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UPenn s Alan Gewirtz on Antisense, RNAi, and Gene Silencing in Cancer

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At A Glance

Name: Alan Gewirtz

Position: Professor, medicine and pathology, University of Pennsylvania Health System

Background: Assistant professor, Temple University — 1984-1990; Postdoc, Yale University — 1979-1983; Resident, internal medicine, Mount Sinai School of Medicine — 1976-1979; MD, State University of New York at Buffalo — 1976; MA, microbiology, State University of New York at Buffalo — 1976; AB, marine biology, Colgate University — 1971

Though trained as a physician, Alan Gewirtz has maintained a long-standing interest in the research side of medicine, including gene silencing. In fact, one of his patent applications has found its way into the IP estate of Acuity Pharmaceuticals.

Recently, Gewirtz spoke with RNAi News about his research and how he is applying RNAi to cancer.

How did you get started with RNAi?

I’ve been interested in gene silencing forever — we’ve been very interested in antisense DNAs and RNAs for a really long time. I think we had some of the first publications, at least with antisense DNA, showing that you could use them to look at gene function in normal cells.

I’m a hematologist — we were looking in normal hematopoietic cells, and were looking at the function of the Myb gene in hematopoietic cells. We had a paper in Science, I think in 1988, using antisense DNAs to dissect the function of the Myb in normal blood cells. You couldn’t do any of that stuff back then; you couldn’t get enough blood cells to do any kind of molecular or biochemical analysis — there just wasn’t enough material. [Antisense] was a way to ask those sorts of questions on small numbers of cells. I think that most of the work we did was confirmed when an actual knockout was done three of four years later [and] was published in Cell.

We’ve been at this a long time: trying to look at or do what’s now [called] informatics-type work — there wasn’t a name for it back then. The segue into trying to make some sort of therapeutics out of it was straightforward for me, because I’m a doctor. It wasn’t exactly a major leap.

How did the transition [from antisense] to RNAi come about?

Well, if you can’t beat ‘em, join ‘em. I think that antisense DNA and all that stuff has been very difficult to apply in live cell systems, and I suspect that some of the issues that revolve around this work are also going to apply to siRNA. That was kind of one of the messages that we had [in a recently published] Nature Medicine paper.

The issues have always been delivery and targeting — not the gene you wanted to target, but where in the messenger RNA could you direct and antisense and expect to get some hybridization. If it doesn’t hybridize, nothing’s happening, at least as it relates to an antisense effect.

So what’s cool from my point of view about siRNAs is that they get incorporated into this multi-protein complex that has unwindase activity. It’s a way of allowing hybridization to take place between the targeting nucleic acid and RNA … and the actual mRNA target with some greater degree of efficiency or predictability. Because the RISC can melt the RNA, there’s a much greater chance of having that hybridization event take place.

Do you at all still work with antisense?

We do. There is this [recently published Nucleic Acids Research] paper [we put together] as an attempt to get at some of these core issues.

The delivery is still a problem, but we’ve been trying to develop ways to physically probe RNA structure as it exists in a real cell so that we could, at least in some predictable manner, try to target something.

Can you give an overview of the NAR paper and the Nature Medicine paper?

[In] the Nature Medicine paper … we used an siRNA approach to silence a downstream signaling protein from BCR-ABL in chronic myelogenous leukemia cells. [Novartis’ CML treatment] Gleevec, [which is a protein-tyrosine kinase inhibitor,] has been unbelievable for chronic myelogenous leukemia. But now we’re starting to see resistance develop.

There are a number of point mutations that have been described that don’t allow the binding of the Gleevec into … the ATP portion of the BCR-ABL [kinase]. When you get these point mutations … [disease] resistance emerges.

There are new inhibitors now that get around some of these issues, but still people are concerned that mutations and the inability for the Gleevec to bind to the ABL is still going to be a problem. So, we looked downstream of BCR-ABL, found this kinase, which seems to be very important in the signaling that BCR-ABL initiates, and tried to knock it out. I guess what was interesting about what we did was that we did this in primary cells. We did it in cell lines also, but we looked at actual patient material — that’s important.

We demonstrated that you could deliver [siRNAs] ex vivo and see an effect. Remember, delivery is a problem and targeting is a problem, so if you could deliver this stuff ex vivo and show that it was effective, … you could begin to apply [it] more or less immediately if you had an approved molecule.

In terms of where the RNAi-based therapeutics field is going, do you see it initially, at least, as an adjunct to small molecule drugs?

I guess it depends. Not every thing is druggable with a small molecule, that’s the first thing and everybody is probably hip to that now. So there’s a world of things that you could try and have some effect on [with an RNAi drug] that you couldn’t do anything about with a small molecule.

The second thing is that [one might use an siRNA] to complement the effect of a small molecule [inhibitor]. … At least as I conceive of this … the number of diseases where single hits are going to make huge therapeutics differences … like with [Gleevec] and CML … may be limited. There’s lots of diseases where it looks like one hit [will translate into a great clinical success], and that’s just not the case.

So it may be that you need to do multiple targeting, and [RNAi] might be a way to complement that in the manner in which I was suggesting.

I also have a bias, in a way, and my bias is that if you start mixing any of these different approaches with things that you know work, it may be difficult to see an effect of what you added. For instance, if you change a chemotherapy regimen for breast cancer, unless it’s really gangbusters, you’ve got to do thousands of patients before you come with the statistics to say that something’s better than another.

I think that if one’s trying to do a demonstration project, it’s probably better to use a cleaner system. So I would be very happy with a study that says, “We delivered an siRNA, we hit our target.” Then, it’s almost irrelevant in a way whether you got a therapeutic effect, because that’ll come later. I think the first thing is to show that you delivered this [material] to a live person and you [hit the intended target].

Could you highlight the NAR paper?

The NAR paper was just looking at different kinds of backbones for antisense DNAs that might be more efficient, and [the paper covered] how to physically target the molecule so that they would work. My sense of the number of papers that are now out there is that if one finds a region within an RNA that you could target reliably with an antisense DNA, then it doesn’t seem to be any better or worse in terms of RNA destruction than what you would get with an siRNA.

In other words, they’re equivalent if you can find a place to direct antisense DNA — they seem to be, anyway, or there’s a bunch of papers out there that suggest that. Intuitively, that makes sense to me. If you look at the catalytic portion of RISC that’s been published, its basically an enzymatic activity that’s very akin to RNase H, which is what you need to cleave a DNA/RNA duplex.

Even the cutting mechanisms look like they’re conserved, [and] a major thing from my point of view is structure and being able to target things.

So where are you now in terms of what’s going on in your lab with RNAi projects?

I’m still not convinced that delivering short hairpins or siRNAs with a viral vector is the best way to go, and so I’m … trying to mix the best of both worlds, antisense DNA [and] siRNA. I’m thinking that if we could deliver short double stands [with a workable transfection technology] … that might be a better way to do it.

So we are going to try to develop this purging application, because from a medical point of view there are diseases where you could use that immediately.

Purging application?

In other words, removing cells from a patient, then delivering the siRNAs to the cells you’ve removed and putting [the cells] back in [the patient]. That restricts the world considerably in terms of therapeutics because obviously you can’t do that with solid tumors, but for hematological malignancies like multiple myeloma or non-Hodgkin’s lymphoma … this is something that might make a difference.

That seems like something you could more or less do straight away.

How far along is this effort? Is this something you envision testing in people within a year?

This could go very rapidly if we had an appropriate partner. We’re an academic laboratory and we can’t make molecules that the FDA would approve — we just don’t have the facilities for that. We would need somebody to partner with who would say, “This is worthwhile and we’re willing to make an effort.”

Typically, in hematologic malignancies, [finding a partner] has been difficult because from the point of view of big pharma, it’s not very many patients. Gleevec may have changed this somewhat, and people may be more receptive to it, but it’s not clear. For chronic myelogenous leukemia, just as a for instance … that’s like 6,000 cases a year — that’s not something people are terribly interested in from a pharmaceutical point of view.

Have you had discussions with any possible partners?

Nope.

Do you envision this being something you need to arrange with big pharma?

To me, this seems like something that some biotech might be more willing to take a chance on in terms of this being a demonstration project, and then trying to get something off the ground that way.

Are you actively looking for collaborators? How does that process work?

Historically, people that are interested come knocking on your door, or my door I should say. But no one’s come knocking on my door yet, so where this is going to go, I have no idea.

It could, I think, go fairly rapidly, because as we said in this Nature Medicine paper, there are clinical grade apparati out there for electroporating molecules into cells — that already exists. So if that exists, and if you had an FDA-certified molecule, then you could put the two together — bone marrow purging, or this ex vivo delivery, is already approved. Everything is there, all the pieces exist. … I don’t think it’s crazy to say that in two or three years it might be possible to have something ready to be tried in the clinic.

 

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