Associate professor, pediatrics
University of Pennsylvania
Name: Terri Finkel
Position: associate professor, pediatrics, University of Pennsylvania; chief, division of rheumatology, Children's Hospital of Philadelphia
Background: Assistant/associate professor, University of Colorado 1990-1999
Postdoc, National Jewish Medical and Research Center 1986-1987
Medical residency, pediatrics, University of Colorado 1984-1986
PhD, biochemistry, Stanford University 1984
MD, Stanford University School of Medicine 1982
When she's not practicing medicine at the Children's Hospital of Philadelphia, Terri Finkel researches the mechanisms of HIV persistence and immune dysregulation, as well as the viral and cellular factors regulating HIV-induced apoptosis, immune suppression, and latency.
Earlier this year, she and her colleagues published at paper in the Journal of Immunological Methods detailing the use of Amaxa's Nucleofection technology to transfect human T-cells with siRNAs targeting the novel cellular gene HALP.
Recently, Finkel spoke with RNAi News to discuss her research, the Journal of Immunological Methods paper, and her efforts to develop an RNAi-based HIV therapy.
Can you give a quick backgrounder on what you are doing with RNAi and T-cells?
We can't make transgenic people, so the human cells present a number of challenges that mouse cells do not. We saw RNAi as a way to effectively make a knockout cell.
In working on HIV, I've been interested in how the virus subverts the ability of the cell to do its normal functions and how it alters T-cell signaling. In the course of those studies I found that the coat protein of HIV, gp120, primes bystander cells uninfected T-cells for apoptosis. Many years ago when I started this research, there was a question as to how the virus was killing as many cells as it was, how it was destroying the immune system. Part of that confusion was because the amount of virus didn't seem to be enough to be causing this destruction. In some people who were infected, the virus wasn't even detected in the blood based on the measurement techniques we had at the time. But the person would [still] progress to AIDS, and opportunistic infections, and then death.
Now we know that, in fact, levels of virus can be quite high. We have more sensitive detection techniques, and the virus was found to reside largely in lymphoid tissues, which were not what we were sampling. But [previously] there was this question as to how the virus was doing its dirty work [at] apparently low levels. So we were intrigued by this bystander T-cell death that we saw happening via the coat protein and reasoned that maybe this was one way that the virus was killing cells that weren't actually infected and could explain the immune system destruction.
This was an in vitro phenomenon, and we wanted to ask whether the same thing was going on in vivo. We looked at lymphoid tissues from both infected people ... and in SIV-infected macaques. We found, to our surprise, that we could see bystander apoptosis, but … we weren't seeing cell death in infected cells. That was surprising because the dogma was HIV kills cells. At lot of my research from that time started asking the question … 'How is it [that] infected cells survive as long as they do?' That was somewhat heretical … but has been borne out by other researchers and in particular by the fairly definitive demonstration by [Johns Hopkins'] Bob Siliciano and a number of other investigators that HIV becomes latent within the cell. So clearly the virus must be able to infect cells and not kill them. How does that happen? How does the virus protect the infected cell from apoptosis either to become latent or at least until it can produce a viral burst?
Jiyi Yin [in my lab at the Children's Hospital of Philadelphia] did a subtractive hybridization of infected cells that were dying and [those] infected cells that weren't dying, and pulled out this gene that we named HALP, for HIV-associated life preserver. We got into RNAi because we wanted to inhibit HALP in vitro and then ultimately in vivo. We subsequently have gotten into some lentiviral techniques encoding [an] shRNA [targeting HALP] … and our goal is to put this into people to direct the infected cells to commit suicide.
So you're developing an RNAi-based therapy?
Exactly. Our goal would be to make this RNAi [agent] tat-responsive. Tat being the critical transcription factor for promotion of HIV, by being tat-responsive this RNAi [agent] would only be expressed in infected cells. It would be a magic bullet to kill the infected cells.
But you were finding T-cells tricky to transfect.
Correct. T-cells float. They're in suspension. They don't sit down and stick nicely on the bottom of the plate, and it's hard to get DNA into them, and our approaches to express the siRNA were DNA-based.
We took advantage of what at the time was a very new technique [from Amaxa] called Nucleofection, which is a black box, as far as we know it's a company secret of solutions and electroporation protocols that allow the DNA to more effectively get into the nucleus. We have unpublished data that shows that pretty nicely. It's only when the DNA gets into the nucleus via this technique that it is expressed.
Where does your effort with this therapeutic approach stand?
We're partnering with a small biotech firm right now to put [a HALP-targeting] shRNA into a lentivirus in a tat-responsive construct that would allow expression solely in HIV-infected cells.
Right now, we're still working in the test tube, we're still doing in vitro studies. But we have plans to take it into the SCID-NOD mouse, and ultimately into a SCID-NOD HIV-infected system and phase I trials in people. We're also collaborating with Carl June [at the University of Pennsylvania] who has done this kind of thing with lentiviral-based shRNA/siRNA. [His] is a different kind of approach where he knocked down one of the HIV gene products to directly decrease the infection.
The approach that has thus far been taken to combat HIV using shRNA/siRNA has been directed either against viral proteins that's most typical, I think or directed against the viral receptors such as CCR5. What we're doing that we think is fairly innovative is to target a survival factor for HIV to eliminate the infected cells and theoretically to prevent latency, which is really the bugaboo of HIV therapy right now.