A growing number of cancers and genetic disorders are having their cover blown. We’re learning more and more about their root causes. We know — in principle — how to correct many of them, but we’re still behind in the delivery of specific therapies. How do you get the right stuff into the right cells at the right time?
Genes can be delivered into our cells by modified viruses, but there are still some serious issues with them. Viruses can cause an immune response, and that means either inflammation or ceasing to be effective. Commonly used viruses like adenovirus and lentivirus integrate DNA into the patient’s chromosomes, and that’s a permanent change. Sometimes inadvertent changes to the patient’s DNA are made, which can cause, for example, cancer.
RNA can be an extremely useful therapy for many reasons. It can interfere with the functions of harmful genes, it can activate or deactivate certain proteins, and it can be used to make new proteins. RNA by itself generally cannot cause any permanent changes.
The RNA version of the COVID vaccine delivers RNA to muscle cells in order to get them to make a fragment of the spike protein. It uses little lipid balls to hold and deliver the RNA (which is quite a story in itself). These aren’t very specific in what cells they go into, but they don’t need to be. As long as the spike protein fragment gets made and the immune system can spot it, the vaccine will work.
But what about specific diseases in specific tissues? It would be great if we could deliver any RNA we wanted to any specific cell type we wanted, and somehow do it without any viruses, instead using only genes that humans already have.
But humans don’t have RNA delivery systems inside them, right?
Actually, wrong. We do, and their recent discovery is leading to ways we can use our own machinery to deliver therapies to our own cells!
A first system to do exactly that has been developed by Michael Segel and several others in the Broad Institute (Harvard/MIT) lab of Feng Zhang. It’s reported in the August 20 issue of Science. (Zhang, by the way, along with Jennifer Doudna of Cal-Berkeley, is one of the early pioneers of CRISPR).
One of the things I really like about this story is that the RNA delivery systems that the human body uses every day were derived during the process of evolution from viruses. A repurposing of something ostensibly bad or useless into something undeniably good.
A great example of this sort of thing is the bird “thumb”. Birds evolved wings from the same hand form that we did, but for birds, the thumb was sort of a weird leftover, just a little spur.
Many birds, including chickens and ducks, have vestigial thumb spurs tucked under their wing feathers that you can get cut on if you aren’t careful. Do they ever actually use these spurs for defense? It’s hard to say whether they have any remaining purpose. But in some other birds, like the bald eagle, the thumb got repurposed from useless to awesome. Now it supports a structure called the alula:
Its positioning on the eagle’s wing sure looks familiar, doesn’t it? That’s because it acts just like an airplane’s leading-edge slat or flap:
This helps the eagle — and the airplane — slow down and glide to a nice landing instead of faceplanting like a doofus on the tree branch. I’d say that’s a pretty good use of the “useless” thumb.
In a similar way, we think of viruses as bad, as “selfish” DNA that exists only to be a parasite. That’s certainly true up to a point, but evolution is never quite that simple.
Pretty much all of us eukaryotes (animals, plants, and fungi) — from yeast up to humans — have remnants of viral genes throughout our chromosomes. An awful lot, actually: About 42% of our genome is derived from retroviruses and their close relatives, retrotransposons. The vast majority of that DNA has lost its virus-like functions, but that doesn’t mean it isn’t useful; a lot of it is used to make repetitive RNA that plays a big role in which genes get turned on and which don’t.
But in the process of evolution, some of those old retroviral genes have been cleverly co-opted to act viruslike in very helpful ways, even essential ways. Let’s do a quick refresher on retrovirus genes so we can appreciate this better.
Just a quick note before I go on. Humans, mice, and viruses all have different rules for names of genes and proteins, but — mercifully — they do all have one thing in common: Gene names are in italics, and protein names are not. Let’s say I have a gene called KOS in my (human) genome. Its corresponding protein will be called KOS. In a mouse, they’ll be Kos and KOS, and in a virus they’ll be kos and Kos. Juuuust to make things complicated….
Anyway, a retrovirus has three prototypical proteins called Gag, Pol, and Env. In short, Gag has the structural components, Pol has the processing components, and Env is the cell-invasion component.
We’re looking at HIV in the figure below.
HIV starts out in a cell as DNA in the genome. The cell uses that DNA to make RNA, and then uses that RNA to make proteins, the same as for lots of its other genes. Two of the proteins that get made by retroviruses like HIV are shown at the bottom of the figure: Gag and Gag-Pol. Gag, all by itself, can assemble into rudimentary spherical capsids, like the one at the bottom of the figure, but Pol helps turn them into luxury spacepods for RNA transport (shown at the top). Parts of both Gag and Pol bind the viral RNA to help load it into the capsid.
That red part of Pol is the protease, which snips Gag into its component parts, each of which then assembles into its own structure: matrix (brown), capsid (green), and nucleocapsid (blue). That’s triple protection for the precious HIV RNA cargo. The other two parts of Pol are reverse transcriptase (light blue), which will be used once the virus enters its new host cell. It turns the HIV RNA back into DNA. Then integrase (gray) inserts the HIV DNA into the host cell’s DNA, and the process starts all over again.
Env is the yellow mushroomy guys. Env goes to the outer surface of the virus particle to help it break into a new cell. That function makes Env a “fusogen”. Retrotransposons have Gag and Pol, but they don’t have Env, so they can only move DNA around within one cell; Env is the key to attacking other cells.
So now we know what Gag, Pol, and Env do, so their evolutionary repurposing will make more sense.
In 2018, Elissa Pastuzyn and others in Jason Shepherd’s lab at the University of Utah showed that the Arc protein, which is made within neurons, and which is clearly a remnant of a former viral Gag protein, encapsulates its own RNA (Arc-encoding RNA, that is) into little rudimentary capsids, just like a viral Gag would do. It’s interesting to note that Arc is required for, among other things, the establishent of long-term memory.
A nice graphic demo of this was made by putting the rat Arc gene into E. coli, producing lots of Arc protein that way, purifying it, and showing that it spontaneously forms structures that resemble virus capsids. And there you are — an old viral gene with some kind of new function:
The Arc RNA inside these capsids gets shuttled from an established neuron into a new, growing neuron to help guide its development. There, the Arc RNA is used to make Arc protein, which does whatever it does in a fledgling neuron. No DNA is ever involved, so in that way it’s like the RNA COVID vaccine: Inject some RNA, let it be used to make protein for awhile until the RNA gets degraded, and that’s the end of the show. No permanent changes.
These capsids are limited, though. They can’t enter neurons or any other cells by themselves like viruses can. They get loaded by donor neurons into extracellular vesicles (little lipid balls), and the new neuron must cooperate and take them in using its own machinery. It’s occurred to people that this could be a way to transfer therapeutic RNA and other things into cells, but not enough is known about the whole process yet to do that precisely and intentionally.
Another recently discovered repurposed viral gene in humans is PEG10. It includes both gag and pol components, but not env. Like Arc, PEG10 also spontaneously forms capsids and binds RNA.
Segel and coworkers note that there are at least 48 real genes in the human genome that are derived from gag; that is, that get made into proteins and are used for … something. Of these, 19 are very similar between humans and mice, implying that they are doing something important and fundamental. They showed that many of these 19 form capsids.
PEG10 has not only Gag, but some of Pol (protease and part of reverse transcriptase) as well, meaning it probably forms luxury spacepod capsids. PEG10’s capsids were also the most full of its own RNA out of the 19 tested, meaning it was the best at packaging specific RNA. This was true of both mouse and human PEG10.
So they were off to a good start with PEG10, but a couple of things were still missing. First of all, no one cares (therapeutically) about PEG10 RNA, but they do care a whole lot about transporting some particular RNA of choice. So the authors figured out what parts of the RNA were needed for packaging and what parts were interchangeable with other stuff. And indeed they showed they could get cells to package pretty much any RNA they wanted into PEG10 capsids, provided it included the little packaging parts.
The other missing thing was Env. A capsid can’t get into other cells without that. So they started by recruiting a viral Env protein called VSVg that had been shown previously to enable fusion of viral capsids with cells. They expressed VSVg along with PEG10 and with a cargo RNA in mouse cells, and indeed, those cells produced capsids packaged with cargo RNA, and it went into recipient cells and delivered the cargo RNA. One of their cargo RNAs was an entire CRISPR gene-editing system (the Cas9-encoding RNA and the guide RNA), and it too got into the recipient cells and edited what it was supposed to.
They swapped out VSVg with another fusogen from mice called syncytin A, and that worked, too.
That gave them a workable RNA delivery system without any viral pieces at all. Human fusogens are the next obvious step, and some of those are known, but as far as I can tell they haven’t been studied in this way … yet. Now we’ve got to find out which human fusogens work effectively on which kinds of cells.
But also: Now that we know there are at least 48 real human genes that are viral gag derivatives, we’ve got to find out what they do, and how they might be useful as well.
Michael Segel said:
That's what’s so exciting. This study shows that there are probably other RNA transfer systems in the human body that can also be harnessed for therapeutic purposes. It also raises some really fascinating questions about what the natural roles of these proteins might be.
We still have so much to learn about ourselves, but the amazing discoveries keep coming. Diseases don’t get cured in a day; it happens bit by bit with advances like this one. So for now, let’s relish our new cannonball…