Instructor, division of infectious diseases
University of Pennsylvania
At A Glance
Name: Prakash Bhuyan
Position: Instructor, division of infectious diseases, University of Pennsylvania
Background: PhD, cellular immunology, University of Texas, Southwestern — 1996
MD, infectious diseases, University of Texas, Southwestern — 1998
BS, biology, Johns Hopkins University — 1991
University of Pennsylvania researcher Prakash Bhuyan spends most of his time in the lab, but also dedicates a portion of his time to the practice of medicine. In the lab he is working on the use of RNA interference to modify the immune evasion characteristics of herpes simplex virus-1.
Recently, he spoke with RNAi News about his RNAi work.
Let's start with an overview of your lab and what your work is focused on.
My [focus] has been to use herpes simplex virus type-1 as a model system, first to develop an in vivo application of RNA interference in a small animal model. So I try to knock down the expression of viral genes first in vitro in what are known as plaque assays and in viral titer assays. The gene that we've been studying is glycoprotein E, which is a viral gene that mediates viral cell-to-cell spread. We were able to identify a siRNA that could knock down the expression of glycoprotein E and change the viral phenotype — change the virus's pathogenicity from a pattern of wild-type spread to restricted spread due to a diminished amount of glycoprotein E. After characterizing that reagent, I've spent probably the last year and a half developing a small animal model where we apply the siRNA, induce RNA interference to shut down glycoprotein E, and look at it in the context of corneal infection with the wild-type herpes virus.
The first step has been to try to develop a good small animal in vivo model. There're a couple of things that are necessary for good gene therapy: you have to be able to deliver well [and] you have to be able to quantify the specific gene knockdown accurately. … The cornea model in the mouse, at least, has had a lot of benefits in that it's directly applicable to topical siRNA delivery, and most of the delivery models use parenteral delivery, so it's either intravenous or peritoneal. We thought this [topical] approach might be a little more feasible for human therapy eventually.
When you knock down the expression of glycoprotein E in the eye, the virus is poorly able to spread from the cornea to infect the trigeminal ganglion, which is where the herpes kind of sets up shop. It migrates from the corneal epithelium and infects the trigeminal ganglion and eventually will establish latency and chronic infection from the trigeminal ganglion.
After the in vitro work, we were trying to establish a good working in vivo model.
You said topical delivery. So these siRNAs were delivered …
This is a lipid cationic complex that was made enzymatically. There are many ways to do [RNAi]: chemically, plasmid-generated, and people use short hairpin constructs. We just did a 21-mer double-stranded RNA fragment specific for glycoprotein E with a diuridine overhang, which is actually the method that Ambion uses. The siRNA solution is then deposited into the surface of the eye.
Now you've actually performed the in vivo experiments?
That's the step we've been working on — to try to work out the dose, [and] to quantify both specific and non-specific effects on both the viral genes and the cellular genes because … it's not a matter of whether there are off-target effects, it's more of a matter of how much. So one of the important things in developing this model has been to be able to characterize how many non-specific gene effects we're having. You can induce interferon — and in terms of a vaccine that might not be a bad thing to do, though, to induce a little bit of pro-inflammatory cytokines … to have a little bit of non-specificity where interferon is induced.
So the animal work is going on currently?
That's correct; we are now applying our animal model.
Do you have any kinds of data coming back at this point?
Even in vitro, when you try to knock down gene expression, you get a spectrum of effect in measuring the phenotype. In our case, the phenotype we were measuring was viral spread from one cell to another. There were some cases where spread wasn't affected at all, some where it was affected to an intermediate level, and there were some where there were absolutely no viral plaques that formed because the virus really was unable to spread from cell to cell. You get sort of a bell-shaped distribution of effect and that's similar to what we're seeing in vivo. We see some animals that behave as if they've not really had a good RNA interference effect induced.
The real proof, though, has to come from measuring messenger RNA. What we're trying to set up is a system where we induce the phenotype change and then measure the messenger RNA to see how well we've been able to knock it down. I suspect that there is probably a variation in the amount of messenger RNA that's remaining that allows the virus to behave either closer to a wild-type virus or closer to a virus that has poor capacity to spread. The in vivo picture sort of mirrors what we've seen in vitro … and I'm actually working on a manuscript right now to describe these results.
Is this the bulk of your RNAi work there, or do you have other projects going on as well?
This has been the majority of my effort. The next step would be to use RNA interference to alter the virus in vivo — in other words, change the phenotype of the virus during an infection. This would be medically significant because herpes simplex causes chronic infections, including neonatal herpes, which is probably one of the most significant manifestations clinically of herpes simplex.
As a proof-of-concept, it would be nice to alter the course of a chronic infection. There are many viruses that cause chronic infection like CMV, HIV, and Epstein-Barr virus … but in any of those viruses they form a latent state and then reactivate, and people tend to get sick when they're immunosuppressed, which can happen for a variety of reasons. In any case, it's difficult to eradicate latent virus, but the crux of my research has been to alter the virus in situ, then allow the immune response to develop a better cognitive response.
Now you're preparing to go work for [a pharmaceutical company]?
That's correct. I'm going to be working in [the company's] vaccine division.
Is there anyone else at the University of Pennsylvania that might continue this work?
Drew Weissman and Harvey Friedman were my mentors during my infectious diseases fellowship at the University of Pennsylvania, and would be best qualified to continue this work.