I was mentioning the number of unusual therapeutic modes that are being explored these days, and one of those is (broadly) RNA-based approaches. The ones that directly feed into coding for proteins get a lot of attention (the mRNA vaccines, for example), and everything that’s currently approved is some sort of antisense or siRNA species. But RNA being what it is, there are plenty of other things to try. Here’s a good review of the noncoding RNA ideas, including a good list of things that are now in the clinic. The number of preclinical programs, I would not even want to guess at. As usual, I have to say that the variety of RNA species, structures, and functions is one of the biggest changes in the biology landscape since my college and grad school days. Who could have imagined all these things, or their importance to the functioning of the cell?
Of particular interest in the clinic are long noncoding RNA and microRNA targets. The lncRNAs are around 200 bases long, and they’re produced in the cell in a very similar manner to the more familiar mRNAs. But they’re not translated into proteins, which was a mystery for quite a while. What good are such RNA species otherwise, was the question. But they have domains that interact with other RNA molecules, with DNA sequence, and with proteins, and they’re large enough to fold into a wide variety of three-dimensional structures. It’s a bit like a parallel universe to proteins – modular linear structures that form an extremely large number of functional molecules with complex shapes. lncRNAs have been shown to have a variety of gene-regulation roles, through several mechanisms (transcription factor binding or blocking, interactions with epigenetic marks on histone proteins, or influencing the stability, further partner selection, or localization of their protein binding partners).
miRNAs are smaller beasts, generally 17 to 25 bases long. They don’t start out that way; there’s a lengthy processing cascade that starts from much longer RNA transcripts which are then chopped down to size by specific enzymes such as Drosha, Dicer and more. All these intermediate forms have somewhat different structures, but the eventual product is a double-stranded species that is unwound by RNA helicase for one of the single strands to be taken up by the RNA Silencing Complex (RISC), which is involved in gene regulation events.
All these RNA species (these two and all the ones directly targeting coding as well) have some general problems to overcome. A big one is immune response: as we have all been learning during the pandemic, the innate immune system is constantly looking for odd RNA species that it doesn’t recognize. There are whole families of receptors waiting to bind such things, single-stranded and double-stranded alike, so if you’re going to dose someone with a spiffy new RNA construct that’s not a vaccine, you’d better give this some thought. This is where all those chemically modified RNA bases (pseudouridines and more) come in. These changes can allow your therapy to fly under the radar. . .or not. And at the moment, there’s only one way to find out, and that’s to do all the preclinical and animal model work you can, then take it into human patients and cross your fingers.
The example of MRX34 will show what that’s like. This is a mimic of microRNA-34a, delivered in a liposomal formulation, to restore the lost tumor-suppressing activity of miRNA-34a itself in some solid tumors. It had shown no immunogenic effects in mouse models, and the same formulation had been used another trial for a different therapy with no sign of trouble. The drug had gone through nonhuman primate studies as well. But in the human trial, there were signs of immune response early on, and as the dosing cycle went further, five patients had severe adverse events, with four of them dying. All of these (enterocolitis, hypoxia/systemic inflammatory response syndrome, colitis/pneumonitis, hepatic failure, and cytokine release syndrome/respiratory failure) were indicative of a severe immune response set off by the drug. Meanwhile, four patients in the trial actually responded to the therapy, but as far as I can tell, it’s still not clear if this was due to its direct mode of action, or was something brought on by the immune response as well.
The review goes into a whole list of ideas to get around this problem – better immune function assays, combinatorial RNA dosing, different dosing schedules entirely, and more. Another approach is to ditch the RNA molecules completely and try to find small molecules that will affect the same pathways, and a ferocious amount of money has been pouring into that area, on the theory that small-molecule drug development at least has devils in it that we’re more familiar with.
All of these, though, have to deal with another overarching worry: specificity. The RNA-based drugs at least have something going for them there, since they have larger, more complex structure, while the small molecules can really be troublesome. Those compounds can have a lot of different modes of action – either directly at the business end of the mechanism, or by interfering with all those earlier processing steps mentioned earlier, and you can’t quite be sure up front if you’re not doing more than one of those simultaneously on different pathways. Inhibiting the processing/maturation steps can especially lead to more downstream action than you were planning for.
If you stick with the RNA-based species, there’s delivery to worry about, which has long been a tough part of all the oligonucleotide-based drugs (DNA and RNA alike). Historically, this has been the biggest reason for failure in the clinic. The recent success of the lipoprotein nanoparticle vaccines is good news, but it doesn’t mean that all RNA delivery problems have been solved. The range of potential RNA species is just too huge for a one-size-fits-all solution. There are many formulations already in use (gold nanoparticles, conjugated RNA species, non-lipid polymers), and the review has a good roundup of many other ideas just getting into clinical trials, which are extremely imaginative and wide-ranging (bacterial minicells, exosomes, bacteriophage carriers, etc.) The amount of time, effort, and money that has gone into this work is huge, and largely unknown to the general public.
This is going to keep everyone busy for some time to come, when you add up all the targets, disease, mechanisms, modes of delivery, and so on. And keep in mind, we still only have rough ideas about a lot of the opportunities in this area. There are some lncRNAs and miRNAs that have pretty solid stories built up around them, for sure, but there are far more about which we know zilch, or have never even encountered at all. It’s the wild frontier!
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