Thursday, May 25, 2006

More RNAi

Some of you may remember that a couple of months ago I wrote about some advances in RNA interference (RNAi) technology. Well, today brought 2 new stories about RNAi and, since they also dealt with mice (with which I am also somewhat familiar) I thought I would elaborate on them.

RNAi has been demonstrated to be a possible epigenetic mechanism. This essentially means that small interfering RNAs (siRNA) can cause a mouse to inherit a mutant trait from its parent (white tipped extremities) without inheriting the actual DNA that encodes the corresponding mutant gene (Kit). Apparently, the Kit mutant gene produces a lot of what are probably siRNA precursors and these siRNAs are contained in the sperm and/or egg that becomes the new mouse – thereby silencing the wild-type gene copies found in the developing pup.

The articles that I read both made big deals of the fact that this mechanism operates outside of classic Mendelian genetics but there are other such mechanisms that have been known for years and this is just one more. (Not that epigenetics is not an important field, but the writers acted like we could toss Mendel in the toilet.)

The more interesting question to me is: would these siRNAs and the associated gene silencing be heritable? Could the next generation also have white tipped extremities? I am unfamiliar with any research that suggests siRNAs can self-replicate, so I doubt it. They would inevitably degrade and, without being replaced by the mutant gene, lose their gene-silencing effect. It is possible the effect could last a generation or 2 but probably not much longer. But the only way to find out is to breed them.

Another interesting point is that, normally, when a mouse has 2 mutant copies of the Kit gene, they die but when they have 1 wild-type copy, they live (but display the characteristic white tipped extremities). Since the mice in this experiment lived (and display white tips), it implies that the siRNA mediated gene silencing in this case is incomplete (otherwise the mice would be dead).
AP, CBC, BBC, ScienceNOW, Nature

The second story today is about research into the potential toxicity of RNAi. In this study, researchers introduced 49 different types of short-hairpin RNA (shRNA) into the livers of mice and monitored for toxicity. 36 were “severely toxic” and 23 caused death within 2 months. It seems the shRNAs overwhelmed the cellular mechanisms that deal with shRNA, siRNA, and miRNA; which is necessary for normal cellular function.

That sounds pretty bad, right? We had better just chuck this whole technology into the toilet with Mendel, right?


While this is an important, cautionary study it does not mean that RNAi will not be an incredibly effective therapeutic tool. A few things to keep in mind here:

1. The researchers used shRNA which appears to be more toxic than siRNA. siRNA has not shown any problems in the very few human RNAi tests that have and are being conducted.

2. High doses of shRNA were used and the toxicity was worse with greater dose. Like most drugs (or anything for that matter), the more you have in your system the more likely you will experience adverse effects.

3. 13/49 shRNAs tested were not toxic. This may seem like “The-glass-is-slightly-over-one-quarter-full” optimism but this result occurred without any attempt to design the molecules to avoid toxicity. That’s not bad. You do not get nearly that high a percentage of small molecules tested as pharmaceuticals that have no toxicity.

The bottom line is that this study has shown that caution is needed in RNAi testing. Overall, that is a good thing and will, hopefully, help avoid any setbacks to RNAi therapy caused by toxicity. That really hurt traditional gene therapy and, with some diligence, hopefully RNAi can avoid that pitfall.
NYT, Nature

All in all, an important set of discoveries regarding RNAi. Keep your eye on this technology, it will be coming sooner than you think.

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