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Brown s Bharat Ramratnam on RNAi-Based Microbicides as HIV Treatments

Bharat Ramratnam
Assistant professor
of medicine
Brown Medical School

At A Glance

Name: Bharat Ramratnam

Position: Assistant professor of medicine, Brown Medical School

Background: MD, Brown Medical School — 1993

AB, chemistry, Brown University — 1989

After receiving his undergraduate and medical degrees from Brown, Bharat Ramratnam went on to conduct his postdoctoral work at the Aaron Diamond AIDS Research Center at Rockefeller University. He made his way back to Rhode Island, however, where he now divides his time between doing laboratory research and practicing medicine.

Recently, he spoke with RNAi News about his work in the HIV field and how RNAi is playing a role.

Could you give an overview of your lab and your medical practice?

Our medical practice is consigned to HIV-infected individuals in Rhode Island and Southeastern Massachusetts. The take-home message is that people are still getting infected, and they're still getting infected despite increased education on the things that people need to do to prevent being infected. People are being infected mostly through heterosexual sex is what we're finding here, though homosexual sex also continues to be a risk factor — but the rates have increased for women getting infected.

This has lead to our interest in novel microbicides — agents that can be applied to mucosal surfaces and thereby prevent a person from being infected by a viral pathogen such as HIV. But if you also look at other viral pathogens that are sexually transmitted, whether it be herpes or human papillomavirus, the same principles apply: we need locally acting drugs that can prevent the transmission of these viruses during sexual intercourse. That is really where our interest in RNAi is right now — to see if RNAi can provide a platform to provide these drugs.

Given your postdoc work and your experience there, you've been working in the HIV field for quite a while. When did RNAi enter the picture?

RNAi entered the picture just three or four years ago. The publications [at that time] and indeed our own work were on using RNA interference towards a potential genetic therapy — that is, transducing lymphocytes and introducing this into the whole body in infected individuals to see whether or not viral transcription could be decreased. This sort of approach, unless there is a major advance in gene therapy, is not something that is going to be doable in the next few years or months. So that is what made us change our focus to see whether short interfering RNAs can be formulated and taken up by mucosal surfaces, and in that way, if they could be used to degrade proteins that are necessary for the transmission of viral pathogens.

The gene therapy work, is that going forward in any respect?

With respect to HIV and our use of RNAi, we've shifted completely to a microbicidal approach and none on gene therapy.

So where are you in the microbicide development effort?

Well, there are a few challenges. Curiously enough, the easiest thing to do is to select targets, and that is the hardest thing to do when you're thinking about HIV-1-specific gene therapy by RNAi because if you're choosing a viral target, I'm not convinced that there is any one that would be a drug target useful for each and all infected individuals. Say you had in your mind that you were going to degrade tat using RNA interference; just the genetic variation in tat across different patients, different clades, different continents is so large that it's going to take [a tremendous amount] of time to come up with one siRNA molecule that is going to effectively target all these different kinds of tats. The challenge is actually much [greater] because if you just degrade tat, as we've shown, you're going to have escape mutations emerge, and you're going to have viral strains that have mutated to render the siRNA useless. So you have to go for quite a few targets.

Whereas for the microbicide work, CCR5 is a perfect example of one target that a human can do without that, if we are able to downregulate on mucosal surfaces, could serve as a potent microbicidal preventative strategy. New knowledge is emerging about herpes co-receptors, and the same sort of principles apply there: if we can design an siRNA molecule against one of these entry molecules that are specific for herpes, then you could make an enormous impact not only for herpes, but [HIV as well.] … Those people infected by herpes secret larger amounts of [HIV] virus, they're more infectious, those people that are HIV-positive, if they get herpetic lesions, the course is worse. All these viruses live in this ecosystem and they help each other out. In the end they can hurt the overall epidemic.

So at this point do you have an siRNA formulation?

We've gotten proof of principle with liposomal formulations, but now we're working with different academic enterprises and a few commercial enterprises to formulate them as microcapsules and sustained-release rings, but that work is still all in the very preliminary stages and we have no results as to whether that will be successful or not.

Have you tried the liposomal formulations in vivo?

We've tried the liposomal formulations in vivo and we do see uptake and we do see knockdown of targeted genes.

What kind of animal?

Rodents. We're just beginning macaque work at the New England Primate Center with a liposomal formulation.

You talked about gene therapy being pretty down-the-road kind of stuff. Where do you see an HIV microbicide in terms of a timeline?

I think the most important roadblock is proof of principle in macaques. If there is convincing data that siRNA can indeed knock down CCR5, and the kinetics and potency of knockdown are sufficient to prevent vaginal virus transmission, then it really eases the next steps, which would be preclinical safety [and[ phase I safety studies in humans. That also is a long process, but here I don't think we need a major technological advance such as in gene therapy to see this go into humans. We have all the tools now, and I'm confident that we will be able to formulate these molecules just with the knowledge we have now.

A big hurdle to [an HIV microbicide] would be compliance issues. Do you have any sense of how long the prophylactic effect might be sustained?

This is a complicated question because it not only depends upon your formulation — the stability of the siRNA molecules — but the turnover kinetics of whatever your target is going to be. So we have no in vivo data yet on CCR5, but the macaque studies will hopefully provide that.

You mentioned your collaborating with different people. Can you comment on who?

Right now, I don't think I have their permission to say who they are.

But they are both academic and industry?

Yes. And we're getting very strong industry support, which is a very nice thing.

Thus far we've been talking about microbicides, but what we're essentially talking about is transient mucosal gene therapy. Transient because we expect the siRNA effect to wear off after either it is destroyed or the gene target turns over. Gene therapy because it is gene therapy — we're silencing a gene. We don't need a viral vector to do this, but if you look at the types of diseases that this could have a potential impact on, an obvious one is inflammatory bowel disease.

We have very nice data in a murine model showing that we could potentially knock down inflammatory mediatorsl. The next step, of course, is to see in inflammatory bowel disease whether such knockdown would impact disease severity.

What targets are you looking at?

The obvious targets in IBD that most people agree on are things such as TNF-alpha and NF-kappa B, for which there are already existing therapies but which are systemic. This would allow a localized anti-cytokine therapy. And that is the sort of disease that an siRNA drug is going to be ideal for.

Is that indication something you are actively pursuing?

Yes. And for [this] we also have strong commercial backing. We are needing milligram quantities of siRNA. And another thing we haven't talked about is that there are a lot of people out there who are accumulating expertise on the structural modifications, the chemical modifications, that could potentially render an siRNA molecule more stable, more potent. That field of study, which we're not directly involved in but [for which we] are tapping into the expertise of our collaborators, is going to be extremely important, as well, as we decide what form of siRNA to formulate.

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