Name: Darrell Kotton
Position: Assistant professor, medicine/pathology, Boston University School of Medicine
Background: Instructor, medicine, BU School of Medicine — 2002-2004
Postdoc, genetics, Harvard Medical School — 2002-2004
Research fellow, pulmonary/critical care medicine, BU School of Medicine — 1999-2002
Clinical fellow, pulmonary/critical care medicine, BU — 1998-2002
Intern, Hospital of the University of Pennsylvania — 1994-1995
MD, Washington University School of Medicine — 1994
BA, psychology, University of Pennsylvania — 1989
Late last year, Boston University School of Medicine researcher Darrell Kotton was awarded a grant from the National Institutes of Health to develop an approach to deliver shRNAs into mice as a tool to investigate the biology of inflammatory lung disorders.
This week, RNAi News spoke with Kotton about his research.
Let’s begin with some background on your lab.
I’m a physician/scientist — a pulmonologist and critical care physician — but 80 percent of my time is spent in the lab. The lab’s focus is stem cell biology and gene therapy, with a focus on applications for new therapies in lung disease.
Where did RNAi come into the picture? With gene therapy?
Anything you want to do in the laboratory, whether it’s understanding stem cell biology or gene therapy itself, requires genetic manipulation of cells, [in this case] in the lung, or in the … dish, with either over-expression or under-expression. The under-expression obviously needs some technology like RNAi, and that’s how we came across it.
There are a number of genes in lung diseases, for examples the NF-kappa B pathway in emphysema or any inflammatory lung disease, that would be nice to inhibit through an RNAi approach.
You’re currently working on developing a delivery approach for RNAi to the lung?
That’s right. One aspect of our lab, more for studying the biology of emphysema, is manipulating the alveolar macrophage — a key cell involved in the pathogenesis of emphysema — and we’ve come up with a lentiviral system where intratracheal installation of this lentivirus naturally targets the alveolar macrophage in a very selective way in vivo.
If you stick a lentival vector down in the lungs, it’s blocked from getting any transduction of the epithelium and endothelium in the lung, but the alveolar macrophages get very efficiently and robustly transduced. So we kind of stumbled upon a system that does specific and selective in vivo transgenetic manipulation of alveolar macrophages in the mouse.
We’ve been successful in over-expressing reported genes in the alveolar macrophages in vivo, and our [next] task was to adapt that for knockdown. That’s what we’re working on: delivering RNAi to the alveolar macrophage in vivo specifically and selectively to knock down genes of choice.
These RNAi molecules are expressed.
That’s right. The approach involves short hairpin RNAs that are cloned into a lentiviral vector that expresses two things. One is a tracking reporter gene like a fluorescent reporter … so we can track and monitor the cells in vivo at later time points if we want to. It also expresses, using a Pol III promoter, a short hairpin RNA.
At this point, have you tried this in animals?
We’ve taken the vector with a hairpin against one portion of the NF-kappa B transcriptional complex — it’s called p65. We’ve delivered it in mice, tracked the macrophages, and seen that the vector goes to where we want it to go, it gets into the macrophages, it has gene expression for several weeks in vivo in the cells we want it to.
So the tracking reporter is expressed, and right now we’re working on trying to define whether the short hairpin RNA does what we want and succeeds in knocking down NF-kappa B activation in the macrophages. We don’t yet [know] if the actual RNAi part of the vector works, but we know that when it is in the vector it will get to the macrophages and transduce the cells in an effective way in vivo.
So it’s getting there, but you need to be sure you’re knocking down your target.
Correct. We’ve tested the vector in vitro in macrophages, and we’re convinced from that data that it is knocking down the target in vitro in macrophage cell lines and inhibiting inflammatory signaling by knocking down NF-kappa B. We just can’t say at this point whether its working in vivo.
How exactly is [the compound] delivered?
The mice are anesthetized, and the concentrated lentivirus is in a blunt-end canula. The canula is placed right at the top of the trachea and squirted down, and the mice wake up with in 30 seconds with this virus in the lungs. It goes throughout the lung but happens to bind to the surfaces of the alveolar macrophages, [where it is] integrated into the genome. … So it’s permanently in the [macrophage] or any progeny if the [macrophage] were to divide.
For applications, you’re looking at NF-kappa B?
Our first step to inhibit gene expression is to … deliver [to] … green transgenic mice … a hairpin against GFP to show how robustly and stably it would knock down expression of GFP in the green mouse. From there, once we understood the kinetics, we would apply it to the actual NF-kappa B RNA. Once we understood the kinetics of how stable or effective the knock down is of that molecule, we’d put the mouse into smoking models to see if the vector effectively inhibits activation of the NF-kappa B inflammatory cascade in alveolar macrophages in a mouse that was exposed to tobacco smoke.
[We’re looking at] smoking-induced inflammation in that kind of model. Because that is a short-term model — within hours the inflammation starts within the lungs of either the person or mouse exposed to cigarette smoke. If it looks promising, the mice can be exposed to tobacco smoke for many months, and that would be an emphysema model. [But] we’re many months away from applying it to an emphysema model. It would be applied more for the short-term [with] smoke-induced inflammation in the lung.
I would say we wouldn’t have any intention of giving lentiviruses to patients in the clinic; [the system] is more to understand the biology. Once we’ve understood [diseases better, the technology] could be more easily adapted to, for instance, make an inhaler that had siRNAs that could do a similar task [as the shRNAs].
So for possible clinical applications, you wouldn’t go with a lentiviral vector. Is that [because of] a general safety issue or something particular to the lung?
It is a safety concern. Lentiviruses are already are used in the clinic for certain kinds of approaches to HIV or immunodeficiency treatments, but I think putting it in patients when you don’t have to … would be desirable. After all, the lentivirus does integrate in the cell’s genome and could cause insertional mutagenesis and problems that wouldn’t happen if you had just given [treatment] with an inhaler and could stop … treatment.
Why make things more complicated than they need to be?
Or more dangerous than they need to be.
Is this work being done in-house or are you collaborating with anyone?
It’s strictly in-house.
So as far as the timing on these experiments, do you have a sense of when you might know whether you’re getting actual RNAi knockdown in the mice?
The project, which we’ve just started, is a two-year project [funded by the National Heart, Lung, and Blood Institute]. We’ve built in the milestones and goals to understand knockdown of both a reporter, GFP, as well as NF-kappa B at the end of that two-year timeline.
How far would you go with this project? Into the clinic?
We’d like to basically understand the pathophysiology of inflammatory lung disease using this as a lab tool. We have no intention of making a therapy out of it in the near-term, so applying to short-term smoking models or longer-term emphysema models, or even some asthma models, would be the goals.
Once the signaling pathways in those disease models are better understood by using this tool, it would be more logical to take the steps in the signaling pathway to develop safe therapies like ligands or RNAi [agents] that you could put in an inhaler, rather than doing it through viral vectors.