Oligonucleotides Flashcards
LO
- Outline the potential uses of oligonucleotides as therapeutic agents for inhibiting the synthesis of individual gene products.
- Describe their mechanism of action and the various modifications that have been used to increase their activity.
Tell me about the central dogma of molecular biology, tell me about the ampliciation cascade involved in this and what binds to each stage of the cascade
What are Antisense oligonucleotides (ASOs)?
Short pieces of DNA (oligonucleotides) which are complementary to a region on a specific mRNA
Tell me the function of the Antisense oligonucleotide RNA-DNA hydrid the use of this hybrid
This RNA-DNA hybrid will block translation of this specific mRNA
If the chosen sequence is unique, then only this gene product will be affected
Universal therapy to target gene products (human or microbe!)
What are the main problems with antisense oligonucleotides?
Stability- oligonucleotides are rapidly degraded by nucleases i.e., half-life in plasma is minutes
Uptake- entry of a polyanion into the cell
How long should the oligonucleotide be?
To produce a stable duplex
To be unique
4 bases
42 = 16 dinucleotides
43 = 64 trinucleotides
44 = 256 tetranucleotides
What is n so that 4n is at least the size of the human genome
What is n so that 4n is at least the size of the human genome (3 x 109 base pairs)?
How long should an oligonucleotide be for selectivity?
416 = 4,294,967,269 possible 16-nucleotide sequences
(Though much of this does not code for RNA)
n > 16 (usually at least 20)
So ASOs need to be > 16 nucleotides in order for their target sequence to occur just once in the genome (but usually around 20 nucleotides for affinity)
What is a substrate for RNase H?
What is the role of this?
RNA/DNA hybrid is a substrate for RNase H
This degrades the RNA (not the DNA strand)
Apart from the RNA/DNA hybrid being a substrate for RNase H, what are some other unexpected bonuses?
Not just steric block on the mRNA, but removal of the specific target
Oligonucleotide is released to target another mRNA molecule – catalytic activity as these aren’t degraded themselves but enhance the degradation of mRNA
What are some antisense stretegies, explain briefly about each one
Antisense strategies
- Steric block/ translation arrest
- RNase H-mediated mRNA degradation- catalytic!
- Modulation of Pre-mRNA processing
Oligonucleotides breakall of Lipinski’s rule of 5, what are these rules?
- No more than 5 hydrogen bond donors
- No more than 10 hydrogen bond acceptors
- A molecular mass less than 500 daltons
- An octanol-water partition coefficient
(not all facotrs of 5)
What are the disadvantages/ problems of using oligonucleotides as drug molecules?
What are the main factors causing these disadvantages?
Need to be present in the right place, at the right concentration, for right amount of time
- Pharmacokinetics
- Cellular uptake
- Accessibility of mRNA target
- Hybridisation properties
- Inflammatory response
Tell me how pharmacokinetics is a disadvantage for oligonucleotides
Accumulate in specific organs and tissues e.g., liver, kidney, spleen, fat cells (BBB provides an obstacle to the CNS)
Poor half-life due to intracellular nucleases (Short half-life (T½) due to degradation by exo- and endonucleases.
Cannot be administered orally, usually through intravenous, subcutaneous, intravitreal (into the eye), or intrathecal injections (200-300 mg/week)
Tell me how Cellular uptake is a disadvantage for oligonucleotides
Poor permeability through hydrophobic bilayer
Tell me how Accessibility of mRNA target is a disadvantage for oligonucleotides
- mRNA secondary structure may inhibit binding
- mRNA is subject to up-regulation of target gene
Tell me how hybridisation properties are a disadvantage for oligonucleotides
Suitable binding and annealing kinetics?
Non-specific interactions with cell-surface or serum proteins (but can reduce renal clearance and increased circulation time)
Tell me how inflammatory responses are a disadvantage for oligonucleotides
Exogenous DNA / oligonucleotides containing CpG steps (e.g., ApApCpGpTpT) can invoke an innate immune response by binding to toll-like receptor 9 (TLR-9) present on immune cells (B lymphocytes, etc.)- TLR-9 recognises CpG and leads to an inflammatory response
(CpG steps are often methylated in human DNA (mCpG) but not in bacterial DNA, consequently the body recognises non-methylated CpG steps and initiates a natural defence mechanism).
Cellular uptake/ targeting of oligonucleotides
Schematic illustration of formulation strategies for peptide-mediated oligonucleotide delivery.
(a) Covalent conjugation between peptide vector and oligonucleotide via a stable or cleavable linker.
(b) Complex formation between peptide and oligonucleotides through electrostatic and/or hydrophobic interactions.
(c) Complex formation between lipid-conjugated peptide and oligonucleotides through electrostatic and/or hydrophobic interactions.
(d) Oligonucleotide condensation by peptide-functionalized cationic polymers.
(e) Lipid vesicle or exosome loaded with oligonucleotides and functionalized with a CPP.
(f) Lipid vesicle or exosome loaded with oligonucleotides and functional- ized with a targeting/homing peptide. CPP, cell-penetrating peptide; ON, oligonucleotide; siRNA, short interfering RNA
Tell me about N-acetylgalactosamine (GalNAc) oligonucleotide conjugate for liver targeting
Whats the receptor?
Asialoglycoprotein receptor (ASGPR)
How does the ASGPR receptor function?
ASGPR functions as a scavenger receptor that removes desialylated glycoproteins from circulation (into liver hepatocytes).
Conjugation of GalNAc to ASO increases its potency 10-30-fold
Where do N-acetylgalactosamine (GalNAc) conjugates target?
Where to target?
Early in the mRNA sequence is best (AUG start codon)
Avoid regions with secondary structures
Can target alternative splice sites
Tell me about N-acetylgalactosamine (GalNAc) conjugates cell entry
What may be some problems with this?
Cell entry
A problem! – as they are highly charged and high molecular weight.
Can be used in liposomes, attaching hydrophobic groups (cholesterol) or cell penetrating peptides (penetratin, HIV TAT peptide, transportan; these are all self-penetrating peptides)
Cell uptake in vivo is less of a problem than in vitro
What % of oligodeoxynucleotides are taken up by cells?
How are they taken up?
What can be done to improve this?
Only 1-2% oligodeoxynucleotides are taken up by cells
Diffusion across bilayer unlikely
Receptor-mediated endocytosis
Improvements:
Can be improved using uncharged/positively charged backbone modifications (see previous slides) or the attachment of hydrophobic groups. e.g., cholesterol
Attachment of cell penetrating peptides e.g, penetratin, HIV TAT peptide, transportan
Tell me about the toxicity of oligonucleotides
Generally non-toxic
phosphorothioate oligonucleotides can cause thrombocyotpaenia
Tell me about the role of CpG oligonucleotides with the innate immune response
CpG oligonucleotides and innate immune response (Toll-like receptors - TLR9) causing release of cytokines (MeCpG) in eukaryotes.
Toxicity
Is it really an antisense effect?
How would you demonstrate that it is?
Scrambled/mutated oligos.
Mutate the target.
Some may act as aptamers (folded structures with specific binding sites).
Automated synthesis (phosphoramidite chemistry)
Deprotect to leave a reactive hydroxyl group
Coupling with reactive phosphoramidite and tetrazole (essentially a catalyst)
Capping (99% efficient but protects unreacted molecules attached to solid support – prevents incorrect coupling in following cycle) and oxidation of phosphite (to generate phosphate)
Repeat
A phosphoramidite nucleoside is a derivative of a natural or synthetic nucleotide, with a N ́N ́-diisiopropyl phosphoramidite group attached to the 3 ́-hydroxyl. It is more reactive than the naturally occurring nucleotides and commonly used during ON synthesis. To prevent undesired reactions, the phosphoramidite nucleosides have their functional groups protected by an acid-labile dimethoxyltrityl or base-labile 2 ́-cyanoethyl groups. The synthesis proceeds via cycles containing the following steps: deprotection, coupling, capping and stabilization. Each cycle results in the attachment of an additional nucleotide from the first nucleotide attached to the solid support made of controlled pore glass (CPG). First, during deprotection, the dimethoxytrityl group protecting the 5 ́-hydroxyl is removed with trichloroacetic acid in an inert solvent (toluene or dichloromethane), leaving a reactive hydroxyl group to attach the following nucleotide. In the coupling step, reaction between the hydroxyl group and a tetrazole-activated phosphoramidite creates a 5 ́-3 ́ linkage to attach the additional nucleotide. After reaction, excess of reagent and tetrazole are washed out. Although this step is usually 99% efficient, a small amount of hydroxyl group remains freely reactive. To avoid further reaction in the following coupling steps, a capping step is performed with acetic anhydride to block the free hydroxyls. The last step in the cycle is the stabilization that results in the oxidation of the phosphite into a phosphate using iodine and water
How can the affinity of oligonucleotides be measured?
Whats the explanation formula for duplex stability?
Depends on base pairing and stacking interactions (length/ sequence) UV/ fluorescence melting allow comparison of melting temperatures (Tm ) between duplexes
Simple drug design forms a W-C duplex
Tell me about the structure types of the DNA-RND hybrid, DNA helices and RNA helices
RNA helices form A-type structure
DNA helices form B-type structures
Whats meant by the ‘sugar pucker’ structure and tell me about how this make the S-type and N-type helices lie?
Sugar pucker is a ribose ring which sugar attaches. It is unable to lie flat
S-type DNA lies below the ring
RNA N-type the 3’ carbon sticks up
More likely to have N-type over S-type as we want the most stable complexes with RNA
The RNA-DNA complex is less stable than the DNA-DNA helix
What are some pros of using antisense oligonucleotides as drug molecules?
- Universal
- Automated synthesis
- Simple drug design
- Easy to adjust affinity/ selectivity
What are some cons of using antisense oligonucleotides as drug molecules?
- Poor targeting/ uptake
- Degraded by nucleases
- CpG can induce immune response
Why is DNA an excellent platform for rational drug design?
Can change the base, sugar, backbone (can replace the atoms to alter the charge distributions, in some you can get rid of backbone all together), and conjugates (e.g., cholesterol, and other which can increase the affinity as well as the uptake)
What do the base modifications of DNA do and what are the different ways in which this can be done?
They increase affinity and duplex stability
- 5-methyl-C
- 2-amino-A
- G-clamp
improved interactions:
- hydrogen bonding
- hydrophobic/ stacking
- charge
- etc.
What do backbone modifications improve and what are the different ways in which this can be done?
They improve nuclease resistance
ways in which this can be done; forming…
- phosphorothioate: changing the oxygens for sulphur atoms
- Methylphosphonate: Me group added
- Phosphoramidate: N added and also can be used in combination where an S is used in place of an O
Tell me a bit more about each backbone modification
1st generation
In general, these modifications generate less stable duplexes due to steric hindrance (e.g., S atom or lack of stereospecific synthesis)
Most common is changing the Oxygens for sulphurs (makes much more resistant to nuclear degradation). Presence of sulphur means it binds more to serum proteins, preventing the clearance of oligonucleotides and being less charged facilitates the uptake into cells
Chiral centre (R and S forms) formed if only one sulphur is swapped in.
All those made for commercial use could be an R or an S form
If charged removed and Me added, these are useful clinically, far less soluble as charged has been removed, restrictions in concs made, no charge means it crosses membrane more easily
N added, could be combined with other modifications i.e., swapping O for S
All these modifications give great resistance against nucleases
Very useful analogues
Don’t activate RNaseH
Methyl phosphonate and phosphoramidate do not activate RNaseH?
Tell me about some sugar modifications, what do they improve?
What are the different types?
Improve nuclease resistance
2nd generation
Add things in order to force the North/ up configuration
Generates a more stable duplex
The different ways:
- 2’-deoxyribose
- 2’-methoxy (O-Me)
- 2’-O-(2-methoxy)ethyl (MOE)
- Locked/bridge nucleic acid (LNA/ BNA)
Strongest way of doing this (not used clinically) is locked/bridged nucleic acids; links 2’ and 4’ carbon with methylene bridge across which forces 3’ carbon into north config., which increases stability. Would want to use in every other position otherwise structural constraint would be too great
Tell me about some nucleoside modifications to DNA
Improve nuclease resistance (and affinity)
3rd generation
Morpholino: sugar is a 6 ring, still P in backbone, expensive, clinically useful structure
PNA: backbone is not a peptide, has extra C, side groups are the bases, based on pseudopeptide structure, no charge, forms strong complexes with RNA targets, downside is it’s not soluble, have to add charged groups to be soluble
The other DNA modifications made them not very RNase H compatible, what can be included to make it RNase H compatible?
Chimeric ‘gap-mer’ oligonucleotides (RNase H compatible)
- Since sugar and backbone modifications reduce susceptibility to nuclear digestion they can also decrease/ prevent RNase H cleavage
- Make modified antisense, the ends are modified and protected, central region is normal DNA (6-10 nts), RNaseH will still recognise and cleave target mRNA
- Gap-mer is part modified and part standard
What are some examples of small-molecular binders that bind DNA-RNA duplexes?
Tell me about each type…
Minor groove binders: Netropsin
Intercalators: Anthraquinone and Pyrene
(flat molecules that stack tightly between base pairs, protect from degradation and increase affinity)
Edge binders: Spermine and Spermidine
(highly charged, common small molecules in all cells that bind to DNA, +ve charge to neutralise the DNA, protonated at physiological PH, increase stability)
You can also attach reactive molecules that chemically modify DNA/RNA, what are some examples of this?
Cross-linking agents
Cleavage agents
Summary table of all of the DNA modifications
ASOs in clinical trials and use today
2ʹ-H, 2ʹ-deoxy; 2′-MOE, 2ʹ-O-methoxyethyl; ALT, alanine aminotransferase; apoB-100, apolipoprotein B-100; apoC-III, apolipoprotein C-III; ATTR, transthyretin
amyloidosis; CMV, cytomegalovirus; CNS, central nervous system; DMD, Duchenne muscular dystrophy; FCS, familial chylomicronaemia syndrome; HoFH,
homozygous familial hypercholesterolaemia; ISR, injection site reaction; IT, intrathecal; ITV, intravitreal; IV, intravenous; LDL-C, low-density lipoprotein cholesterol.
MHLW, Japanese Ministry of Health, Labour and Welfare; PMO, phosphorodiamidate morpholino oligomer; SMA, spinal muscular atrophy; SC, subcutaneous.
TTR, transthyretin; VLDL, very-low-density lipoprotein. All drugs are modified with phosphorothioate linkages, except for the PMOs. All 2ʹ-MOE chemistries include
2ʹ-deoxy sugar residues to support RNase H1 activity, unless specified as fully modified. Information in the table adapted with permission from ref.48, Elsevier.
Global RNA based therapeutics market
Tell me about the antisense oligonucleotide Vitravene (fomivirsen, ISIS 2922)
The first antisense oligonucleotide to be licensed for clinical use.
Active against CMV retinitis (AID patients who were susceptible to this virus due to compromised immune system, leading to blindness with this virus)
21-mer phosphorothioate (has sulphurs in each of the links)
5’-GCGTTTGCTCTTCTTCTTGCG-3’
Targets cytomegalovirus (CMV) mRNA encoding for the CMV polymerase
CMV causes retinitis in AIDS patients
Administered intravenously (intraocular- direct injection into the eye)
Approved by the FDA and available commercially since 1998
Not used now (still works well), but other therapies for AIDs are so successful that the incidence of CMV retinitis is almost zero so it’s not required
Structure has a mixed chirality as it has R or S forms
What can be used for exon skipping and exon inclusion?
Hybridisation of ASOs to splice sites, enhancer elements within the pre-mRNA transcript can be used for exon skipping
Whilst hybridisation to silencer elements can be used for exon inclusion (i.e., restoration of a splicing pattern)
Tell me about Exon exclusion by Exondys 51®
Tell me about Exon inclusion by spinraza®
Explain the process in which dsRNA acts by endogenous RNA interference
Natural long RNA molecules which is degraded and cut at the ends
dsRNA in RISC complex is separated into two strands; guide strand (useful), one released as passenger strand and is degraded
RISC finds complementary sequence to mRNA and binds to it, recognising the target
The RISC then leads to degradation of RNA target so its removed
Guide strand is degraded, RISC with guide can find another mRNA with the same sequence leading to degradation of RNA target
RNA is less stable than DNA. When RNA is in ds form it is relatively stable
Tell me some of the rules but also the disadvantages for the antigene oligonucleotides recognising genomic DNA by triplex formation
Rules:
- Simple rules that C binds to GC and T binds to AT
- The C has to be protonated though when binding
Cons:
- The PK increases from 4 to about 5-6 but this is still physiological- unstable complexes
- Cytosine derivatives have the same bonding pattern but higher PKs that work close to physiological
- strand targeting only contains purines
- Limited by PH and the sequences with what they can target
With Antigene oligonucleotides facing various issues with triplex formation, what could also be done to address oligonucleotide chemistry, why does this work better than AGOs?
- be addressed using oligonucleotide chemistry
- These are less stable than if just recognising a long string of purines
- Works at physiological PH
- Once they bind and form a 3-strand complex, is it silenced? TF cannot bind and cannot be read. Non-physiological complex formed in 3-strand (selectively switching off this gene)
Tell me the effects of antigenes in mice
- Vasquez et al. showed TFOs can induce mutations at specific genomic sites in adult mice
- Mutations introduced due to recognition and mis-repair by DNA repair pathways (e.g., NER)
- Mice were given daily intraperitoneal injections with 1 mg/day of oligonucleotide
- 5-fold rate of mutation in the cells
- Other target genes are mutated to a lower degree
- Shows it gets to sequence it supposed to (whether it’s used clinically will be told in time)
How are aptamers produced?
Created by in vitro selection from a large random sequence pool by SELEX (see over page but don’t need to learn in detail)
Tell me the process for how you generate aptamers via systemic evolution of ligands by exponential enrichment (SELEX)
- Generate a synthetic piece of RNA which has constant regions at either end and a random region in the centre
- Every strand that is made has a unique sequence at a low concentration. However, even though there is a low concentration of each unique sequence the overall pool has lots of combinations
- Out of all of the combinations, maybe one will bind to our target and the rest will fold into structures which are irrelevant and not useful
- Once you add the target, one should bind and those which are unbound should be washed away as it is now useless material
- You take the sequence that bound to the target and reverse transcribe the RNA into DNA
- go thorugh a thorugh sequences of PCR in order to generate more of the wanted sequence
- Then sequence it (might be lucky if you get more than one sequence that binds to the target)
- The one which binds to the target will bind with a very high affinity
Compare the engineering of antibodies vs aptamers
Compare the identification of antibodies vs aptamers
Compare the production cost of antibodies vs aptamers
Compare the reliability of antibodies vs aptamers
Compare the shipping of antibodies vs aptamers
Compare the affinity of antibodies vs aptamers
Compare the specificity of antibodies vs aptamers
Compare the detection systems of antibodies vs aptamers
Compare the benefit of antibodies and aptamers as drugs
Compare the reusability of antibodies vs aptamers
Tell me about the Thrombin-binding aptamer
- sequence
- structure
- DNA sequence that binds to thrombin (involve in blood clotting)
- Short sequence which binds selectively and tightly to thrombin
- Folds into G-quartet and quadruplexes
- Has a metal ion in the middle of the quartet
- This sequence folds into unique structure which has a pocket which binds to region of thrombin
What is VEGF (vascular endothelial growth factor) critical in and what is this?
VEGF is a signalling protein that promotes the growth of new blood vessels
VEGF is critical in angiogenesis and can lead to ocular degeneration
Angiogenesis: this is the formation of new blood vessels. This process involves the migreation, growth and differentiation of endothelial cells, which line the inside of wall of blood vessels
This process is controlled by chemical signals in the body
Macugen (peganatib)
