An Interview with Dr John Rossi
| ||Dr John Rossi is Chair and Professor of the Department of Molecular and Cellular Biology at the Beckman Research Institute of City of Hope (Duarte, CA). His research focuses on the biology and applications of eukaryotic small RNAs and, in particular, their therapeutic use in HIV/AIDS and cancer. We recently had the opportunity to talk with Dr Rossi about the field of RNAi as therapeutics and his own path in science. |
What do you see as the future applications of DsiRNAs?
Dicer-substrate technology has progressed over the years. They have certain advantages over 21mers and we’re starting to realize that quite a bit in our own applications. We know that they get incorporated more efficiently into RISC because they are going through the microRNA pathway where they have to be “diced” and then handed off directly. That is one advantage and makes them potent at lower concentrations. We have been using them just for regular RNAi-mediated knockdown but also connecting them to other molecules so that we can hopefully get them processed off of aptamers and maybe even antibodies in the future. We have been successful with aptamers and CpG-containing oligonucleotides which get taken up by the immune cell where the Dicer substrate is processed off. If you use the 21mer it doesn’t work at all, but if you use the Dicer substrate it works great.
I’m hoping that Dicerna (see below) will be able to develop these into anti-cancer therapeutics and that will hopefully be where the field will have its first big impact. What targets and diseases they go after will have to be a business decision, but I would love to see them be applicable in cancer therapy.
Dicerna Pharmaceuticals is a private, venture capital-backed, RNAi-focused biopharmaceutical company developing novel therapeutic agents and related drug delivery systems in multiple disease areas based on its proprietary Dicer Substrate Technology™ platform and Dicer Substrate siRNA (DsiRNA) molecules. Dicer Substrate Technology is a second generation RNAi approach that results in greater potency, longer duration of action and enhanced delivery potential, differentiating it from other RNAi approaches. Dicerna is based in Watertown, Massachusetts and has major alliances with global pharmaceutical companies as well as other collaborations. www.dicerna.com
How are you using aptamers to target HIV?
Aptamers are a favorite subject of mine. They have been around for a long time, but the idea of using them for a delivery vehicle had not received much attention until the paper that came out from a group working at the University of Iowa and Duke University .
HIV puts its envelope out to the surface of the cell as part of the packaging process when it infects a cell. The envelope is present on the cell membrane before the virus assembles. So we thought that maybe that could act as a receptor and the DsiRNA might get internalization through pinocytosis. We tested that concept with a published aptamer and that aptamer was internalized very nicely in cells that expressed the envelope. But the aptamer really wasn’t as potent as
we wanted, so we developed our own aptamers by in vitro selection and came up with one that had a very broad host range (it effectively targeted a number of different HIV isolates). It was internalized nicely and, when we tagged it with a Dicer-substrate siRNA, we got good delivery, processing of the siRNA in cells, and target knockdown.
We went on to prove that all cells that expressed the envelope were getting targeted by the aptamer and then began an in vivo set of experiments with mice that were humanized. The mice had hematopoietic progenitor cells infused in them which can mature into T cells, monocytes, and macrophages. The mice can then be infected with HIV. We showed in a recent publication that we can inject the aptamer with the siRNA into the tail vein and see inhibition of the virus. The aptamer alone was a pretty good inhibitor but didn’t give long-term inhibition. But, the aptamer tagged to an siRNA targeting a viral gene gave us better long-term inhibition of replication than the aptamer alone. Amazingly, both gave one million-fold inhibition of viral replication. We had basically undetectable levels of virus in the cells. The virus did come back, but a subsequent injection knocked down the viral load again.
Dicer-substrate RNAs (DsiRNAs):DsiRNAs are chemically synthesized 27mer duplex RNAs that have increased potency in RNA interference compared to traditional 21mer siRNAs. DsiRNAs were developed as a collaborative effort between John Rossi at the Beckman Research Institute of the City of Hope and IDT.
Passenger strand: Before processing, the siRNA is unwound into two strands—the passenger strand and the guide strand. The passenger strand will be degraded while the guide strand will be incorporated into the RISC complex.
Aptamers: Oligonucleotide or peptide molecules identified to bind to specific target molecules through successive rounds of in vitro selection from large sequence pools. These molecules are used in both basic and clinical research and have played an important role in drug discovery.
What was the most surprising discovery made in the field?
To me, the discovery that small RNAs would activate Toll-like receptors was a surprise, but that has been several years ago. That level of activation was an alarm to the field but, if you look at the level of interference, it was actually really small compared to what other things do. Whether or not that is really a danger or not, I don’t know. I don’t think it is.
In a sense, the surprises are important findings because they dictate where to go next but also because they show that maybe we can couple some of the innate immune responses with the potency of the target knockdown. On the other hand, we know that you have to be careful, you don’t want to overexpress these sequences because you end up having toxicity.
We’ve been finding microRNAs in the nucleus of cells. It may be that they are doing something there and maybe siRNAs are going to the nucleus as well. We think that we see nuclear-mediated RNA interference. We are definitely keeping our ears open and being open-minded about the field. I think we’re going to see some interesting surprises.
What was your path into scientific research?
I was interested in biology in high school, but in environmental biology more than experimental biology. Rachel Carson’s Silent Spring was a really important book for me. When I went to college, I started with a forestry major but then decided that I didn’t really like what forestry was doing because, at the time, it was more concerned with producing lumber than with environmental concerns. So, I switched to environmental biology.
By the time I became a junior and had taken more advanced classes like genetics and cell biology, I realized that I really liked research. I had done a little bit of research in my senior year, but not much. I got accepted into a couple of good graduate programs and decided to go to Connecticut because that was my home state and they had a good genetics program. And there, I became fascinated with the molecular aspects of genetics.
The turning point for me was a postdoc at Brown. It was just at the time recombinant DNA and DNA sequencing technology was developing, so I was involved in all of that—mapping genes, identifying new genes, looking at gene expression at the molecular level—and that was incredibly interesting.
I started at the City of Hope as a postdoc but, once I got a grant of my own, it was converted into a staff position. The initial idea was to learn how to use synthetic oligos which were, in 1980, just in their infancy. The technology for putting oligos and genes together was just starting to be established. I became involved in learning how to use synthetic DNA for site-directed mutagenesis, as probes, for building genes.
My interest in how we can apply oligos led naturally to studies of ribozymes and then RNAi and all sorts of small RNA applications. At the City of Hope, we also have a very good climate for translating basic laboratory findings into the clinic and we developed a program for HIV. It was first antisense but then we quickly switched over to ribozymes. When I wrote my first progress report, I thought it was going to get shot down, but it turned out the NIH person who was reviewing my grant thought it was fantastic and she established a whole RFA in the ribozyme field for HIV. So, my work actually led to that whole development of ribozymes for therapeutics. We still use ribozymes, actually, but RNAi is so much more powerful. When I heard about how RNAi works in mammalian cells, I became intrigued as to how we could use it for HIV. We were able to make one of the first expression constructs which didn’t make a hairpin, it made separate sense and antisense transcripts. It worked very well in knocking down HIV replication. To this day, I still think it is a really useful tool because it doesn’t have some of the problems that hairpins have. But, it just hasn’t caught on.
The Dicer substrate was a completely accidental finding. We were trying to get rid of an effect we were seeing with in vitro transcribed RNAs that were triggering Type 1 interferon and, to make a long story short, we started to make our RNAs longer so we could clip off an extra G that was at the 5’ end. We found that we got rid of the interferon response and they worked much better than the corresponding synthetic 21mer that we had tested against. Everything else has been more directed but that was just a purely accidental finding.
Where do you see the aptamer/siRNA field moving in the next few years?
We have been trying to develop the idea that we could just kill the HIV-infected cells with the envelope-targeting method because it is very selective. So far we haven’t found an siRNA that basically wipes out HIV-infected cells. We can reduce cell viability quite a bit but it is not a permanent killing. But, that is with a single treatment. A Dicer substrate siRNA that is targeting an essential cellular gene would be a good strategy. But, the question is, how will the cells respond? Do they go through apoptosis? Do they just stop growing? We seem to have some indication that they do go through apoptosis, but we need to give multiple injections. That is something we are following up on.
We think this could be a real treatment for the disease–for patients who are not able to take their drugs anymore or have reactions to their drugs. And, in the end, it could actually be a stand-alone treatment for newly HIV-infected individuals. The idea is to purge their viral reservoirs by continually killing cells that have the virus in them, but only those cells. In an in vivo setting we might be able to illicit more immune response in these patients. I think we could think of a cure as an outcome of this. That is the long term goal, but maybe not that long term. It could happen soon, we’d just have to partner with someone who is willing to do the toxicity testing that needs to be done before this could go to a clinic.
We also have other projects going that are targeting B cell lymphoma and autoimmune diseases, like lupus. And, we are starting projects with collaborators that are targeting hepatocarcinoma and targeting pancreatic tumors. We would like to find dual-function aptamers that will block signaling but also deliver siRNAs to trigger either the immune system to go out and kill or block proliferation of the cells.
Have you found tissues that you aren’t able to target with aptamers?
Not yet, but we haven’t tried a lot of things yet. I would say we have been really lucky, at least initially, because the first two aptamers we ever tried to evolve were both highly functional. I have to attribute that to a really talented person in the lab who has done the aptamer selection. She just has good experimental technique. It does take a good experimenter to be able to develop these and follow through. I’m sure we’ll run into things that we won’t be able to target aptamers against. But, it is the technology that seems to be the issue. We were initially trying to evolve the Baf receptor aptamers using bead-bound receptor and ended up getting high-affinity aptamers for strep-avidin and for beads, but not for the Baf receptor. We went to the old-fashioned way which is just to bind these on filters which is very, very difficult and takes someone who has a lot of patience. But, she was able to get this done for three different aptamers now. We could probably find amongst the ones that have been evolved even higher affinity aptamers, we just haven’t had the time and resources to do that.
Have the successes in the field (RNAi as therapeutics) come faster or slower than you would have expected when you started?
I think the field has moved relatively fast. The rush to the clinic may have been premature. People have done the wrong things, like using naked siRNAs to try to treat diseases and finding that they are triggering Toll-like receptors or not really getting into the tissues. And, delivery vehicles have been a real problem because most of the liposome or lipid-based vehicles end up in the liver. That means that you could target liver disease, but if you’re going to target liver disease and go after it, go after something that is important, like Hepatitis C. I haven’t really seen huge efforts in that regard.
I think it is moving at a reasonable rate. It is certainly a tool that everyone uses; in terms of its use in mammalian genetic studies, it has been remarkable. But, we’re starting to discover some of the dark side of all of this, like some of the off-target effects, interferon induction, problems with design algorithms. But it’s nucleic acids, there have always been issues with RNA/RNA, RNA/DNA, DNA/DNA interactions. We’re finding things that, I think, are predictable. I’m pretty positive still. I see it as the only real powerful approach for targeted genetics. Antisense oligos are more diffusion dependent and I don’t see them as working by a mechanism that is going to give the efficiency you get with RNAi. With RNAi, you engage the cellular machinery which is very efficient at doing its job and that is really, to me, the big attraction. I feel that we need to understand a little more about the biology we are dealing with and we will come up with much better drugs in the future.
The Dicer substrates definitely have some advantages. I think what is going on at IDT is really important because Mark Behlke and his group study the backbone modifications that stabilize these. When we think about using the Dicer part of this and putting something at the other end of the molecule that allows delivery, it could be huge. If you could put a small molecule that would deliver it to specific receptors or specific proteins on the cell surface, there are all sorts of possibilities, it’s just a matter of identifying what small molecules you can attach and get them to be delivered. I see target-specific delivery as a really big area where Dicer substrates trump the other siRNA formulations right now.
What has surprised you about the development of this field?
The recent events were the big pharma companies pulling out. Roche, Pfizer, and Novartis all have backed off a little bit with their excitement in the field. That is probably partly the economy but there may also be some negative impressions that people in the pharmaceutical industry have had about the field due to the lack of a drug being out that is really efficacious. It has been a surprise that some of these companies have backed out after their investment has been pretty substantial.
Do you have any advice for students in the field of genetics? What are some of the current challenges in making a career in science?
My advice is to do what you love. If you do good work and you understand that science really is the search for the truth, as the Greek word translates, it will come to you, you don’t have to create anything.
There are various opportunities now for young people that didn’t exist when I was coming through. They aren’t limited to being academic scientists or at pharmaceutical companies—there’s biotech, there’s law, there’s a need for people with PhDs in genetics in a variety of fields, like genetic counseling. I think the genomics era is upon us and we’re going to have huge amounts of information to evaluate because of high-throughput sequencing. We’re going to need people that are trained in genetics and biology to be able to understand what these mutations mean and what SNPs in genes are all about in terms of disease diagnosis. I think we’re really going to open up whole new areas of personalized medicine that will need support from good basic scientists. That’s an area that I feel has a lot of future potential for young people.
1. McNamara JO, Andrechek ER, et al. (2006) Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nature Biotechnology, 24(8): 1005–1015.
Author: Jaime Sabel is a Scientific Writer at IDT.