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CRISPR Editing in Primates

There’s some really interesting CRISPR news out today, and it’s likely to be a forerunner of much more news to come. A research team has demonstrated what looks like robust, long-lasting effects in a primate model after one injection of the CRISPR enzymatic machinery. There have been plenty of rodent reports on various forms of CRISPR, and there are some human trials underway, but these is the first primate numbers that I’m aware of.

The gene they chose to inactivate is PCSK9, which has been a hot topic in drug discovery for some years now. It’s a target validated by several converging lines of evidence from the human population (see the “History” section of that first link). People with overactive PCSK9 have high LDL lipoproteins and cholesterol, and people with mutations that make it inactive have extremely low LDL and seem to be protected from a lot of cardiovascular disease. There are several drugs and drug candidates out there targeting the protein, as well there might be.

It’s a good proof-of-concept, then, because we know exactly what the effects of turning down the expression of active PCSK9 should look like. It’s also got the major advantage of being mostly a liver target – as I’ve mentioned several times  on the blog already, many therapies aimed at gene editing or RNA manipulation have a pharmacokinetic complication. The formulations used to get such agents intact into the body (and in a form that they can penetrate cells) tend to get combed out pretty thoroughly by the liver – which after all, is (among other things) in the business of policing the bloodstream for weird, unrecognized stuff that is then targeted for demolition by hepatocytes. Your entire bloodstream goes sluicing through the liver constantly; you’re not going to able to dodge it if your therapy is out there in the circulation. It happens to our small-molecule drugs all the time: hepatic “first pass” metabolism is almost always a factor to reckon with.

But the liver is also a major organ involved in cholesterol homeostasis – that’s where VLDL lipoproteins are produced (which are turned into LDL out in the circulation), and where HDL is produced as well. So if you have some fancy new treatment that’s not going to be able to make it past the hepatic shredder, you might as well roll with it and target something in there anyway. That’s what the first RNAi therapy from Alnylam did, targeting a liver amyloidosis. Indeed, Alnylam also was working on a PCSK9-targeting RNAi (inclisiran), which they’ve now licensed to The Medicines Company for further development.

And that’s what we’re seeing from this CRISPR work now, which features the use of an adenine base editor that will come in and change A to G at a specific location (based on the guide RNA included in the package). One nice thing about these is that they don’t induce the double-strand DNA breaks of the original CRISPR technology, which is expected to allow for much cleaner editing – the homology-directed repair of a real double-stranded break can be a messy business. A screen of possible guide RNAs in human cells narrowed down the possibilities – the team was targeting an adenine residues were near splicing site of exon 1 and intron 1 in the sequence. Changing that to a G should make the subsequent protein start to include part of the nearby intron, which itself included a hard-stop codon near the beginning. So if you send the transcriptional machinery down that track, the whole thing comes to a halt, and you make completely hosed-up and inactive PCSK9.

The actual therapy is a long mRNA encoding the sequence of the base editor (with all the appropriate modifications to make it express well – this is very much like making an mRNA vaccine, just with a very different payload. The mRNA vaccines just make an antigen protein, but this one will of course produce a functional enzyme that is itself capable of modifying DNA. That mRNA and the guide mRNA (to tell the newly produced enzyme where to go) are encapsulated in a lipid nanoparticle formulation (again, similar to the vaccines and the existing RNAi therapies).

Exposing human hepatocytes to this did indeed knock down PCSK9 expression substantially. Infusion into mice did the same (albeit with some modifications the guide RNA to take the mouse sequence differences into account). Then the main event: infusing cynomolgus monkeys with this therapy at a dose of 1 mg/kg showed an 81% decrease of PCSK9 in their blood and an accompanying 65% reduction in LDL. Tissue studies showed that the base editing took place almost entirely in the liver. Modified sequences could be detected in the spleen and adrenal glands, and hardly anywhere else. Similarly, liver samples showed very low levels of off-target editing in their DNA sequences – the guide RNAs picked from the hepatocyte experiments performed just as hoped for. There were no adverse health effects, other than a transient rise in liver enzymes (which you would expect with any liver-targeting drug of any type, to be honest). Control experiments showed the enzyme elevation to be due to the lipid formulation, rather than the base-editing process. But the lipids from the nanoparticle formulation were almost completely cleared after two weeks, and levels of the injected mRNA itself declined rapidly over the first 48 hours in liver samples and were nearly gone at the 1-week point.

A longer-term study used four monkeys dosed at 3 mg/kg. In these, LDL cholesterol levels have apparently stayed at about a 60% reduction for eight months (and counting) after a single infusion. A worry before the study was that there might be an immune response to the new base-editing enzyme protein, but if that’s happening at all (no evidence for it) then it’s sure not lowering the efficacy. In fact, the authors estimate from the observed lowering that they may well have edited basically all the hepatocytes in the livers of the test animals. These levels match or exceed the LDL lowering seen with all other existing PCSK9-targeting therapies, and as the authors make sure to point out, these range in dosing from once a day to once every six months.

More work needs to be done on this before human trials than just a demonstration in four monkeys. But this looks to be a very promising demonstration indeed, both for PCSK9 editing and for base-editing CRISPR therapy in general. There are a lot of disorders, even just ones restricted to liver function, that could be potentially targeted by such genetic fixes, and most of them frankly have no real therapeutic options at all as things stand. We are getting closer and closer to being able to work down that list, to reach into the body and fix such defects by a technology that (if you’d shown it to people a few decades ago) would have been hard to distinguish from magic. These are great times.

56 comments on “CRISPR Editing in Primates”

  1. metaphysician says:

    First thing to pop into my mind: what are the known harmful side effects of inactive PCSK9? Not in terms of this therapy specifically, I mean. It would suck if preventing heart disease this way screwed up, say, cholesterol handling in the brain. Or is this one of those “closely conserved” systems where a mutation in this one gene has most of its changes compensated for elsewhere in the system ( minus the nonharmful reduction in ldl )?

    1. Derek Lowe says:

      There are already humans who are born with inactive PCSK9, and many others who are inhibiting its actions with other therapies. So far, so good.

      1. JohnBoy says:

        I posted this a week or two back and you deleted it.

        I was just wondering why?

        As of 30 April the number of deaths associated with the three vaccines given to Americans.

        PFIZER 1577
        MODERNA 1835
        JANSSEN 258
        TOTAL 3837

        As to thrombocytopenia we have:

        PFIZER 119
        MODERNA 106
        JANSSEN 75

        Readable VAERS data can be downloaded from http://preearth.net/data/2021-04-30-covid19-vaers.txt.gz

        1. Sean Fhearsalach says:

          preearth.net is a crackpot pseudoscience website. If these figures are true I would want to see a more reliable source.

          1. Pedantic Speaker says:

            You forgot to mention that “reliable VAERS data” is an oxymoron.

          2. WST says:

            “Readable VAERS ” was the wording….

            VAERS data are raw reports, need confirmations before it can be used in any meaningful way.

    2. PCSK9 guy says:

      PCSK9 may be needed in case of liver damage. As an example, it has a critical role in mouse liver regeneration. See Hepatology. 2008 Aug;48(2):646-54. doi: 10.1002/hep.22354. PMID: 18666258.

  2. Stephanie says:

    “…very low levels of off-target editing in their DNA sequences…” oy.
    But hail to the lab nerds, my heros, honestly. They’re the quiet, unheralded saviors of human beings, and I am grateful for their passions and funding.

  3. Thomas Salomis says:

    Can endogenous RNA act as guide RNA and contribute to off target editing?

    1. Eric K says:

      I love this question.

    2. Not-an-epidemiologist says:

      No. You need a very specific RNA secondary structure to interact with Cas9 (the protein that allows a guide RNA to bind its target).

  4. Arkham says:

    This is what they put in the COVID “vaccine”. God only knows what genes they changed.

    1. Tony says:

      The mRNA-based COVID-19 vaccines do not change any genes.

      1. Arkham says:

        Or so they tell us

        1. Daniel Correia says:

          It appears to be possible to rule this out simply on dose – each vial of vaccine would need to surreptitiously contain about 10 times as much material for such a regimen to work!

          The Moderna and Pfizer vaccines have between 0.03 mg to 0.5 mg total mRNA, about 3.5 mg of lipids and sundry, and 43.5 mg sucrose (total about 0.8 g per 10 dose vial).

          The dose used in this study was between 0.5 and 2.0 milligrams *per kilogram of body mass* – a whopping 40 milligrams for an adult, *plus* >3 mg/kg of dexamethasone (necessary for the effect, see figure 6 in https://doi.org/10.1038/mt.2009.208, inhibits inflammation).

          In addition, the vaccines are injected into a muscle, rather than intravenously directly into a vein over the course of an hour! I believe experiments using radiotracers have demonstrated that the majority of the vaccine resides almost entirely within the arm it is administered into; I believe very little of that small dose could even make it to the liver or other organ.

          The previous trial (Conway 2019) says 0.05 mg/kg (4 mg total for adult) could be ~1/3 as effective as 2.0 mg (I don’t know what the dose-response curve is for this, I’m not an expert), but the dexamethasone seems to be a deal-breaker.

          As far as I am aware, the sucrose is absolutely necessary to keep the LNPs (both mRNA or CRISPR) from breaking in the freezer; it also strains credulity that none of the dozens of countries doing independent testing of their batches would fail to notice that their vials contain 10 times less sugar!

          1. Rahviwub says:

            Okay but we’re going to need more doses, I saw an article where it said that people might need boosters every 8 months. You can’t just shut down a question with a second-in-time answer like that.

          2. Daniel Correia says:

            Dear Rahviwub:

            Great point! However, I think that dose-response curves of drugs generally mean that 10 administrations of 1/10th the dose (spaced beyond the residence times) do not produce anywhere near the same effect.

            The claim was that the specific technology described in this paper is used in the vaccine. Looking at the dosages and timeline used (pre-treatment with Dex via IV, then IV CRISPR NPs), it does not seem possible to me that this could be so, never mind the number of doses.

            Perhaps some technology could exist that would work with much less of a dose. However, if a pharma company wanted to surreptitiously CRISPR a population, it would seem much easier to use a vaccine that can be injected intravenously, like the tuberculosis inoculation[1].

            The concern is, of course, understandable. How can we “trust, but verify” if we cannot determine the vaccine’s contents ourselves? It would probably be relatively easy to verify for yourself that the vaccine does not contain a CRISPR mix – bring a spent vial to your local university’s GC/MS machine, or buy a cheap RT-PCR kit (<$300 nowadays).

            Every country independently tests the received material with tests that would easily pick up a CRISPR mix ("lot release programs"). You can see a transcript of a conference on lot-testing here [2]. A conspiracy would seem to rely on every single country's government participating – given global politics right now, that seems like it would be pretty hard to pull off.

            [1]: https://www.upmc.com/media/news/010120-bcg-iv-vaccine
            [2]: https://www.who.int/biologicals/publications/meetings/areas/vaccines/lot_release/Final%20Rep%20Mtg_lot%20release%20March%202007-2.pdf?ua=1

    2. JDK says:

      My vaccine genetic changes seem to be limited to making me look younger and hotter. YMMV.

      1. Belgian Grad student says:

        Touché

      2. TallDave says:

        that one comes out in 2041 (mammary firming options extra)

      3. theasdgamer says:

        Now all the female baboons won’t leave you alone.

        1. haha says:

          I’m guessing they give you a wide berth

      4. johnnyboy says:

        I don’t want to worry you but since I’ve had the vaccine I look in the mirror and every day I look more and more like Bill Gates.

        1. Jim says:

          Which vaccine did you get? I look more and more like Joe Biden.

    3. Derek Lowe says:

      The vaccine does not change your genes. This CRISPR experiment is *directly designed* to do that, but the mRNA vaccine has no such ingredients.

      1. TallDave says:

        really surprising prevalent that myth has become

        after all practically the whole selling point of mRNA is to confer immunity *without* any risk of touching the nucleus

      2. theasdgamer says:

        Do you know if anyone has looked at mRNA being RT’d into the DNA of people infected with HIV?

        1. Thomas says:

          Why? That RT risk would exist for any simple viral infection such as a common cold. Oh and native human RNA fragements as well. The vaccine RNA is nothing special, but there’s surely less of it.
          So it is a silly question you’re asking.

  5. VectorGuy says:

    Base editing is the latest cool CRISPR thing, but Penn has already done this in monkeys more than 3 years ago.
    https://endpts.com/jim-wilsons-team-hands-precision-bio-a-big-proxy-win-for-its-pcsk9-gene-editing-tech-with-3-year-monkey-data/

    1. Derek Lowe says:

      The current paper explicitly compares itself to that work in its discussion section, I believe. . .

  6. Anon says:

    What might be especially effective genes to target CRISPR for liver cancer?

    1. Sam says:

      Por que no P53?

      1. Nesprin says:

        Activating a mutated gene with as many splice variants as p53 sounds horrifically complicated, especially when there’s dominant null and true null versions of p53 floating around

        My vote would be for something like Kras for pancreatic or ER or Her2 for breast cancers, where knocking out function of a known tumor driver protein blocks growth and is hard to do efficiently enough with pharmaceutical inhibitors.

        1. Anon says:

          Appears Alzheimer’s might be Target table through the liver as well. CRISPR technology might be an all purpose tool to cure everything.

          1. Derek Lowe says:

            You must know more about the mechanism of Alzheimer’s than I do!

          2. John says:

            I assume you are referring to the production of gamma secretase that gets promoted after telomere degeneration? That secretase cuts amyloid proteins into beta-amyloid units which conglomerate to form beta-amyloid plaques that penetrate the blood brain barrier and are a known cause of Alzheimer’s. Fun fact, they also build up in retinal tissue and are believed to be a main cause for age related nonexudative macular degeneration.

          3. John says:

            And now that I think on it, I’m not certain that CRISPR would work for telomeric degeneration. And stopping the production of those secretases by preventing the reading of that gene would affect the beta-secretase production which seems like it would affect neural pathways. IDK am I missing something?

          4. anon says:

            The fox and the hare.

            Liver is the origin of brain amyloid. PMID: 32250293

          5. John Mabrey says:

            You need to read the articles involved instead of just calling me a liar. Do your research asshole.

  7. Kent Matlack says:

    Why did they choose the approach they did – with the intron – rather than mutating the promoter so that no unusual protein is made?

    1. Not-an-epidemiologist says:

      Probably because nobody’s promoter-bashed this gene to the level where a single base change is known to disrupt expression. (And I doubt you’d find a candidate even if you tried — gene regulation is pretty redundant in most cases, and the best you could hope for would be a reduction in expression.)

      The intron sequence, OTOH, was especially fortuitous, as there was an in-frame stop codon only three codons downstream from the splice donor site. It’s pretty-much your dream scenario for editing.

      1. LdaQuirm says:

        Do you happen to know how many residues long the truncated protein is? PCSK9 has 13 exons right? So it could ~50 AAs long?

        1. Not-an-epidemiologist says:

          It looks like the edited transcript yields a 72aa peptide (69aa’s from the first exon + 3 additional aa’s from the intron sequence).

      2. Derek Lowe says:

        It truly is. I assume that people are looking out for possibilities to do CAG or CAA (glutamine) to TAG or TAA (amber or ochre stop), or flip one of the tryptophan TGG codons to TAG or TGA (amber stop or opal stop).

  8. Barry says:

    What fraction of all hepatocytes must have been infected to achieve the effects reported?

    1. Derek Lowe says:

      The authors think that nearly all of them were, given the numbers.

      1. Barry says:

        yeah, isn’t that extraordinary? ‘Til now, gene therapy has relied on transforming a few cells.

  9. Crni says:

    Okay I am super confused is there a pictogram of all this somewhere? CRISPR without breaking the DNA double strand but how does it edit DNA then? Is this thing getting into the cell nucleus at all?

    1. sgcox says:

      It is the link to the paper Derek inserted in the middle of the post.
      I think it is in open access.

      1. Crni says:

        Unfortunately not 🙁

          1. Bannem says:

            Okay, a (probably stupid) question. Reading the blog posted linked to above, I see that as well as editing the target base on the desired strand, the opposing base on the complementary strand is also ‘edited’, to remove the base-pair mismatch caused by the first edit.
            What happens if this region of the complementary strand is also expressed as a gene? Is this known?

          2. sgcox says:

            If repair process removes this new C-to-U base, cell simply maintains its old gene variant. If it replaces the complementary G to A, you will have the desired effect. As I understand, it is 50-50 usually (kind of expected). To bias toward the desired effect, people reengineered Crispr to introduce the nick in the unmodified strand to mark it as “bad” one. Then repair process makes what you want in 80-90% of cells.

  10. TallDave says:

    good news

    and with AlphaFold2 we are much better at predicting protein structures

    you may yet live to inject mods coding for designer proteins that will extend your life, or at least improve the decline

  11. Tony says:

    I’m reminded of the scene in Star Trek IV where McCoy is in a modern hospital and is absolutely disgusted by all that he sees. I comes upon an elderly woman who’s in for dialysis. When he hears the word, he mutters something about the dark ages. He gives her a pill, says to take that, and if she’s not feeling better soon, just call him. Perhaps that’s not to be such far fetched science fiction for long.

  12. John says:

    Derek, I would like to thank you for the information that you have given out during this uncertain time involving the pandemic. As a native Arkansan, I have been friends with another native Arkansan who did his post grad work in organic chemistry at MIT. Together, using your information, we have successfully sent the credible information you have provided to a friend ours’ who is an internal medicine specialist at Baptist. He was put in charge of finding a treatment regimen that was successful due to your information as well as others fighting this pandemic. The treatment that was made was a part of your blog post here. I would like to personally thank you, as well as many of your Arkansan residents who benefited from your scrupulous posts involving treatment during this time. I firmly believe that your blogging has saved many Arkansan lives.

    From the bottom of my heart,
    Thank you

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