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City of Hope s John Rossi on Using RNA Interference to Fight HIV-1

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At A Glance

Name: John Rossi

Dean, Graduate School of Biological Sciences at the Beckman Research Institute of the City of Hope Cancer Center

Chairman, division of molecular biology at the Beckman Research Institute

Background: Professor, Beckman Research Institute — 1996-present

Chairman, division of biology at Beckman Research Institute — 1992-1994

Research Scientist, Beckman Research Institute — 1990-1994

Postdoc, Brown University — 1976-1979

PhD, microbial genetics, University of Connecticut — 1976

MS, microbial genetics, University of Connecticut — 1971

 

John Rossi has spent most of his professional life working at the Beckman Research Institute studying RNA. That work has led him into the field of RNA interference, and its potential use against HIV-1, and also landed him on the scientific advisory board of Australian RNAi firm Benitec. Rossi recently spoke with RNAi News about his work.

How did you first get involved with RNAi?

I’ve had a long-standing interest in RNA function in the cell, starting out with the role of pre-mRNA structure and small nuclear RNAs in the process of pre-messenger RNA splicing. I became interested also in autocatalytic mechanisms of splicing, and then in particular, I became very interested in how RNAs can function as enzymes. I was studying small ribozyme function for a number of years — hammerhead ribozymes being the focus of my work. At the same time, I realized that this had potential therapeutic applications since these ribozymes could be engineered to Watson-Crick base pair with any RNA target of interest, and then functionally inactivate that.

So, we developed a program in my lab to target HIV with ribozymes … and recently, because of the more potent activity of RNA interference, have switched a lot of focus on RNA-mediated mechanisms to RNA interference.

So, that’s where I stand at this moment. We’re actually trying to develop RNAi as a therapeutic modality for the treatment of HIV infection in a gene therapy setting.

Can you talk a little bit about the work on HIV?

What we’re doing, in particular, is trying to utilize the idea that you can genetically modify hematopoietic stem cells … with a lentiviral vector that expresses short hairpin RNAs that would generate siRNAs inside the cell — the idea being that we could actually put several different siRNA genes targeting HIV, and also one of the co-receptors for HIV, on the same viral backbone, and use that to introduce the genes into hematopoietic precursor cells that you could isolate easily from patients. [We could] then re-infuse those cells back into patients under conditions where the cells can engraft in the marrow and then continually produce T-cells and macrophages and other lineages that would be susceptible to HIV infection but would now become resistant.

[The] idea [is] that the long-term impact of this could be complete treatment for HIV infection and basically eliminate the need for chemotherapy or could be able to be used as what we call an adjuvant chemotherapy, where the doses of the standard drugs could be lowered considerably because the gene ther- apy would actually come into play.

What is it about HIV that makes RNAi a good approach?

The reason I like RNAi and HIV as a combination is that when you target the virus with any of the chemotherapy agents, which are small-molecule drugs, they’re very effective but the virus mutates very quickly and it becomes resistant to these drugs. That’s one problem. The second problem is that the drugs are also somewhat toxic to the patients that have to take them, and if you have to take these drugs for the rest of your life — some of them are ... immuno-suppressive in their own right, so it could ultimately lead to some downstream lymphoma or leukemia.

So that’s the problem: you have this virus that mutates rapidly and escapes any kind of drug you throw at it, eventually, and what do you do next? The reason I like RNA interference is because the whole virus genome is a target; that is, there’re 9,000 bases of information in that virus and any stretch of 21 bases is a potential target for RNA interference. So, we can make multiple RNAs against multiple targets, and that way we can evade the resistance problem because siRNAs are small. [Therefore] in a gene therapy setting we can multiplex these — we can put up to 10, 20, who knows how many, in a single vector and have a very effective way of evading viral resistance and, because this is such a powerful mechanism in terms of its knockdown, we’ve been able to observe up to a 10,000-fold inhibition of viral replication using RNA interference, which is as good as you can do with any of the conventional drugs.

Can you comment on any particular hurdles for the technology in HIV?

The hurdles are really going to be putting viral vectors into human stem cells, because there is, right now, a feeling that this is not completely safe, primarily based on some negative results that occurred in a trial conducted in Europe with children that had X-linked immunodeficiency. There were actually 15 patients in this trial and two of them came down with T-cell leukemia, which was directly related to the insertion of a retroviral [vector] bearing the corrective gene into an oncogene area, turning on this oncogene.

That kind of created a negative atmosphere for gene therapy, in general. That’s changing. It’s realized that [the European trial] was a special situation, although that cast a shadow over the entire field of gene therapy. So, I think that’s the major hurdle. We can actually get hematopoietic progenitor cells out of patients very easily; we’ve already done this with ribozymes. You can mobilize these into the peripheral circulation, isolate large numbers of these, genetically modify them, and put them back in the patients.

Under the proper conditions of gene transfer, these cells should engraft in the marrow and start producing progeny cells that should be T-cells and macrophages and other cells that are potentially a target for HIV, but now will become resistant for HIV infection.

But, the main challenge is overcoming this reluctance of the regulatory agencies to readily approve stem cell gene therapy trials.

It should be pointed out that our first approach is going to be to do a T-lymphocyte trial. We’re actually going to go to the recombinant advisory committee next spring and propose a trial in which we isolate T-cells from HIV-infected individuals, transduce in RNAi-expressing vectors, expand those T-cells ex vivo several million fold, and then put them back in. This is a trial that will not be received with any kind of skepticism because It’s T-cells, it’s not stem cells. This will be our first in-road into the therapeutic application [of RNAi].

When do you hope to start the trial, and how long do you think it will take to start seeing data?

We hope to start the trial within a year and a half … and the data will start coming in quite quickly — a month after the trial is initiated. We will have no problems getting patients to voluntee;, that’s not a problem at all, because there are plenty of patients who are drug failures [and] who feel that they can’t take the standard chemotherapy agents. So, this would be a fairly straightforward process, to enroll patients in this trial. So I think that we could start very soon and get data with it. [In] two years we should be getting significant data.

Is this being done alone or with partners?

We have an academic partnership with Carl June at the University of Pennsylvania, who is an expert in T-cell biology. He will primarily be involved in doing the T-cell expansions and analyzing T-cell function. We also have an academic collaboration with Hans-Peter Kiem at the University of Washington Primate Center, who’ll be doing some work with primates using RNAi. This would help us model a stem cell approach.

And then we have two corporate partners. One is Benitec, which is an RNAi company. … And the other partner is International Therapeutics Incorporated, or ITI, Seattle, Washington. They are interested in T-cell therapies for HIV infection.

We’ve actually also just finished submitting a grant to the NIH to conduct this trial, which included all these partners.

You chair Benitec’s scientific advisory board. How did you get hooked up with these guys?

I have actually known [Benitec’s director of research and technology] Ken Reed for years. He was a postdoc at the City of Hope. His daughter was actually treated for leukemia at the City of Hope, before she died a number of years ago. And his son was a postdoc at the City of Hope. I have known Ken from all his associations with the City of Hope for years and years, and when I found out he was involved in RNA interference, I was actually pretty shocked. I didn’t realize he was interested in that field. …

I think [the company’s] technology, which is patented, is very interesting and perhaps would dominate, at least in the gene therapy world, the application of RNA interference. … Since my interest is using gene therapy with RNAi, I felt this was a good marriage.

What things are you doing with RNAi aside from HIV?

We’re very interested in mechanistic studies. The things we’re looking at are cellular factors that are involved in the RNAi process, and we’ve actually discovered that a protein called La is a key component in the RNAi pathway. It’s an RNA binding protein that is known to be essential for Pol-III transcript processing and maturation. It’s involved in the translation of hepatitis C virus — the internal ribosomal entry site requires La associated with this complex, although the functional role of it is not clear. In our hands, it’s part of a complex of several proteins which binds siRNAs and mediates their unwinding, and hands off the unwound siRNA to the RISC complex.

This work is actually in submission right now to the EMBO Journal.

We’ve also been analyzing other components of this complex, which is continuing work.

One of the things we’re very interested in is if [RNAi] is a process that occurs naturally in human cells. It must be doing something important — I know that microRNAs are involved in gene regulation, but [is] the RNAi itself involved in any kind of functional role? One of the tests that we’re asking is if its involved in maintenance of the stability of retrotransposons in human cells. If you interfere with RNA interference, then you activate retrotransposon movement, and this could eventually lead to chromosomal instability. So, we have a very strong interest in that area as well.

The other area I’m interested in is the role of RNAi in transcription gene silencing. That is, are RNAs — either small RNAs or longer RNAs that are utilized in components of the RNA interference mechanism — involved in chromatin silencing, or possibly even DNA methylation.

Do you have big plans for the Thanksgiving holiday?

Just family plans. I have grown children that will come and visit. … I like Thanksgiving because there are no gifts, no frenzy. It’s just being together, lots of good food — and football, of course.

 

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