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.