Wednesday, August 24, 2016

Going with the Flow in Replicons

RNA replicons are an exciting new platform for synthetic biology.  Basically, you take a virus and you remove the part that makes it infectious, replacing it with whatever synthetic system you want to put into the cells instead.  You leave the part that replicates inside the cell, though, so that even if only one or two pieces of RNA get into the cell, they’ll self-amplify and express your system really strongly.  It’s great because you can get expression as strong or stronger than you’d get from editing it into the cell’s DNA, but it’s safer to use because it doesn’t get into the DNA.

We’ve already shown that you can precisely engineer unregulated expression from replicons.  Regulating expression, to actually control which parts of your system are running when, is trickier.  Even though people have been able to get repressors like L7Ae working on replicons, they are much less effective than with DNA, even though they’re being expressed more strongly.  This is a problem if you want to make controlled replicon systems, and seems like it really ought to be solvable.

Digging into this problem, I found that indeed, it looks like the problem is that when you set up a system to work on DNA, it’s likely to work poorly because it’s going to end up fighting against the natural dynamics of the replicon.  Turn that around and go with the flow, however, and it looks like it should be possible to get even better performance on a replicon than you can on DNA.

In my talk at IWBDA this year, I showed my conclusions about why things go wrong when you just go straight from DNA to replicon: since everything is amplifying exponentially as the RNA replicates, low levels of expression get raised a lot higher, making the system “leaky,” and outputs rise before there’s enough regulator to shut them down.  Big problem.
Model for L7Ae repression of mVenus from replicon, providing an explanation for observed poor performance.
Once you’ve identified the problem, though, some good paths to try towards fixing it appear as well.  In this case, it turns out that decreasing the dose of repressor and increasing the rate at which the output decays looks like it should radically increase the efficacy of repression.  Will it really do so?  We won’t know until it can get tried out in the lab, but the model’s based on things that have worked before and there’s a nice broad area of high performance to shoot for, so I’m awfully hopeful this will work.
According to the replicon repression model, the best performance comes when both L7Ae dose and mVenus degradation time are moderately reduced.
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