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Dana-Farber s Carl Novina Discusses Starting a Lab and Working With RNAi

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At A Glance

Name: Carl Novina

Position: Assistant professor, Dana-Farber Cancer Institute

Background: Postdoc, Center for Cancer Research, MIT — 2000-2004; MD, Columbia University College of Physicians and Surgeons — 2000; PhD, immunology, Tufts University — 1998; BS, biology/BA, English, Rutgers University — 1990

After obtaining degrees in biology, immunology, and medicine (not to mention English), and completing a postdoc under Phillip Sharp at MIT, Carl Novina has struck out on his own at the Dana-Farber Cancer Institute.

Though busy putting together his lab, he took the time to speak with RNAi News this week about his work with RNAi.

How did you get involved with RNA interference?

I came to Phil Sharp’s lab as a postdoc and I originally began my work hoping to investigate mechanisms of transcription DNA recombination coupling. I was hypothesizing that I could identify a factor that was required for nucleosome remodeling, specifically at the immunoglobulin locus. This is an area that Phil has had a continuing interest in for a long period of time, but he was also at that time interested in developing and working on small RNA-directed processes.

So I was one of the first two people to start working on that in his laboratory after Tom Tuschl had left the laboratory, and after he and Phil Zamore had both gotten independent positions. So Phil [Sharp] was looking to fill up his laboratory. This created an opportunity for me to work in an area that was not as well developed — a lot less was known — and was very, very interesting both from an intellectual standpoint [and] potentially a therapeutic and experimental standpoint.

Those are things that appealed to my intellectual side, from an intellectual curiosity standpoint, [and] also my practical side, [given] the fact that I’m a physician and I’m interested in developing small RNAs as a potential molecular therapeutic. These things kid of got married at that one time and I changed my emphasis from mechanisms of transcriptional silencing — which now we know may actually have a small RNA-directed mechanism — studying nucleosome remodeling and nucleosomal event to just studying small RNAs in general.

You’re doing this in your own lab now. You’re in the process of setting that up at this point?

My laboratory is going to be dedicated mostly to investigating the basic mechanisms of small RNA function in mammals — I would say that’s 85 to 95 percent of my laboratory. But some 10 percent or 15 percent will also be investigating the applications of small RNAs in mammals.

What sort of things does it take to put together a lab? It must be pretty time consuming.

It is horrendously time consuming. It is very, very time consuming. You have to tend to a lot of scientific issues — What am I going to study? Which areas am I going to focus on first? — to the very practical — Who’s going to do it? How am I going to get money for it? What’s the space going to look like? Can I get all the licenses?

It’s a dazzling amount of effort that has to go into getting a laboratory running.

Can you get into what you’re planning on doing and the role of RNAi?

Are you interested in how I’m going to go after and investigate basic mechanisms or are you interested more in the applications of RNAi?

Both.

Okay. There are a few different ways to investigate the basic mechanisms. There’re huge efforts in many laboratories to identify the factors that are involved in small RNA function, and those things are evolving in numerous organisms: tetrahymena, worms, flies, plants, and yeast, as well as mammals.

There are some common themes that are emerging from these other eukaryotes, such as the involvement of PAZ and Piwi domain-containing proteins — the so-called PPD proteins. These are conserved across the phyla, and they seem to be involved in RNAi and small RNA-directed phenomena in mammals.

One way go about this is to do it in a hypothesis-driven approach, where you look at some of the factors that have been implicated in other organisms. You test whether they are implicated in mammals either by knock out, by direct gene silencing, [or] by making epitope tags on these factors and trying to see if you can identify other factors that are necessary and sufficient for RNAi in mammals.

So one effort is going to be using a sort of hypothesis-driven approach, and that sort of the way that many of these laboratories are working — Phil Zamore [at the University of Massachusetts Medical School], Tom Tuschl [at Rockefeller University], Greg Hannon [at Cold Spring Harbor Laboratory], Phil Sharp [at MIT]. It’s a more traditional way of going after some of the factors and the small RNAs themselves that are responsible for RNAi in mammals.

In addition to that, and I guess one of the larger focuses of my laboratory, is to develop not a hypothesis-driven approach but a more discovery-driven approach, and that’s to use a reporter system. So you have cells — for example, human cells like HeLa cells, that have a reporter such as GFP or luciferase or an endogenous gene — and you can silence those genes by RNAi, by making hairpin RNAs that silence luciferase and GFP. What you do is use genomic approaches, such as a chemical genomic library, which is one thing that I’m doing in collaboration with Brent Stockwell at Columbia University. We’re using a library of hairpin RNAs against every single gene in the human genome to see if you can reduce the ability of the reporter to silence its target.

Another way that I’m going to go about doing this is using a genetics-suppressor element approach, using a fragmented cDNA library to find dominant repressors of RNAi in mammals.

These are ways that you can identify not only the genes that are responsible for RNAi, but you may also be able to enrich for the small RNAs themselves that are active in these cell types.

That’s on the basic mechanism side, and you can see that takes quite a bit of my effort. That’s the majority of my focus: using both hypothesis-driven approaches, as well as discovery approaches, on the basic mechanism side.

Part of my effort is wanting to use and develop RNAi as a tool and on that front I am working with a group of Boston-based laboratories that have formed the RNAi Consortium (see RNAi News, 4/9/2004). These are groups whose focus is to make hairpin RNAs to every single gene in both the mouse and human genomes. I am representing one group [that is joining] several other laboratories around Boston — Brent Stockwell, who is also part of the group, has made it down to New York [from MIT] — that are focused on developing the best reagents that can be used widely by scientists to silence whatever gene they want to be able to investigate that gene’s function.

That’s the consortium headed by David Root?

David Root is heading that up, and other laboratories include [those of Massachusetts General Hospital associate professor] Nir Hacohen, [MIT’s] David Sabatini, [and assistant professor at Dana Farber] Bill Hahn. [Additionally], Eric Lander, [director of the Center for Genome Research], is helping, as is Sheila Stewart, who was one of the co-authors with me on that RNA paper from which this project has grown out of. She at Washington University in St. Louis.

There are many laboratories that are contributing to this effort.

The paper you referenced, can you give an overview of it?

That was a paper that was published in RNA last year. We were amongst the first groups to develop retroviruses, specifically lentiviruses, which are able to infect non-dividing cells, … to deliver cassettes — these are short-hairpin inserts that express hairpin RNAs to silence target genes. We were the first group to use them to silence genes in dendritic cells, but there were several other groups that published right around the same time who were using other retroviruses, specifically lentiviruses, to silence genes in mammalian systems.

You mentioned interest in possible therapeutic applications of RNAi, and being a doctor gives you a unique perspective on that. What’s your take on the sector, and is involvement something that you’re looking into?

I don’t have a license to practice medicine so I’m not going to be using [RNAi drugs] directly myself, but because I do have training in medicine I am afforded a little bit of a broader view. So I would like to collaborate with physicians, and find unique and plausible targets that we can efficiently deliver small RNAs to and have them silence these genes so that there can be a therapeutic and beneficial outcome.

This is definitely something that I would be involved with in a collaborative capacity, but not directly and solely myself.

You done some work with HIV, correct?

Yes, and that was done in a collaborative effort.

With Judy Lieberman [at the CBR Institute for Biomedical Research]?

That’s right.

Can you talk briefly about that work?

This was a proof-of-concept paper, just like the RNA paper. We were, along with John Rossi and his group [at the Beckman Research Institute], the first to use small RNAs to silence both viral genes … and a cellular gene, and have that reduce the ability of the virus to replicate intracellularly.

The idea there is a proof of concept that this can be used as a therapeutic. In silencing [the] … cellular gene … CD4, we were able to block the virus entry into the cell. The notion there is that there’s a group of Northern European males who have a homozygous deletion in the extracellular domain of CCR5 — it’s a chemokine co-receptor for HIV. These patients are refractor to HIV infection and going on to full-blown AIDS, and they do not have a major or appreciable immune dysfunction. So this is the combination of having a viable cellular target, CCR5, and the right target cell, for example a macrophage, where you may be able to have a therapeutically viable method of using RNAi.

So your outlook on the RNAi therapeutics field is …

It’s positive. RNAi has many therapeutic advantages that other nucleotide-based therapeutic modalities don’t have. For example, if you use antisense — antisense is not an endogenous and normal pathway within the cells, whereas RNAi is. Almost all cells are capable of doing small RNA-directed gene silencing. What you’re doing with RNAi is co-opting an endogenous pathway, which is one of the reasons why it looks like small RNA-directed silencing is so potent and so specific.

There are many obstacles to overcome, but these are obstacles that are similar to other nucleotide-based therapeutic options. That principally revolves around the issue of delivery: How do you get these things to their target tissue? So many of these things that we’ve already learned from other approaches can be applied to RNAi to delivery siRNAs directly.

Do you have any interaction with companies are groups that work in the therapeutics field?

No. I don’t. I have been focused principally on working on the basic mechanisms. My collaborators, and also my postdoctoral advisor [Phil Sharp], have had these connections. So most of my work has gone through those avenues to the companies, although other companies have … approached me about wanting to discuss possible arrangements or possibly getting me to be a consultant.

But [I have] no [company affiliations] directly, and none yet.

Is that something you’re considering currently, or do you have you hands full?

Not actively. You leave your options open — never say never — but 110 percent of my time is already accounted for. I’d have to find some more time somewhere else, and that might only happen after I’ve established my lab.

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