RNA therapeutics Flashcards
what 2 big discoveries questioned the dogna: gentic info flow from DNA -> RNA -> protein through transcription + translation?
catalytic RNAs
RNA interference
give example of catalytic RNA?
RNA can have catalytic enzymatic activity - ribozymes
What is RNA interference?
RNA molecules inhibit gene expression or translation by neutralising targeted mRNA molecules
what does RNA play an important role in?
regulating geen function.. needed to maintain cell health
Why is RNA targeted in therapeutics?
RNA translation is involved in the pathogenesis of many diseases so can be used to downregulate disease.
what do rna based therapeutics often manipulate?
transduction of rna signal + offer therapeutic effects
T/F
all rna based therapeutics are rna oligonucleotides -> polymers of nucleotides
True
What determines what bases are linked together in Watson-Crick?
specificity
The distances between the bases.
A = U
G ≡ C
what are the types of RNA therapeutics? 5
antisense oligonucleotides ASOs
RNA interference RNAi
ribozymes
aptamers
RNA vaccines
how do antisense oligonucleotides work?
inhibit mRNA translation
RNAi form RNA induced silencing complex w a protein (Argonaute) how?
=RNA inhibitors of gene expression by degradation of mRNA
How do ribozymes work? catalytically active RNAs
cleave covalent bonds in the target mRNA molecules next to a recognition sequence = destroying RNA sequences
similar to protein ribonucleases
How do RNA vaccines work?
Introduce mRNA sequence coded for a disease specific antigen into cell to build up an immune response.
How do aptamers work? protein binding RNAs
length of RNA that binds to a protein (like a small mol inhibitor)
What is an antisense oligonucleotide? (ASO)
Short strand oligonucleotide that hybridises with complimentary mRNA in a sequence-specific manner via watson-crick base pairing.
approx how many bp long are the short strand oligonucleotides used in ASOs?
20 bp
What does the entry of ASO into a cell cause?
protein knockdown in the cytoplasm or the nucleus
What does the binding of ASO to mRNA (cytoplasm) cause?
bind to mRNA
– triggers ribonuclease (RNase H) activity
OR
– inhibits ribosomal mRNA translation by steric hindrance.
what happens when ASOs bind in the nucleus?
regulates mRNA maturation (via 3 mechanisms)
how can ASOs regulate mRNA maturation in nucleus?
– inhibition of 5’ cap being formed OR
– inhibition of RNA splicing OR
– recruits ribonuclease (RNase H)
these -> knockdown of target genes so target protein knockdown by preventing mature mRNA from forming
what are some examples of RNA interference?
siRNA
RNAi
RNA interference- what happens if the AGO siRNA is
- perfectly complementary
- partially mismatched
- mRNA cleavage
- association-mediated repression
describe process of RNAi
- siRNA enters cell as duplex, incorporated into Argonaute (AGO) to form part of RISC
- in RISC, siRNA is unwound, passenger strand removed, guide strand left to find desired target RNA in cell
- target mRNA bound in AGO then cleaved and digested/ silenced
whats AGO argonaute
protein in cells
whats RISC?
RNA induced silencing complex
whats the difference between ASO’s and RNAi?
ASO delivered as ss, finds target alone
siRNA duplexes taken up by AGO (part of RISC)
ribozymes: catalytic RNA enzymes used to inhibit gene expression
whats their enzymatic mechanism similar to?
that of protein ribonucleases
What is an aptamer and how does it work?
RNA sequence that binds to a protein, preventing it from functioning.
what do aptamers act similarly to?
small mol inhibitor
when may RNA vaccines not be safe to administer?
for patients susceptible to autoimmune response
an mRNA vaccine introduces an mRNA sequence from disease specific antigen -> px cells. what does cell do then?
translates this RNA to produce antigen then triggers immune response + antibodies made against this antigen
-> allows body to build up immune response to protect from original diseasw
rna production very rapid. what does this mean for mRNA?
can be altered easily
half life of RNA and implication?
short half life = fast degradation
How can you design ASOs for a therapeutic beneft?
Can design the RNA sequence of the ASO to fit the mRNA of a specific gene
just make complementary base pairs in right sequence
What does the strength and stability of interactions between ASOs and complementary mRNA target depend on?
Thermodynamic stability
Secondary structure
Proximity of hybridisation state to functional motifs on designed transcript
why is Thermodynamic stability something to consider for designing ASOs?
how tightly bound is RNA to ASO?
how stable is complex?
what about the Secondary structure should be considered for designing ASOs?
which sites are accessible to ASO?
what does RNA look like in 3D?
what about ‘Proximity of hybridisation state to functional motifs on designed transcript’ should be considered for designing ASOs?
strategy can use Proximity to translation start factor eg AUG initiation codon or 5cap. block translation
or can block splice points in nucleus
How do researchers increase the ‘hit rate’ in discovering therapeutic ASOs?
Predicting secondary structure of the target RNA.
Identification of preferable secondary local structures.
(use software)
3D shape of RNA is V important
the ASO can be designed to bind at which 5 sites? of 10 or more consecutive nucleotides?
terminal end
internal loops
joint sequences
hairpin
bulges
the bp in the nucelotides that make ASOs have great impact on stability of ASO-mRNA complex and mRNA knockdown.
what does presence of CCAC, TCCC, ACTC… motifs do?
increases their mRNA knockdown effects
what does presence of GGGG, ACTG, AAA, TAA… motifs in ASO do?
weakens its activity
some motifs (unmethylated CG sequences) are common in bacterial, not eukaryotic DNA so may trigger what?
immune response
GGGG can form intra-strand tetraplexes, what is this?
single structures of 4 strands
.. which have high affinity for proteins so can also cause bad side reactions/ effects
why is GC content strongly correlated with thermodynamic stability of the complex and RNase H activity?
in design of antisense oligonucleotides
- GC bp have 3 H-bonds, while AT bp have only 2. GC stronger + more stable
- Oligonucleotides w higher GC content more stable and less prone to degradation
- RNase H: enzyme that degrades RNA in sequence-specific manner when it is bound to a DNA/RNA hybrid molecule.
what is efficiency of RNase H activity influenced by?
length, sequence, and thermodynamic stability of the DNA/RNA hybrid.
for a potent ASO designm binding energy of ASO and mRNA should be …
change in G 37 degrees more negative than
-8kcal/mol
what is the MAIN disadvantages of RNA therapeutics?
their delivery to site of action
3 disadvantages of RNA therapeutics?
- enzyme degradation - activity of ribonucleases (cleavage by DNase and RNase)
- fast excretion - unmodified oligonucleotides have a poor PK due to weak plasma binding = filtration thru the kidney
- poor absorption/bioavailability= poor cellular uptake
why RNA therapeutics have v poor cellular uptake?
typical ASO: 20bases long, charged backbone of -19
how to chemically modify ASO in mRNA vaccines to try + decrease inflamm response/ enhance translational capacity/ increase stability of mRNA by changing untranslated regions?
use nucleoside-modified mRNA (eg pseudouridines)
chem modifications of ASOs must retain ability to do what?
recognise their target mRNA by watson-crick bpairing
also modifications should not reduce efficiency of mRNA cleavage by RNase H (regardless of binding affinity) to use ASO in same way
why are chem modifications needed for ASOs?
stabilise ASOs and make them resistant to ribonucleases
should improve ASO’s:
- PK
- PD
- retain/ improve RNase H efficiency
- reduce immunogenecity
to what 3 sites can chem modifications be made to ASO?
sugar
base
ASO backbone
in terms of PK why do you want to chemically modify ASO?
enhance nuclear resistance + increase tissue half life
- want to prevent nucleolytic degradation of ASO BUT NOT RNase H activity on mRNA
need enough stability too avoid metab of ASO before gets to correct cells
what type of tox would you want to decrease when chem modifying ASOs?
non-sequence specific tox
- design ASO so wont hit anything off target
- small ASOs less than 20 bp
how do you prevent ribonuclease attacking RNA therapeutics?
chemically modify the antisense oligonucleotide
2 chemical modifications you can add to ASOs/ DNA/RNA?
Thiophosphoramidate
PNA
Phosphoramidate
why is thiophosphoramidate used in chemical modification of ASOs?
improve stability, binding affinity, immunogenicity, PK, and reduce off-target effects, making them more effective as therapeutic agents.
what are generation one modified antisense oligonucleotides?
PS-modified ASOs
non bridging O atom of phosphodiester bond replaced by S (phosphorothioate bond)
what are the advantages of PS-modifications?
1st gen mASOs?
increase nucelase -resistance
thus increase BA (less cleavage of ASO)
what are the disadvantages of PS-modifications?
1st gen mASOs?
reduces binding affinity (Tm reduces by 0.5degrees per nucleotide)
increases non-specific effects by interactions w cell surface + other cellaulr proteins
what are generation two modified antisense oligonucleotides?
changes to 2’ position of sugar
eg:
2’-O-MOE
2’-O-methyl
2’-O-Fluoro
what are generation two modified antisense oligonucleotides?
2nd gen
increase resistance to nucleases
tight binding between ASO and RNA
but reduce RNase H activity
how do you overcome the disadvantages of using generation 2 modified antisense oligonucleotides?
ASOs w central 10-PS-modifies 2’deoxynucleotides flanked by 5 2’OMe/O-MOE nucelotides each side
what are generation three modified antisense oligonucleotides?
ASOs w changed in sugar region, developed w non-charged backbone
name a gen 3 ASO other than PNA?
locked nucelic acids LNAs conformationally restricted nucelotide containing a 2’-O-4’-C-methylene bridge in b-D-ribofuranosyl configuration
what are the advantages/disadvantages of Peptide Nucleic Acid (PNA)?
- higher binding affinity to complementary RNA and DNA than RNA-RNA and DNA-DNA duplexes
- more stable towards nucelases + peptidases
- good stability in vivo
- can bind DS DNA -> transcriptional arrest: stop RNA made in nucleus
but: not substrates for RNase H, thus only produce effects by steric inhibition of mRNA translation
what are the advantages/disadvantages of Locked Nucleic Acid (LNA)?
- increased affinity towards target mRNA
- resistant to nucelase degradation
but not processed by RNase H, but can be incorporated into RNA + DNA as w PNA, which then reintroduces RNase H activity
What affects ASOs excretion from the body?
The interaction of ASOs with plasma proteins prevents their excretion in the urine.
- Notably, binding to high-affinity plasma proteins does not lead to better antisense activity.
All factors need to be balanced
what affects affinity of pp to ASOs?
type of chemical modifications made to ASOs.
Single stranded, PS-modified ASOs exhibit high affinity for plasma proteins and prevent excretion.
what oligonucleotides have low pp binding?
Uncharged oligonucleotides
PNAs and morpholino
have low plasma protein binding and are rapidly cleared.
T/F
ASOs DO NOT affect proteins that alr expressed, instead PREVENT protein expression
true
think abt mechanism
what 2 things can ASOs target both
cellaulr (host) RNAs and viral RNAs
possible to detect changed in RNA lvls how long after admin ASOs in wohle organism work 3-4hrs in cell?
12-24hrs
why 2nd gen ASOs allow weekly and biweekly dosing? advantageous as allows admin by injection
produce effects 10-15 days after single admin
disadvnatages of rna therapeutics?
RNA therapeutics, such as siRNA and antisense oligonucleotides (ASOs), have revolutionized the field of gene therapy. However, they have several limitations, including:
- Poor stability: RNA is prone to degradation by nucleases, which limits its stability and half-life in biological fluids.
- Limited tissue penetration: RNA is relatively large and hydrophilic, which makes it difficult to penetrate cell membranes and reach its target site.
- Immunogenicity: RNA is recognized by the immune system as a foreign substance, which can lead to unwanted immune responses.
- Off-target effects: RNA can interact with unintended mRNA targets, leading to off-target effects.
