Gene therapy and RNA therapeutics 3 Flashcards
siRNAs
Exogenous- cell doesnt make them, they get into the cell somehow.
Produced by organisms in response to dsRNA(double stranded) which is an immune repsonse
dsRNA is produced by DICER
One strand of duplex incorporated into RNA inducing silencing complex. (RISC)
This binds to RNA (fully complementary) then cleaves the RNA
Noncoding RNAs
siRNAs- Short interfering RNAs
miRNAs
piRNAs
IncRNAs (long non-coding RNAs=not well undertsood)
eRNAs (enhancer RNAs)
How do siRNA function?
- siRNA is complementary to the target mRNA delivered into the cell.
- RISC binds to siRNA and selects ONE strand
- Selected siRNA bijds to exact complementary sequence in target mRNA
- Ago2 in RISC will then make a single cut where the target mRNA is.
- Target mRNA is destroyed.
siRNA used therapeutically.
siRNA that targets specific mRNA thus having less mRNA and less protein.
Examples in therapeutics can be the damaging mRNA mutant TTR. A build up of this overtime can lead to failing of the organs.
So you can target that specific mRNA to decrease this in the cells.
Overexpression of oncogene, you can target this theoretically and decrease proliferation in cancer cells but not reseacrhed yet.
**But you cannot target specific mutant alleles **, such as in Huntingtons.
Difficulties with siRNA
Specifity- off target binding
Efficiency- Do not completely knock out damaged gene, will have to keep giving it to them all the time to keep knocking it out.
Viruses evolve and prevent sirna CLEAVAGE
Delivery- Some organs are easier than other, live ris easier than brain
For cancer treatment on siRNA to cancer cells, not normal cells surounding the tumour
Maintenance is also difficult.
Look at lecture again for shRNA dicer on siRNA delivery slide
How do you deliver siRNA?
Mamilians have an innate immune response, any dsRNA longer than 30base pairs induces signalling processes with downstream effects.
Synthetic siRNAs are too short to activate these mechanisms so can be used.
Can also enocde them in a vector as a short hairpin RNAs (shRNA) which can be processed by dicer.
Thus allowing viral delivery and being able to target specific organs.
Oligonucleotide delivery
Need to achieve stability and entry into cells
Encapsulation in lipid nanoparticles
Chemcial mod:
* Phosphorothioate mod of backbone increases stability
* Ligand conjugsation to enhance uptake
* GaINAc binds to asialoglycoprotein receptor on heptaocytes allowing specific targeting opf oligonucleotide drugs to the liver
First approved siRNA drug.
Hereditary transthyretin amyloidosis (h-ATTR) with polyneuropathy.
Accumulates in other organs and eventually fatal
Patisiran- Targets TTr mRNA both WT and mutant and is encapsulated in lipid nanoparticles requiring lengthy infusio of every 3 weeks.
2022 GaINAc-conjugated siRNAs (Vutisiran) allows efficient delivery to liver and SC delivery every 3-6 months
Tegsedi- Targets WT and mutant TTR mRNA by gapmer ASO which recruits RNAse
How are miRNAs made?
Encoded in the genome
Folds to form hairpain
Drosha protein recognises this and chops hairpain to give shorter pre-mRNA (single strand)
This is then recognised by dicer to form a duplex.
One strand is incorparated into RISC, the other strand is degraded.
Look at slide for diagrams to get better undertsnading
microRNA activity
Chemically identical to siRNAs, differneces are due to target binding.
Complete base pairing to the site anywhere in mRNA, RNA cleavage followed by RNa DEGRADATION which is the PREDOMINAT MECHANISM IN PLANTS.
Partial base pairing to 3 UTR sites is translation repression RNA degratdation in P bodies
Do not induce RNA cleavage.
Which is the predominat mechanism in animals
MicroRNA targets
60% of all human mRNA have target sites but its still difficult to predict targets due to imperfect complementarity so need to test experimmentally.
Look at lecture slides
How do microRNAs repress protein synthesis? (LESS UNDERSTOOD)
- Translation repression
- mRNA decapping, mRNA deadenylation
END GOAL= LESS PRODUCTION OF ENCODED PROTEIN
Summary of both siRNA and miRNA
- Both 21-23nt RNAs, processed from larger dsRNA precursors by DICER.
- siRNAs derived from external dsDNA (viral)
- miRNAs encoded in the genome
- Both cleave exactly complementary target RNA
- Most animal miRNAs have imperfect complementarity to target sites in 3 UTR of mRNAs and repress gene expression at the level of translation and/or RNA stability.
siRNA vs miRNA
Design siRNA carefully, to bind to only single strand (Destroys mRNA leading to reduction of encoded protein)
miRNA, having lots of targets but having smaller effect (reduces but does not abolish protein production thus modulates gene expression)
miRNA therapeutics
Regulation of broad range of cellualr processes
Specific expression patterns
* Tissue
* Differenation stage
Important in diseases like cancer, CVD, diabetes, neurodegeneration and viral infection
Increases or decreases miRNA, can inhibit via antagomirs which are chemically modded oligonucleotides thus preventing target binding.
Or can overexpress a beneficial miRNA (tumour suppressor) but due to them being chemically identical to siRNAs you still have the same problems with delivery.
