Here's a question we get all the time: I was wondering if the protein needed can be delivered another way, gene therapy, or via bacteria perhaps?

Many people who are new to fragile X ask us about protein replacement strategies of one kind or another; it's an excellent question, and it's one we've been thinking about for a long time. After all, people with fragile X are only missing a single protein---isn't the simplest and most effective possible treament to just put that protein back in there? When FRAXA first started funding biomedical research, we were especially interested in exploring the possiblities of protein replacement therapy because this type of treatment was being commercialized by several biotech companies for several inborn errors of metabolism, like Gaucher's Disease.

However, we've subsequently found that there are several problems unique to fragile X that make a protein replacement approach very difficult, perhaps impossible. It all has to do with the nature of FMRP itself---this protein is a key regulator of dendritic protein synthesis whose own translation is very tightly regulated, bother spatially (where) and temporally (when). We have tried to add the protein into cells in a number of ways, and it is always quite toxic, because the protein needs to be at very specific places, only at very specific times---putting FMRP into cells in any way that does not involve natural regulatory mechanisms is not good for those cells, and it certainly doesn't fix fragile X.

One way to add some natural regulation to the process of restoring FMRP to cells is through gene therapy---add a copy of the gene, and let the cell make its own FMRP. This can actually be done in vitro, and even in the mouse model, and it works---sort of. The problem is that the version of the gene added to cells in this manner isn't exactly the same; it typically is packaged in some kind of viral "vector" and has some kind of viral promoter to get the gene going. This produces FMRP, but not the same way as it's naturally produced. However, this is a relatively minor problem. The major problem with gene therapy technology right now is that it still doesn't allow for delivery to the whole brain. The gene therapy experiments that have been done in fragile X have been done by injecting the vector directly into small areas of the (very small) mouse brain with a needle; this is completely impractical in humans.

We have been waiting for a technical advance that would allow delivery of gene therapy vectors (viral or synthetic) globally, because we know this is necessary for fragile X. The whole brain is affected, not just one small area (even conditions like Parkinson's Disease which involve relatively small areas of the brain still can't be treated by gene therapy.) Once the technology advances to the point that life-threatening conditions like Tay-Sachs can be treated by global CNS gene therapy, we should be able to adapt those techniques to gene therapy for fragile X. For now, we're still waiting.

Lastly, the idea of gene re-activation is especially attractive in fragile X. Most people with fragile X have a trinucleotide repeat expansion in the non-coding promoter region of the gene---this means that their mutation leaves them with their genetic code intact to potentially produce perfectly good FMRP. The only problem is that the gene is transcriptionally silenced. This means that it is densely methylated and wound around packing proteins called histones, which are de-acetylated to stabilize the DNA in a compacted form. There are other mechanisms of gene silencing and regulation of transcription that we are only now learning about, so turning a gene back on is far from simple. Turning only one gene on in any kind of targeted fashion is entirely impossible at this time---the technology simply doesn't exist, though lots of scientists are working on this.

We frequently get simplistic proposals to use chemical demethylating agents and histone deacetylase inhibitors as a way to get people with fragile X to start making FMRP again. This is another one of those things that looks easy if you're studying cells in a dish, but it's much, much harder to do in a whole brain. It's potentially very dangerous, because any chemical that can reactivate FMR1 will almost certainly reactivate many other genes. Furthermore, at this time there is no way to test any reactivation strategies, because we don't have any animal models with a transcriptionally silenced trinucleotide repeat expansion. Many attempts have been made to construct a "knock-in" (KI) mouse, but no matter how big the section of CGG repeats scientists introduce, the KI mice don't silence the gene the way people do. So, we have knockin mice that are excellent models of FXTAS, but they don't have fragile X. This is a problem we've been attacking by funding development of mice with human fragile X neural stem cells grafted into their brains, but it's been slow going. Until something like that is available, reactivation strategies are dead in the water.

Can eating broccoli cure autism (or fragile X)?

A fascinating new study is getting a lot of attention lately, so I decided to give it a closer read. A group of MGH, Harvard, and UMass autism researchers tested a broccoli sprout extract containing the antioxidant and apparent active ingredient sulforaphane in a double-blind, placebo controlled trial (read the full text here).

The first caveat regarding this study is that the number of subjects is quite small; indeed, there were only 14 completers in the placebo group (using a 2:1 drug:placebo ratio), which could give rise to all kinds of misleading things. One thing I noticed was that there was no appreciable placebo response. An excessive placebo response can doom a trial, but when you see no placebo response at all, a red flag should go up in your mind. In small studies like this, the superiority of drug over placebo can result from an unusually small placebo response---a statistical fluke in the randomization process, really. If we then compare the placebo group showing no response to the drug group showing a typical placebo effect, but no actual treatment effect, it can appear that there is a big difference. This is essentially what happened in the first Novartis trial; the seven fully methylated subjects just happened to show no placebo response at all; in the larger study of the drug, this effect disappeared (in fact, the fully methylated group had an extra-large placebo effect.)

It was somewhat reassuring to see that ABC scores in the treatment group increased significantly 4 weeks after discontinuing, while still blind to treatment status, though there were numerous dropouts at this point, complicating interpretation. This kind of on/off effect is what you like to see, and is generally indicative of a true drug effect (of course it also means there is little carry-over effect, but most drugs do stop working when you stop taking them!) The effect of the broccoli sprout extract was significant, resulting in a 20+ point drop in ABC scores (note that the outcome measures were all the same as those used in recent fragile X trials.) However, the placebo response in the Novartis Phase IIb/III fragile X trials was actually quite similar in magnitude, so this could all be a statistical quirk.

Nonetheless, this is an intriguing result, especially since broccoli sprout extracts are widely available as nutritional supplements. But what about dosage? Can you actually get the same stuff used in this trial, and how much would you need? The study drug was a custom preparation which is not available anywhere, and the amount of sulforaphane given each day in the trial would be the equivalent of 20 or more capsules of the commercially available broccoli sprout extract, by my calculations. In addition, the potency of the extract was carefully monitored and maintained, implying that the compound is not entirely stable, and the pills you get at GNC might not even be as potent as they say (always an issue with unregulated nutritional supplements.) Still, 20 pills a day is possible as a treatment strategy. At this point, the evidence seems a bit weak, so I'd recommend waiting before trying this, but keep an eye on the broccoli story!