ASOs - AntiSense Oligonucleotides: what they are and how they work

Making sense of AntiSense Oligonucleotides: what there is to know about the ASO! First thing is that there’s not just “an” ASO - there are lots (and could be an infinite number), but “ASOs” didn’t rhyme with “know” - and they can do different things. Broadly speaking, each binds to a specific RNA and alter its processing and usage and/or triggers its destruction.
blog form: bit.ly/ASO_biochemistry
The “what they are” is hidden in jargon in the name. They are short (oligo) strands of chemically-modified nucleic acids (think DNA or RNA but stabilized) with sequences that are “antisense” to a target - meaning they’re like that “other strand” in double-stranded DNA - they have a complementary sequence to a target sequence, and therefore will bind to it. But they’re binding to RNA, not DNA. But they don’t just bind “any” RNA - instead each ASO is designed to bind one - and only one - specific site. They can do this because their sequence is designed to complement (and thus bind to) the sequence of their target.
As we’ll get into, this RNA is usually pre-messenger RNA (pre-mRNA) or mature mRNA. So a quick review:
When a cell wants to make a protein it first makes (and edits) messenger RNA (mRNA) copies of the original gene recipe (written in DNA). The copying part is called transcription and it produces pre-mRNA. A big part of the editing leading to mature mRNA is a process called splicing in which regulatory regions called introns get removed. Once it’s mature, the mRNA is handed off to the protein “chefs” - protein-making complexes called ribosomes. The ribosomes travel along the mRNA and (in a process called translation) link together the amino acids (protein letters) that the mRNA tells them to in order to form a protein.
ASOs can be designed to target and bind to the pre-mRNA (including in intron regions) or the mature mRNA. What happens next is determined in part by where the ASO is designed to bind and what modifications the ASO has, as we’ll get into. But for now just know that these are the main possible outcomes:
* ASO binds to pre-mRNA and alters its processing
* could prevent maturation or, what it’s commonly used for
* alter splicing patterns (much more on this so hang tight)
* ASO binds to mRNA
* Blocks ribosome binding or otherwise physically impedes translation
* Leads to degradation caused by cleavage by RNase H1 (which recognizes & cuts the RNA in RNA-DNA hybrids)
Let’s start with the mRNA-targeting
Once the ribosome does its thing (makes one protein from a mRNA) it can do it again - and again - or it could go off and make a different protein from a different mRNA. Because there are lots of mRNAs around vying for the ribosomes’ attention. If you reduce the number of mRNA copies for a specific protein, it’s less able to compete because it’ll get drowned out by more abundant ones. Which is one way ASOS can function, by selectively “turning down the knob” of problematic proteins by intercepting and degrading specific mRNAs - WITHOUT touching the original gene.
This is in contrast with “gene editing” with things like CRISPR which, when used to “knock out” genes, effectively breaks the knob off altogether - there’s no coming back because you don’t have backups and that DNA will get passed down to every cell made from that cell. As a result, unlike the original DNA copy, which must only be messed with with EXTREME EXTREME CAUTION, the mRNA copies, although important, are more safely tampered with (but still very cautiously because they have effects - which is why this is a treatment strategy in the first place).
It’s easy to see how an ASO binding to a ribosome’s “space” (such as a ribosome binding site) could hide/block it translation. And this is largely how people thought they would work. But turns out the main way they typically work on mRNAs is actually by triggering the mRNAs’ degradation!
But they can’t do this alone! Instead they basically just act as a sort of “flag” on the sequence that draws the attention of RNase H. “RNase” stands for RiboNuclease (hence the a not being capitalized which always trips me up…) and it tells you you’re dealing with an RNA cutter or chewer. There are different RNAs with different cut preferences (specificities). In the case of RNase H it cuts the RNA strand in RNA-DNA hybrids. And when it does so, it exposes unprotected RNA ends to exonucleases (chewers that dig in from the ends). (Normally the ends of mRNAs are protected by cap and tail modifications which I get way more into in another post).
For reasons that aren’t clear (even to the scientists studying ASOs), RNAse H prefers cutting certain places over others. In order to find the best sites, researchers will often do an ASO “walk” where the design and test ASOs spanning a range of the mRNA (since it doesn’t matter where exactly gets cut).
Finished in comments

[@musakhanmedicalenginee890]

I'm really excited to watch your helpful lecture, which I have never watched before. Plzzz keep it up. 😘

[@musakhanmedicalenginee890]

How I can get full course?

[@thebumblingbiochemist]

Thanks! hope this helps: th-cam.com/video/6ER0gy_IRWA/w-d-xo.html

[@thebumblingbiochemist]

RNase H is cutting the RNA, not the ASO, so that ASO can have another go! Meaning it can go target another mRNA, which is good because the cell keeps making them (you’re not messing with the actual gene remember).
But the ASO still has to worry about other nucleases that could degrade it, as well as some inherent instability of normal nucleic acids. So instead of just injecting strands of regular DNA to act as ASOs, the ASOs are chemically modified to improve their stability and nuclease resistances. We’ll get into some of these modifications later. But for now, know that they can kinda hide themselves from nucleases by making themselves look different than RNA or DNA. But this is a problem when you want to actually get RNase H’s attention! Then you have to actually look like DNA! Well, at least part of you does…
Gapmer ASOs take the strategy of bookending a window of DNA with modified nucleotides (5 or so on each side). In addition to improving stability, these modifications can increase the affinity (binding strength). And that increased affinity is sometimes desperately needed because it can be hard to get a bunch of ASOs where you want them. Which brings us back to the stability issue which we will return to after we talk about splice-switching ASOs. So bear with me (or jump forward!)
ASOs binding pre-mRNAs to act as splicing modulators
This section is an abridged version of a much more comprehensive post you can find here: blog form : bit.ly/spinrazasma ; TH-cam: th-cam.com/video/sK9NldpQC-M/w-d-xo.html  
 
Here’s the gist. To help explain things, I’m going to highlight a real-world, life-saving ASO, Nusinersen (Spirnraza™️) which was the 1st FDA-approved drug to treat SMA. SMA stands for Spinal Muscular Atrophy. In SMA, the gene form the “Survival of Motor Neuron” (SMN1) protein is mutated . SMN is important for the nerve cells that talk to your muscles (motor neurons), so patients with SMA, unable to make functional SMN1, have progressive muscle weakness & respiratory failure. Nusinersen works by altering the RNA splicing pattern of a “backup” version of the SMN gene (SMN2) to produce functional protein to make up for a protein SMA patients lack.
Remember that genes are often broken up into parts; some parts (exons) have instructions for making different parts of the protein. In between these EXpressed exons are INTerrupting introns. Introns don’t have “building instructions,” instead they’re like “margin notes” that provide regulatory info like when to transcribe a copy. These introns get edited out through RNA splicing, which turns pre-mRNA (which has exons & introns) into mature mRNA (exons only)(to fully mature mRNA has to get a 5’ methyl-G cap & 3’ polyA tail that tell the nucleus it’s okay to let it out and help it get translated once it gets out). 
 
Because exons are spaced apart, they can be spliced together in different ways (e.g. you can splice out (exclude) some exons together w/introns) to make different mRNAs & therefore different proteins from the same gene. Such alternative splicing can be really useful. e.g. maybe your robot doesn’t need x-ray vision for this task, so don’t waste resources including it this time. BUT sometimes it can “make mistakes” & accidentally cut out important exons &/or fail to cut out introns. more on splicing here: bit.ly/altsplicing  
 
Normally, you have 2 copies of each volume of manuals (chromosome), one inherited from each biological parent, so you have 2 copies of instructions for each robot. SMA is a recessive disease meaning that BOTH the instruction manuals for a robot are defective - there are mutations in both of the SMN1 genes.  
 
Sometimes in the course of evolution, genes get duplicated so that you have multiple DNA copies of an instruction manual. These duplicated versions are called paralogs & they can get altered subtly or dramatically to make new proteins. SMN1 has a paralog called SMN2. It’s almost identical, and if a full-length version of it’s made, it can compensate for missing SMN1. BUT a single nucleotide substitution (a swap of 1 letter in the instruction manual) causes one of SMN2’s 8 exons (exon 7 (E7)) to be spliced out most (~90%) of the time (even in healthy people). This gives you a truncated version of mRNA that, when translated, leads to a truncated protein (SMN2Δ7) that’s recognized as defective & degraded by the cell’s “quality control.” 
 
What determines whether E7 gets included? RNA splicing is carried out by a protein/RNA team called the spliceosome, which is like an editor & it gets help from your cell’s “autocorrect” helpers. These helpers are stretches of the pre-mRNA (like words) which can act as silencers or enhancers by making it fold in ways that hide or “unhide” splice sites (cis-regulation) and/or recruiting additional protein helpers (trans-regulation). Activators recruit the spliceosome (hey, over here!) & repressors help “hide” the site so the spliceosome ignores it (nothing to see here, move along… ) 
 
The combination of these positive and negative cis & trans factors tells the spliceosome where (and where not) to cut. Nusinersen binds to an intronic splicing silencer (ISS) site named ISS-N1 located in Intron 7 (In7) just next to the “end” of E7. This alters RNA folding (cis-regulatory effect) & prevents a repressor from binding (trans-regulatory effect) -> splice site is now recognized -> exon & intron are split up -> when intron gets removed, exon stays put. 
 
Since the drug binds an intron, it’s removed when the intron’s removed and thus doesn’t get in the way of exporting the mRNA into the cytoplasm or making the protein. And since its binding is sequence-specific, you don’t get much in terms of off-target effects.  
 
BUT you have to target lots of pre-mRNAs that keep being made. So some chemical modifications have to be made to the drug to make it last longer… 
Which brings us all back together with the first part of the post!

[@thebumblingbiochemist]

More on the modifications, as promised
One of the reason DNA, not RNA, is used for long-term storage in our cells is that DNA is “deoxy” in a way that makes it more stable - it lacks the -OH group ribose has in its 2’ position (it just has an H). Might seem like no big deal, but oxygen is nucleophilic (positivity-seeking) so it can attack the phosphorus in the neighboring phosphate group and break the chain off - especially if it gets help from RNases. To prevent this from happening in ASOs, but still allow for increased binding by being more “RNA-ish”, modifications can be put on an RNA letters’ normal 2’ -OHs for fluorine (so 2’-F) or capped it with a methyl (CH₃) group (so 2’-OMe). Nusinersen has an -O(methoxyethyl)(MOE) group(-O-CH₂-CH₂-O-CH₃), which protects it from nuclease degradation
It takes 2 to tango - the 2’ O and the phosphate (a phosphorus atom surrounded by 4 oxygens), so another another way they helped stabilize it is by swapping out some of the normal phosphodiester linkages (in which 2 neighboring sugars are linked through 2 of a phosphates’ oxygen groups) for phosphorothioate (PS) linkages (basically the same thing but with one of the “non-bridging” (not involved in the sugar-sugar bonds) phosphate oxygens replaced with a sulfur). Nusinersen has these. All these modifications confuse other molecules in the body, preventing them from recognizing the ASO as foreign nucleic acids, so it can slip past our body’s antiviral and antimicrobial defense systems.
Even with protective modifications, however, they can’t last forever, so repeated treatment is needed.
Modifications can also be introduced to target ASOs to specific tissues or cell types. For example, some ASOs (and other nucleic acid drugs) are designed to target liver cells.
To accomplish this, it can covalently (strong-bondedly) linked to a ligand with 3 N-acetylgalactosamine (GalNAc) residues (triantennary GalNAc). GalNAc is a modified version of the sugar galactose, and “ligand” is a term we use for a binding partner - and in this case, GalNAc is a binding partner for asiaglycoprotein receptors (ASGPRs). ASGPRs are abundantly expressed on the surface of hepatocytes (liver cells) - but not other types of cells - so they can be used to target the liver specifically.
ASGPRs are aka Ashwell-Morell receptors, and they normally serve roles including removing “glycoproteins” from the bloodstream. A glycoprotein is just a protein linked to sugar molecule(s). They do this via clathrin-mediated endocytosis, which is a way in which cells pinch in a piece of their membrane to swallow stuff that’s bound to the outside of it. So if the GalNAc-bound ASO binds the receptor, it will get swallowed and released into the cell.
If you look into RNA or DNA based therapeutics, you’ll see that most of the research so far has been done on diseases of the liver and central nervous system (CNS) - these are “easy” (definitely not actually easy!) targets for different reasons.
The CNS is protected by the blood-brain barrier - so you can confine treatment just to those cells and most of the cells in your brain are “post-mitotic” - they don’t carry out mitosis to copy their DNA, then split in two like most cells in your body do. This is one of the reasons brain injuries can be so devastating, because you can’t just make more brain like you could make more skin to heal a wound. But the non-dividing nature of brain cells also means that if you stick an ASO in there you don’t have to worry about it getting diluted out through cell divisions - though you still need periodic treatment because even with all the modifications, the ASO can get degraded. Liver cells *can* divide, but the liver is a great target because of the ASGPR thing and the fact that basically everything that goes in your body goes to the liver (it’s responsible for detoxing).
There’s currently a lot of work being done to try to target ASOs to other tissues, improve their uptake (which is often through lipid nano particles or with the help of cationic (positively-charged) polymers).
Here’s a review article (open-access): Zhu, Y., Zhu, L., Wang, X. et al. RNA-based therapeutics: an overview and prospectus. Cell Death Dis 13, 644 (2022). doi.org/10.1038/s41419-022-05075-2
And here’s a helpful seminar: Antisense Oligonucleotides- Mechanisms of
Action and Rational Design by Robert MacLeod th-cam.com/video/jkVPsfN5r60/w-d-xo.html
more on splicing and alternative splicing: bit.ly/altsplicing & th-cam.com/video/lKl87g66Rrk/w-d-xo.html
more on mRNA capping & tailing: bit.ly/mrnalife & th-cam.com/video/DCoVp_8zM-M/w-d-xo.html
more on RNA interference (RNAi): bit.ly/microRNARNAi & th-cam.com/video/7XHXF0x2uKA/w-d-xo.html
more on genetic knockdown & knockout: bit.ly/knockdownvsknockout & th-cam.com/video/YgCiYfjQzxw/w-d-xo.html
          
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com