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BU's Jim Collins on Developing a Tunable Genetic Switch Using RNAi

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Name: Jim Collins
 
Position: Professor, biomedical engineering, Boston University
 
Background: Scientific co-founder, Cellicon Biotechnologies — 2000
- PhD, medical engineering, University of Oxford — 1990
- BS, physics, College of the Holy Cross — 1987
 

 
Late last month, researchers from Boston University published in Cell details of a tunable genetic switch that combines repressor proteins with an RNAi target to turn a gene of interest on or off, with repression levels exceeding 99 percent, in mammalian cells.
 
According to the paper’s authors, the switch “can be used to explore the functional role of various genes, as well as to determine whether a phenotype is the result of a threshold response to changes in gene expression.”
 
This week, RNAi News spoke with Jim Collins, senior author of the Cell paper, about the technology and its potential.
 
Let’s begin with a little background on your lab and research areas.
 
Our [applied biodynamics] group at BU was formed in 1990 when I joined the faculty. Initially, we focused on whole-body dynamics, studying things such as human balance control and human locomotion.
 
Starting in the late 1990s, we shifted towards synthetic biology and systems biology using engineering approaches to both forward engineer and reverse engineer gene regulatory networks. Most of our efforts in both areas focused initially in microbial systems. Recently, in synthetic biology and systems biology, we’ve moved into mammalian systems.
 
Our lab, which includes bioengineering students, bioinformatics students, and molecular cell biology students, really focuses on three broad applications in synthetic biology/systems biology. One is anti-infectives — developing novel antibiotics on the basis of some of our network analyses of bacterial cell-death pathways. Two is mammalian disease from a systems biology and synthetic biology standpoint, and within that also mammalian biology from a functional genomics standpoint. Most recently, we’ve gotten involved in bio-energy and efforts to engineer microbes for bio-energy applications.
 
Systems biology is a term that gets used a lot and seemingly has various definitions. Can you give me your definition of it, as well as synthetic biology, which isn’t quite as well-known?
 
I view systems biology as the integration of experimental and computational approaches that utilize high-throughput data to analyze biomolecular networks and pathways.
 
Synthetic biology I view as a new discipline that uses principles from engineering and physics to model, design, and construct synthetic biomolecular systems, in particular synthetic gene networks with a number of applications in mind [including] biotech, as well as gaining additional biological insight into the functioning of natural systems.
 
And that’s where this genetic switch comes in.
 
Yeah. This fits squarely in synthetic biology.
 
Can you give an overview of this technology?
 
As I mentioned, we started in synthetic biology in the late ‘90s in microbial systems, and there our initial effort was to design a bacterial toggle switch. This system … consists of two co-repressive genes — so gene 1 is trying to shut off gene 2, and gene 2 is trying to shut off gene 1.
We found that we could design such a system so that it could be bi-stable; that is, it could exist stably in either one of two states with [the first] being gene 1 is on and gene 2 is off and [the second wherein] gene 1 is off and gene 2 is on. The nice thing with that system is that you could flip between the states by delivering an inducer for the currently active gene.
 
We were interested in moving this into higher organisms, specifically mammalian systems, but found that in order to build a functioning toggle switch to our specifications, we needed very tight repression — repression that was much higher than what was being offered by repressor proteins at the time. So our initial efforts in the mammalian space to build a toggle failed quite miserably because we had too much leakiness and not enough repression.
 
So Tara Deans, who is a PhD student of mine, and I sat back and said, “Okay, we need to get tighter repression. What can we do?” We came up with the idea of combining repressor proteins with RNAi to create a sort of two-pronged attack to shut off a gene of interest.
In many ways, the present work, which I think is a novel functional switch in its own right, really originated as half of a toggle switch. In this scheme, we have a tunable modular genetic switch that involves both a repressor protein and an RNAi component that can be used to control the expression of any gene of interest.
 
While with the repressor protein we might get on the order of 80 to 85 percent repression, and similar levels with RNAi, when we combine the two we got greater than 99 percent repression. With the way we designed it, it is an inducible system so you can, through the application of an inducer, flip it on.
 
Is this system able to do partial gene inhibition?
 
Yes. We can titrate the inducer and tune the level of expression of a target gene. So [the gene is] not only expressed at two levels — either off or on — we can actually express it at multiple levels and tune the expression.
 
This has a number of interesting applications from functional genomics and disease study standpoints in that we can now look to see if there are threshold responses for a given phenotype to the expression of a gene of interest.
 
Is the primary advantage of this over RNAi exactly how much inhibition you are able to get?
 
Yeah, I would say so. That’s probably the prime one. We can get a very tight off [state] so you can do, in principle, multiple reversible knockout studies because you can have this so tightly off and sit there quite stably in the off state.
 
For example, you could study the effect of a gene, whether it be on or off, during the developmental cycle using this switch by first keeping [the gene] off and then flipping it on. Then, if you wanted to see what happens when you remove [the expression], you can flip it off. And vice versa.
 
Given there is an RNAi component to the switch and that off-target effects are an issue there, did you encounter any off-target issues?
 
Certainly off-target effects are a very big problem for RNAi and it’s an area we’re beginning to address from the systems biology standpoint. Here, we’re able to avoid off-target effects because we’re not designing the RNAi [agent] for the endogenous gene; we design it to hit a specific RNA target that we tag onto the gene of interest.
We designed it, basically, on E. coli beta-galactosidase. In doing so, there was no homology for the 19-nucleotide sequence that we used with other genes [and] we were able to minimize, if not avoid, any off-target effects.
 
This work was done in vitro. Are there plans to try to get this to work in vivo?
 
Yes. We’re in discussions with some groups at Harvard Medical School to get this into mice and to see how effective it is. We’re quite confident that it’s going to be very effective.
 
We are now charging ahead on a number of additional in vitro studies. We’re actually combining [the switch with] our systems approach, where we’re able to identify genetic mediators for biological processes in diseases and, using our switch then to control the expression of the identified mediators, test and validate their phenotypic effects in given cell lines.
 
Have you formalized any collaborations at this point or are discussions still ongoing?
 
We’re still discussing. Since the work was published, we’ve been contacted by dozens of labs interested in the constructs, so there is clearly an interest and need for these types of switches, which we were very happy to see.
 
What about industry people? Are there companies looking to commercialize this technology?
 
We’ve been contacted by a few that are in the business of developing and selling reagents for biotech applications and are now looking to evaluate this switch as a possible product. There are also some synthetic biology companies that have expressed interest in utilizing this as part of library construction.
 
Can you comment on which companies have expressed interest?
 
I probably shouldn’t.
 
Undoubtedly this has appeal to people doing basic research. Do you expect that there is a therapeutic potential for the switch?
 
Absolutely. I’m of the belief that eventually, if not soon, we’re going to see cell therapy and gene therapy becoming part of medical practice. In each case, we first need to work out safe means to get the constructs of interest into the patient.
 
Once we do, we’ll need very effective means to regulate the expression of transfected genes, and I think switches such as these, by themselves or perhaps in combination, to create a toggle switch could be really quite effective for control.
 
It’s really the main application that got us excited to get into synthetic biology in the first place.