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.
Thursday, October 23, 2014
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?
Posted by Michael Tranfaglia MD at 12:26 PM