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Oligonucleotides And Their Discontents

I remember being at a chemistry meeting in New Jersey back in around 1990 or 1991, where a speaker mentioned in passing that most of the people in the room would probably soon be offered a chance to move to California and work for some small company trying to develop antisense drugs. There was a wave of laughter, especially from people who’d already had such headhunter calls, and it really was a bit like that for a while. There was a great wave of enthusiasm for the idea of controlling gene expression this way – antisense oligonucleotides seemed to open up a whole new mode for therapy, promising near-magical powers. The exact same enthusiasm drove investment in the various RNA-interference companies years later, and is currently behind the enthusiasm for mRNA approaches and for CRISPR.

Although some of these trends can get irrational, the motivations behind them aren’t. You can see the common themes: control of gene expression, and thus control of protein expression. The obvious application is for the many diseases, many of them rare, that can be traced back to a defective gene sequence that in turn cranks out defective proteins that can’t function well enough, aggregate in cells over time, and so on. Start with sickle cell anemia, which thanks to Linus Pauling and co-workers is the original molecular genetic disease, and work your way on up through cystic fibrosis, Huntington’s, and many others. But there are plenty of normal proteins whose expression and cellular abundance we’d like to be able to alter at will, too. And for that, see the enthusiasm over targeted protein degradation, in oncology and other therapeutic areas.

But getting access to those control panels is not easy. Regulation of gene expression and of protein levels is accomplished in the cell by a completely bewildering mass of overlapping, interlocking, multilayered mechanisms, as well they might be. The work of the Department of Redundancy Department is very much in evidence as you look closer, too, which makes it hard to tweak just one thing without something else making up the difference. As it is with higher-level stuff like feeding behavior and reproduction, all the living creatures in which these pathways could be easily hijacked or broken are long gone. Our ancestors were the other guys.

Another big challenge, if you choose to operate at the DNA or RNA level, is that there are very, very few good small-molecule openings for therapy. That’s what led, by the way, to another burst of enthusiasm in the late 1990s and early 2000s for nuclear receptors, and a bit later on for epigenetic enzyme pathways. Both of those promised a small-molecule route into messing around with gene expression, but both of them, despite a few successes, proved too wooly and multifaceted for easy manipulation. So if you really want to step in and gum up the expression of this particular gene right over here and not much else, you’re forced into playing as the away team on the oligonucleotides’ home field.

That was the idea behind antisense. Come in with a complementary oligo strip to the mRNA that corresponds to a gene of interest, and it would stick to it and block up the works. What could be easier? But plain old oligonucleotides make terrible drug substances. The body does not look with favor on big stretches of unfamiliar DNA and RNA wandering around loose. There are lots of enzymes whose job is to rip such things to shreds on contact, and lots of innate-immune tripwires that are constantly looking for signs of viral infection (which is a main reason a living organism would suddenly experience such oligonucleotide events). What’s more, you obviously have to get into cells for these things to work their magic, and cell membranes are not geared to let them in very easily, either.

Thus the huge amount of work that’s gone into packaging of RNA and DNA molecules over the years – work that was, very fortunately, at a mature enough stage by now to allow the quick deployment of the mRNA vaccines. The famous lipid nanoparticles are a culmination of attempts to get the mRNA constructs to survive long enough in vivo and to actually be able to enter cells in decent numbers. The few siRNA therapies that are out there use very similar packaging, and even then are often targeted at diseases involving the liver. That’s because when you inject these things into the bloodstream (rather than giving them as an intramuscular dose like the vaccines, where you just want to hit the local tissue and a few lymph nodes), the lipid nanoparticles and their RNA payloads will invariably have to go through the liver (since our entire circulatory system loops through there) and there they will come to a screeching halt, never to emerge on the other side. The RNA and antisense ideas that aren’t targeted at the liver are generally targeted to the eye (retinal degeneration and the like), because the eye is a protected compartment where such exotica can linger without being degraded so quickly. Pharmacokinetics constrain us, big-time. Did you have some therapeutic oligonucleotide ideas that don’t involve the liver, the eye, or the lymphatic system? Better raise some more money, because you’ll need it. If you would like (for example) to target an antisense drug at neurons in the brain, then to re-use a favorite line of mine, you’re going to have to break out the Black and Decker, because for now that’s the only way you’re getting that stuff in there.

That is more or less how a potential antisense therapy for Huntington’s has been dosed, via intrathecal injection (right into the spinal column and thus into the cerebrospinal fluid space). Ionis, basically the sole survivor of the early 1990s antisense days, has been working on this for some years now as HTTRx, now known as Tominersen after Genentech stepped in with a deal to develop it. The drug is a complementary stretch of modified oligos that binds to a 20-residue stretch of mRNA produced by transcription of the HTT gene, and the protein produced by it, huntingtin, is absolutely the problem with Huntington’s disease. Wild-type huntingtin has a variable number of glutamines repeating down at the N-terminal region. If you have up to 26 of them, you’re completely normal. Up to 35 of them? Less common, but you’re still going to be OK. 36 to 40 glutamines means that you may or may not develop symptoms – that’s the grey zone. But more than 40 (the repeats can get up to over 200 in some cases) means that you will develop Huntington’s disease, and I am aware of no exceptions that have been found. Now, we’re not completely sure of all of the huntingtin protein’s functions, but losing it completely is embryonic lethal, and it has a wide profile of interacting proteins that mark it down as a big player in neuronal development and function. There’s a whole list of such polyglutamine repeat disorders, and they are all very bad news. The jury is still out on which is causing more damage – the repeated oligonucleotide CAG codons or the repeated glutamine residues in the protein, but the repeats in general are where you want to be if you want to fix Huntington’s. And it would thus seem to be a perfect candidate for an antisense therapy.

In 2018 and 2019, early stage human trials from the 2015-17 period were reported. The drug was administered in four injections, four weeks apart, and then patients were monitored for several months afterwards. CSF samples were taken to both quantify levels of the antisense oligo, and to measure reductions in mutant HTT. The therapy appeared to be well tolerated, and dose-dependent reductions in mutant HTT were observed, all of which sounded promising.

Well, that makes the news this week even harder to take. The data monitoring committee for the ongoing Phase III trial reviewed the results so far, and decided to stop things based on risk-to-benefit ratio. The press release goes on to say that no new safety signals were picked up, so that makes you think that there was little or no clinical benefit showing up, or at least not enough to justify giving people periodic injections into their spines. Intrathecal dosing as a therapeutic mode has always been problematic – you see it used as a one-shot in (for example) anesthesiology, but doing it all the time, even every few weeks, is another matter entirely. The company will continue to follow the patients who have already been dosed, but the drug itself is now abandoned.

I really am curious to hear more about what happened. You would have thought that reducing mutant HTT would have helped, but if this trial followed the Phase I/II results, then they reduced it (at least to some degree) without seeing any real benefit. Do you need to knock it down even more than Tominersen could achieve? Or are we learning something about Huntington’s that will complicate its etiology even more? This might end up adding more evidence to the line of argument that I blogged about before (“jury still out” link above), which was this paper’s focus. It presented evidence that it’s the snarled DNA with all those CAG repeats that is the fundamental problem, and in that case everything is downstream, including using antisense on the mRNA. If that’s the case, is it going to be DNA editing or bust? That’s going to be a significant challenge to do in the CNS. We shall see!

51 comments on “Oligonucleotides And Their Discontents”

  1. ezra abrams says:

    I swear this is true
    In 1990, anti sense companies had powerpoint decks where there was a slide that said
    “Traditional drug industry: 10 years, 500 million”
    “Antisense, 2 years, 50 million”
    [or whatever the numbers were; I forget exactly, but you get the point

    Then antisense sorta crashed
    Then years later, I swear the RNAi people took out the exact same slide deck and simply replaced the words “antisense” with “RNAi” and they were good to go . . .

    and all the cranky old grouches like me said, maybe but “phage display” and “combinatorial chemistry”

    1. jule s says:

      And now it is the Crispr people and they still don’t know how to target their constructs.

    2. mymagoogle says:

      There were also a few too many slide decks entitled “Making Sense of Antisense” or such back in the day.

  2. Gareth says:

    Intrathecal delivery is far from ideal but not unmanageable. Nusinersen, an ASO for spinal muscular atrophy, has been approved for a few years now. Which, depressingly, suggests lack of efficacy in HD is the problem here.

  3. An Old Chemist says:

    Celgene’s Mongersen belonged to this class of drugs! Unfortunately, it failed in a large phase-3 trial for Crohn’s disease, which led to celgene stock gong down in a spiral, and ultimately celgene got acquired by Bristol Myers Squibb, and mongersen got canned.

  4. sgcox says:

    Jennifer Doudna and colleagues are working hard on antisense drugs for Covid:
    What can go wrong ?

  5. luysii says:

    What is so remarkable about the ‘triplet diseases’ (e.g. expansion of a single codon) is that a strong majority of them are neurological. Although some cause trouble outside the brain (myotonic dystrophy) most do not. Even inside the brain only a small subset of cells is affected (Huntington’s chorea, many of the spinocerebellar ataxias). Theories abound, but I’ve found none of them convincing.

    1. /df says:

      Unlike the basic cellular machinery (“refined by 3.5 billion years of evolution”), the human brain has had less than 10 million years and probably a lot less less than 1 million in the form that we see today. Is it surprising that some aspects of its design are prone to error, especially if such errors act so slowly as to have little effect on reproduction?

      1. Charles H says:

        OTOH, consider that “refined by 3.5 billion years of evolution”. What percentage of those ancestors would have been affected by something that took more than 30 years to be detrimental? It’s not like we’re descended from giant theropods.

        There’s probably lots of genes we’ve got that act sub-optimally for those over 30. E.g., I can’t think of any evolutionary benefit to baldness, and I can think of several detrimental effects. But what proportion of our ancestors lived long enough to even notice effects that delayed? (That was the first non-neurological example that came to mind. I’m sure you can think of others.)

        1. sgcox says:

          , I can’t think of any evolutionary benefit to baldness,

          When you get hit by a stone axe, less organic material get embedded into your sculp and hence less chance of fatal bacterial infection ?

        2. Jenn says:

          Maybe baldness prevents some of the older, less genetically ideal men from continuing to procreate?

          1. Marko says:


          2. Cynic says:

            Hang on; wouldn’t that be the opposite of what’s true? Given that older men would still be sound genetically and reproductively, and that they’ve been healthy and clever enough to live to that age?

          3. ted says:

            only if they lack the red sportscar allele…


  6. exGlaxoid says:

    I remember working on an anti-sense target many years ago along with a west coast company also. We were assured that the nucleosides were very safe and would be targeted to only one DNA sequence, so that it had to be safe. We even tested them in several in vitro tox. assays and found no issues. After a year or more of work, we found sequences that could bind to the target DNA with extreme selectivity and potency.

    I spent 2 months preparing several hundred mg of the modified (non-natural bases) DNA oligomer, and then we tested our compound in mice. The first test all the mice died quickly. We kept going down in dose until the mice died very slowly, all from liver tox, as their livers turned into mush. The only group that might be interested now might be Decon, but I expect that it is too toxic for them… So don’t think that modified DNA is all harmless, as I know it is not. Turns out that in-vitro Tox assays don’t have livers…

    1. metaphysician says:

      Did you ever find out why it was liver toxic?

  7. JumboHC says:

    While the PR emphasizes “no new safety signals” were detected it is not true that there were “no safety signals” in the prior trials. Elevations in neurofilament light protein and enlargement of ventricles were both seen. While NFL was transient, it was nevertheless reported as dose-related. While lack of efficacy may be part of the problem, “risk/benefit” still may indicate an imbalance between the amount of benefit seen and the level of AEs (at whatever dimension). It is frustrating to have to wait to get more clarity (especially if you work at WAVE).

  8. Heywood Jablomi says:

    Is this the thread where the cranky old traditional chemists gloat about a trial failure involving a non-small-molecule drug? That thread where they get smug in the face of thousands of disappointed patients suffering from a horrible fatal illness that’s been utterly refractory to traditional small molecule approaches? Is this the one?

    1. Derek Lowe says:

      I hope not. I think this is the one where all of us who are working on new therapies commiserate about how tough it can be, and talk about how even things that look like they should have obvious benefit don’t always work. God knows that applies to small molecules, too.

      1. Heywood Jablomi says:

        Thank you Derek, that was very well said. My snark was based on a) years of experience in non-small-molecule drug dev and b) years of experience reading your blog 🙂

        1. Derek Lowe says:

          Oh, I can’t guarantee there there might be a few crusty comments thrown in by various readers (!) But personally, I’ve tried over the years to avoid becoming the guy who sits in the back of the conference room and says “Ah, that’s not going to work” to everything that comes up on the screen. I mean, most of the time you’d be right – most things don’t work – but what good that does that do anyone?

          Anyway, the humungous molecules being developed for protein degradation and other such tricks don’t give us small-molecule people much of a high horse to get up on these days. . .here’s hoping the delivery of oligos, of weirdo peptides and conjugates, and all manner of bizarre stuff keeps improving!

          1. Double standards says:

            Don’t worry! Derek reserves those comments for AI efforts. He says it’s because the AI companies make overblown claims and, credit where it’s due, he’s right. But it would be interesting to compare against the claims of RNA and CRISPR companies when they were trying to get funded, and which Derek accepts as reasonable attempts.

    2. young medchemist - not yet so cranky says:

      Rest assured that us chemists would be happy if any effective treatment for Huntington’s was found! If Tominersen had been successful, it would have also in part justified all the medicinal chemistry jobs lost as a big junks of R&D resources was moved into oligos. If the oligos don’t work out in the clinics, I’m afraid, it will not only disappoint patients and scientists in general, but also create a few more cranky traditional small molecule chemists….

  9. sgcox says:

    Could not find Tominersen sequence. If it is specifically targeted to triplet expansion full of CTG repeats which are know to form all sort of hairpins and mismatched helices which can produce all sort of side effects. Ionis are not fools of course and most likely targeted outside sequence which of course will degrade normal allele too.

    1. sgcox says:

      Typing too fast.. I mean antisense oligo would be full of CTG repeats.

      1. Heywood Jablomi says:

        Tominersen does not target repeats. It targets another region in the HTT CDS.

        1. sgcox says:

          So it should be relatively safe as AS goes. Then probably it simply does not drop the level of toxic RNA/protein enough to show benefit while being still safe.

          1. Heywood Jablomi says:

            The main issue is most likely poor distribution to the deep brain via IT dosing.

          2. Just curious says:

            Does anyone know of any drug delivered via IT that swims upstream to the brain compartment and permeates into nucleus or cytoplasm of neurons, astrocytes, microglia, what have you?

  10. Anon says:

    A lot of newer “breakthrough” therapies increasingly face drug delivery hurdles above all. The field of Pharmaceutics which is focused on the formulation, drug delivery and pharmacokinetic part of the drug development landscape plays a pivotal role in transforming a lot of these exotic molecules from intellectual curiosity to approved product status. A lot of startups ignore this vital role and end up crashing and burning in the clinic.

  11. Blaine White, M.D. says:

    I smiled at Derek writing, “But getting access to those control panels is not easy. Regulation of gene expression and of protein levels is accomplished in the cell by a completely bewildering mass of overlapping, interlocking, multilayered mechanisms, as well they might be. The work of the Department of Redundancy Department is very much in evidence.” Yes indeed, that’s what we get after 3-1/2 billion years of cellular evolution. God only knows how many LINE1 retrotransposons we’re carrying. And even at the very basic level of translation initiation in eukaryotes, redundancy managed to go from 1 (in yeast) to 4 (in us) eIF-2-alpha kinases. And a couple of those play in cell suicide mechanisms. With genes we’ve learned to read, but with the overlapping complexity it’s a helluva lot harder to write.

  12. Howard Rosenberg says:

    Sad about the Huntington trial especially for the patients. I hope there are more effective treatments on the way, but I would not write off Antisense just yet.

    Aside from Nusinersen mentioned above, Biogen and Ionis are working very hard on a revolutionary set of treatments for ALS and many other drugs. The first ALS drug targets patients with a SOD1 mutation and is expected to read out P3 results later this year. Though the results are still blinded Biogen has recently posted a job description for an EAP Associate Director with a focus on ALS. Seems like Biogen thinks things are going well. And Chris Snow, a patient diagnosed with the disease and his wife are active posters on Twitter. His significant progress makes them think that he is one of the lucky participants receiving the drug.

    In addition to the SOD1 trial, Biogen is running 2 other ALS trials. The first for ALS with C9orf mutations is scheduled to read out P2 data later this year. The third drug targets most other forms of ALS. It is currently in a P1/P2A trial. Also Ionis is starting a P3 registration trial this year in a drug that targets the FUS mutation of ALS. Again do you start 3 or 4 different trials unless you are pretty confident in the results of the first trial?

    Yes Antisense has had its issues over the last 30 years. But isn’t trial and error how you make progress especially in something as complex as disease treatments.

  13. Jon Moulton says:

    “Ionis, basically the sole survivor of the early 1990s antisense days” But what about Sarepta Therapeutics, founded as AntiVirals Inc. on January 1st 1980?

  14. E. M. Unfred says:

    I work in oligo manufacturing, so I get to see how much interest there is in the field. Over the last year, we have been flat out with work. Every major pharma has products in development. As for the problem of delivery to specific tissues (which reminds me of the work done in the 1980s to synthesize ligands for things like Tc99m which would concentrate the probe in the heart or in the liver, etc.), when I started the oligos were naked or coupled to PEGs. Now it is all about the GalNAc ligand, which gets you into the liver. But I am not seeing much of other targeting chemistries.

    These things are hard enough to manufacture. Dr. Dowdy at UCSD thinks that oligos modified with to enhance their escape from endosomes is the way forward, but damn those will be painful to make by the kilo.

  15. Daniele Merico says:

    Three possible reasons, IMHO in order of likelihood:
    – insufficient knockdown, considering that (a) you can’t aim at a complete knockdown (unless you target only the mutant transcript like WAVE is trying to do) because some HTT is required, and that (b) the basal ganglia are important for HD and the hardest brain region to reach with the PS gapmer ASO chemistry used by Ionis (NHP + Malat1 is already optimistic and knockdown in those regions is minimal)
    – the neurodegenerative process in HD is not really reversible, it’s a train wreck that can’t be stopped
    – there is more than mutant transcript and protein expression; some authors suggest a genomic instability aspect

    PS: please don’t give us a hard time about ASOs/siRNA not working; get real and look at spinraza, at the string of approvals by Alnylam – the 1990s were 30 years ago, the backbone chemistry has evolved, the ligands for delivery too (GalNAc is great, but there’s more)

  16. david says:

    “Intrathecal dosing as a therapeutic mode has always been problematic – you see it used as a one-shot in (for example) anesthesiology, but doing it all the time, even every few weeks, is another matter entirely.”

    If the therapy worked, the repeated intrathecal injections would be replaced by an indwelling ventricular catheter connected to a subcutaneous pump and a reservoir. Not an insurmountable hurdle.

    Shame it didn’t work, and I sincerely hope they can find a way to fix the failure.

  17. albegadeep says:

    “you’re going to have to break out the Black and Decker”

    Nearly ruined my keyboard with tea when I hit that line…

  18. Sunyilo says:

    The greatest challenge for antisense programs is to find applications where these therapeutics are efficiently working in our bodies. Most therapeutic ideas are around treating diseases attributed to gain-of-function mutations just like the Huntington’s case you detail here. Alas, for such diseases typically less or even much less of the therapeutic molecules are doing what we want them to do – binding to their targets and have those degraded – and 90+% of therapeutic molecules are just trafficking around and overloading lysosomes everywhere they can get to potentially preventing normal lysosomal function on the long run. Coupled with the fact that gain-on-function diseases typically need significant (>>50%) knock-down of target transcripts it’s very hard to reach efficacious doses without side effects. Needing chronic dosage does not help either.
    In specific instances though antisense is viable approach to treat loss-of-function diseases where boosting the expression of a paralog of the faulty gene even by a few % can be clinically beneficial at relatively low doses (see the success of nusinersen). The lesson is, no approach will be successful without detailed understanding of disease mechanisms and by blindly going after disease-associated molecular signatures – at least there are astronomical odds stacked against success.

  19. David E. Young, MD says:

    As an oncologist, I can remember the anti-sense bcl 2 studies in the 1990s for melanoma: ” Bcl-2 Antisense (oblimersen sodium)”. Ultimately the randomized study did not show benefit or at least not enough to compell the FDA to approve it. Made by Genta. It is not approved. Apparently there are still some studies going on.

    1. David E. Young, MD says:

      Clinical trials. gov lists 40s studies, all completed or terminated.

  20. Steve says:

    The slide I remember from the ’90s:

  21. Trueprogress says:

    Let’s face it. The CDC sucks.

  22. Ken says:

    For some reason every time Derek mentions the liver, I’m reminded of Captain Ahab in Moby Dick. It does seem that organ is the great nemesis of drug designers.

  23. Howard Rosenberg says:

    And today there was good news for Antisense. Ionis unveils positive PhII data for in-house rare disease drug, paving way to blockbuster rivalry with Takeda. “The trial, which tests IONIS-PKK-LRx in hereditary angioedema, is small with 20 patients. But the results were unequivocal: When you compare the 14 adults who received an 80mg monthly dose of the drug to the six who got placebo, you see a mean reduction of 90% in the number of monthly HAE attacks through weeks 1 to 17 in the study, meeting the primary endpoint (p <0.001). From weeks five through 17 (one of the secondary endpoints), the mean reduction in monthly attacks rose to 97% (p=0.003).

  24. Daren Austin says:

    “without seeing any real benefit”. It’s a twenty-year PoC study in asymptomatic newly diagnosed family members. How does one reconcile (relatively) short-term changes in approvable endpoints with possible long-term pharmacology necessary to move the needle on such diseases? AD, PD, DMD, HC there is a pattern here.

    The half-life of an intrathecally administered antibody is about five hours. That’s been known for more than thirty years. Keeping levels high is B&D stuff indeed.

    1. Alex D says:

      Especially with the cost, Huntington’s will be a tough one to reconcile. For the others though, especially involving muscles like DMD & DM1, the end points should be much more easily defined through the clinical process. Marked muscle improvement mixed with reduced splicing errors in muscle proteins.

  25. Andre Brandli says:

    Regarding antisense oligonucleotide chemistry, most FDA approved oligos are MOEs. What’s the opinion of the “In the Pipeline” readers on morpholino oligonucleotides? They work great in zebrafish and Xenopus to disrupt gene functions in vivo, if handled correctly.

    1. Jon Moulton says:

      Delivery into cells is still the primary hurdle for therapeutic applications of Morpholinos. The cell-penetrating peptides conjugated to Morpholinos (PPMO) work pretty well and are now in clinical trials (SRPT-5051). For papers describing research with PPMOs you might start with the work of Hong Moulton, the inventor (pubmed Moulton HM), currently at Oregon State University. For a broader compilation of Morpholino publications, see

  26. BTDT says:

    I like them, especially considering there are great ways to make then such as:

    1. Andre Brandli says:

      Here the notice to the paper your were referring to: “This paper was retracted on May 30, 2019 (J. Am. Chem. Soc. 2019, DOI: 10.1021/jacs.6b08854).

      Did I miss something?

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