antithrombin, it restores the balance.

How to target liver cells for haemophilia?

Use GaINAc ligands - high affinity for ASGPR receptor on hepatocytes

targets the antithrombin mRNA

binds to glycoprotein receptors on liver cell → taken up by receptor mediated endocytosis → endosomal escape → siRNA binds to antithrombin mRNA → RISC destroys it → reduced production → restoration of balance between clotting and anticlotting factors.

Shown to work in mouse models.

How did siRNAs help with ebola?

siRNA designed for new strain of virus

used lipid nanoparticle (LNP) approach for systemic delivery

showed efficacy on monkeys

virus mutates quickly but can redesign in response to this for an effective countermeasure.