Things like this make medical science worthwhile: A drug to treat genetic diseases due to nonsense mutations

There are times when, as a scientist, I look at an idea and its execution and simply stand in awe. It's particularly satisfying when it's a relatively simple idea that could conceivably do a lot of good for a lot of patients. Oddly enough, whether it's because I've been out of the loop or because it hasn't garnered that much attention in the blogosphere (not even here in ScienceBlogs), but I only just heard of it now. It's a new drug in phase II clinical trials that has the potential to obviate or reverse the effects of a wide variety of genetic mutations that cause human disease:

A pill that can correct a wide range of faulty genes which cause crippling illnesses should be available within three years, promising a revolution in the treatment of thousands of conditions.

The drug, known as PTC124, has already had encouraging results in patients with Duchenne muscular dystrophy and cystic fibrosis. The final phase of clinical trials is to begin this year, and it could be licensed as early as 2009.

As well as offering hope of a first effective treatment for two conditions that are at present incurable, the drug has excited scientists because research suggests it should also work against more than 1,800 other genetic illnesses.

PTC124 targets a particular type of mutation that can cause very different symptoms according to the gene that is disrupted. This makes it potentially useful against a range of inherited disorders.

Basically, this drug works against what are called nonsense mutations. DNA provides the information necessary to produce proteins. It is made up of individual building blocks called nucleotides, while protein is made up of individual building blocks known as amino acids. The way that the genetic code in DNA is translated into proteins, which form enzymes, structural proteins, and proteins that carry out virtually every function necessary for life, is through large protein complexes called ribosomes. Ribosomes "read" the DNA, whose code is based on three-nucleotide sequences called codons, each of which codes for a different amino acid. Given four nucleotides and triplets, there are 64 possible codon combinations for 20 amino acids, which means that the genetic code is "degenerate"; i.e., most amino acids are coded for by more than one codon. There is a set of three codons, however, that do not code for any amino acid. They are known as stop codons, because when the ribosome encounters them it interprets it as a signal that the protein chain should end. When a mutation causes a codon to change from a normal codon that codes for an amino acid to a stop codon, it is known as a nonsense mutation, and it results in the termination of the protein chain wherever the mutation occurs.

There are a number of genetic diseases where the inherent defect is a nonsense mutation that causes the premature termination of the synthesis of the protein chain of the protein that is the cause of the disease. Among these are Duchenne's muscular dystrophy and cystic fibrosis, both big killers. Cystic fibrosis, for example, results from mutations in a protein (CTFR) that cause impaired or nonexistent function of a chloride channel. The end result of this malfunctioning channel is the thick, sticky mucous that causes all the pulmonary problems such as recurrent pneumonias, as well as GI and pancreatic problems. In the case of muscular dystrophy, there are a number of mutations than can cause the disease in varying degrees of severity, among these nonsense mutations, and approximately 13% of muscular dystrophy is due to nonsense mutations. If a way could be found to "read through" the aberrantly produced stop codons responsible for these and many other genetic diseases, it might be possible to greatly reduce or even eliminate the symptoms of as many as 1,800 genetic diseases. In replacing many of the proteins involved in these diseases, 100% efficiency is not needed, because in the case of some diseases if the normal protein can be restored to only 1-5% of its normal level, considerable improvement in symptoms could be caused.

But how might it be possible to cause the cell to "ignore" stop codons produced by It turns out that there is a pathway, known as nonsense-mediated mRNA decay (NMD)), by which messenger RNA containing premature stop codons is selectively destabilized and degraded. As Neu-Yilik et al describe in a recent review:

Nonsense-mediated mRNA decay (NMD) is a specific pathway for the degradation of mRNAs that have premature termination codons (PTCs) in their open reading frames (ORFs). Its importance is highlighted by its conservation in all eukaryotes. NMD counteracts the potentially harmful impact of mRNAs that have PTCs as a result of errors at various levels of gene expression, such as nonsense and frameshift mutations, transcriptional errors and faulty splicing. Thus, NMD serves as a 'cellular vacuum cleaner' that protects the cell from the potentially harmful effects of truncated proteins by eliminating mRNAs with PTCs in a sequence of events that is not yet fully understood.

Investigators from PTC Therapeutics in New Jersey, the University of Pennsylvania, and the University of Massachusetts used a high throughput screen to look at 800,000 low molecular weight compounds to identify ones that promoted nonsense mutation suppression. This is some heavy duty medicinal chemistry and a very impressive effort. One compound that they identified (PTC124, chemical structure pictured below) was the most potent and selected for further screening.


This compound was very potent at promoting readthrough of nonsense mutations in cell culture using primary cultured muscle cells harboring a nonsense mutation in the dystrophin gene, mutations in which can cause Duchenne's muscular dystrophy. It was then tested in a mouse model of muscular dystrophy. These mice, known as mdx mice, have a mutation in the dystrophin gene. Lack of functional dystrophin results in increased susceptibility to contraction-induced injury, leading to ongoing cycles of injury and regeneration, inflammation, and necrosis, with the ultimate destruction of the involved muscles. Treating these mice, either with oral feeding or intraperitoneal injection of PTC124, significantly reversed the functional abnormalities normally seen in the muscles of mdx mice associated with increased dystrophin production to levels approximately 25% of what is observed in normal mice. I can see why this paper was published in Nature.

The drug has already made it through two phase I clinical trials, with minimal toxicity. The most troublesome side effects were transient flatulence that did not recur even if dosing was continued (I kid you not), headache, nausea, and dizziness. In addition, some patients had mild reversible elevations in their liver enzymes and more signficant elevations of muscle enzymes (creatine kinase) in three subjects. However, the investigators could find no correlation with dosing of the drug and concluded that these elevations were probably due to exercise-induced muscle injury. Better, no evidence of nonspecific readthrough of normal stop codons was observed. Presently, a phase II study is being completed, and, according to reports, will be published soon; initial descriptions suggest that there are "promising" results in patients with muscular dystrophy and cystic fibrosis.

As has been pointed out elsewhere, although this drug gives children with muscular dystrophy and cystic fibrosis hope that their condition might be effectively treated at its cause, rather than just treating the complications, it is important to emphasize that one critical aspect of this new class of drugs is that they will make it imperative that a patient's specific gene mutation has been identified by sequencing. PTC124 only has the potential to work for nonsense mutations; if the gene mutation involved is not a nonsense mutation, PTC124 will not work.

I also have to voice a word of caution here, much as I have done with dichloroacetate (DCA), the small molecule cancer chemotherapy drug that was hyped as a cheap "cure" for cancer. Although this drug has made it much farther than DCA has thus far, having gone beyond animal studies, it still might fail. We will have to await the final results of phase III clinical trials, but, given that nature of this treatment, such trials can be carried out considerably faster than cancer trials because overall survival does not have to be measured to show that the drug is efficacious enough to be approved. Improvement in symptoms and demonstration of the return of the production of the full-length protein with the nonsense mutation, and likely that would be enough, with long term trials looking at improvements on survival to be done later.

It's very likely that PTC124 will be only the first among an entirely new class of small molecule orally available drugs to treat genetic diseases. If it proves successful in clinical trials, we may be on the cusp of a new age of therapies that make genetic diseases, once considered untreatable, into manageable diseases. True, it would require patients to take a pill every day for the rest of their lives, but, compared to the ravages of diseases like cystic fibrosis or muscular dystrophy, that's a small price to pay.


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This is a very interesting and cool development. As an interested layperson, I have a basic question: How does the chemical differentiate between nonsense mutations and normal stop codons?

Also, in your discussion above that the genetic code is "degenerate" I think you should mention that there are 20(?) possible amino acids that can be coded for from the 64 possible codon combinations. Otherwise your statement, "most amino acids are coded for by more than one codon," doesn't seem to follow.

One might expect that a molecule that decreases nonsense mediated decay might have long term side effects, like carcinogenesis. Several labs (most notably the Radman lab in Paris) have hypothesized that that bacterial cells increase their mutation frequency when they have an increase in messed up DNA repair proteins (during starvation for example). There's some data to support the hypothesis, but it's not been proven. Nonsense mediated decay cleans up the messed up DNA repair transcripts that may make dominant-negative isoforms of the protein. I think the type of side-effects that you might expect from such a drug wouldn't show up during the relatively short period of a phase I trial.

That said, getting cancer at 40 certainly beats the alternative for a cystic fibrosis patient (who if I remember right, don't generally make it past 20).

Another worry would be neurodegenerative disease with long-term therapy. Because neurons do not divide or die, they are particularly vulnerable to accumulation of "junk" -- any kind of protein that cannot be handled by the normal protein salvage and degradation pathways. Presumably, people taking this drug would have a lot more abnormal protein produced--truncated proteins due to RNA that escapes nonsense mediated decay but where the ribosome correctly terminates translation at the stop codon, as well as abnormal proteins in which the stop codon is missed and the ribosome appends a bunch of junk onto the end. The worry would be that these would build up gradually in nerve cells and cause degeneration after years or decades.

Of course, if you have a disease that is going to kill you or destroy your quality of life in the short term, such long term concerns take a back seat. Still, it will be only after we have used this drug for decades that we will have a clear idea of its safety.

The problem with neurodenenerative diseases is (in my opinion) more due to the natural shut down of the normal "housekeeping" pathways rather than generation of something indigestable. In other words, as you point out, how can a nerve cell prevent gunk from accumulating for 65 years and then all of a sudden it accumulates?

I would think the side effects of this could be quite idiosyncratic, depending on what stop codons get read through in what cells. Probably for a lot of them there is a down-stream back-up QC system, ubiquination most likely. This may increase metabolic load by increasing protein turnover, but that is likely going to be tiny because most proteins are made in only small amounts.

Good post, Thanks!

I think Neomycin does this too. For a long time people have noticed that it will push translation past stops. What an interesting idea to harness the effect.

From the excerpt:

"Thus, NMD serves as a 'cellular vacuum cleaner' that protects the cell from the potentially harmful effects of truncated proteins by eliminating mRNAs with PTCs in a sequence of events that is not yet fully understood."

So we don't know yet how it works. But I second Big C, it'd be fascinating to learn how it "knows" which stops are bad ones.

Surely the mdx mice were dosed, observed, and their cognitive skills assessed until death.

It would be truly interesting to research the relationship of the action of this drug and the formation of P-bodies, which have been demonstrated as sites for mRNA storage and degradation.

I can't help but hope...might this work for the treatment of ALS - Lou Gehrig's Disease?

it's also my understanding that a stop codon within 50 bases of the exon junction complex is ok, whereas if it's outside of that range, NMD is activated. There have been some recent articles on ultraconserved regions and the speculation that splicing in the 3' UTR is rare because of this pathway. I also understand that the mechanism of the drug is a general slowing down of translation, obviously not enough to cause many deleterious effects, but just enough to reduce the activation of NMD upon reaching a PTC outside of the EJC window. Disclaimer: this is from memory and citation-free so no knives from ribosomephiles please.

I guess this will only work in cases where adequate suppressor tRNAs mediate the incorporation of amino acids that will not disturb folding or activity of the mature protein.

The problem with neurodenenerative diseases is (in my opinion) more due to the natural shut down of the normal "housekeeping" pathways rather than generation of something indigestable. In other words, as you point out, how can a nerve cell prevent gunk from accumulating for 65 years and then all of a sudden it accumulates?

Neurodegenerative disease can occur because your cells aren't taking out the trash properly, or because the trash is being produced faster than it can be disposed of, or because you are producing trash that won't fit in the garbage can. It is unlikely that the gunk accumulates "all of a sudden." More likely, the trash is gradually accumulating all along. The cell can tolerate a certain amount of this, storing it away in cellular inclusions and the like, but eventually the neuron's capacity to find places to put the junk is exhausted, and it dies. The nervous system can tolerate quite a bit of this, too, but eventually the neuronal loss becomes too great and function begins to suffer.

Presumably, like most new medical treatments, if this one makes it through phase II and III trials, it will be released on the general public and if negative long term side effects show up, it will be withdrawn again. It's a neat idea though.

Question: If we already have a read-through mechanism, why does it not protect against nonsense mutations in the first place? Or is the protective effect one of degrees, which PTC124 raises to a clinically efficacious level?

What about B-thalassaemia? Isn't that a stop codon mediated problem in one variant?

"Thick sticky mucous." I thought mucous was the adjective and mucus the noun.

By Justin Moretti (not verified) on 01 May 2007 #permalink

"Thick sticky mucous." I thought mucous was the adjective and mucus the noun.

Come on, dude! Give me a break!

Spelling flames are really, really lame. If you want to annoy me, that's sure enough one way to do it. Given the amount of verbiage I produce, I'll put my typo record against pretty much anyone else's.

Ribosomes "read" the DNA, whose code is based on three amino acid sequences called codons, each of which codes for a different amino acid.

I think this might be more critical than the spelling flame. Don't you mean "based on sequences of three nucleotides"? BTW, I used the phrasing "sequences of three" because "three X sequences" can be read as "three sequences of X" or "sequences of 3 X." The geek in me just had to disambiguate that, quite aside from correcting "amino acids" to "nucleotides."

Given four nucleotides and triplets, there are 64 possible codon combinations

Also, don't you mean "64 possible codons" here, since a codon is a triplet?

(Sorry, it's possible that I have undiagnosed Aspergers, and unclear language drives me buggy.)

Sorry, it's possible that I have undiagnosed Aspergers, and unclear language drives me buggy.)

You don't say. I never would have guessed. ;-)

I'm actually surprised no one noticed the "amino acid" gaffe before. I'v fixed it. As for the "codon combinations" thing, well, just chalk that up to my tendency to use two words when one will do.

factician: While I know the New York isn't exactly the NEJM, according to an article it ran a few years ago, cystic fibrosis patients at average centers can expect to live 30 years, patients at top centers can expect to live 46 years. At the top treatment center in Cleveland, there are patients living into their 60s. It looks as though cystic fibrosis is slowly moving into being a "manageable" disease with the right treatment protocol. Dying at 40 would be a step back for a lot of CF patients. (The same isn't true for muscular dystrophy, of course...)

"headache, nausea, and dizziness"

Doesn't EVERY drug have this as possible side effects? I, at least, have yet to read an information packet about a drug that doesn't mention those three, given that (IMHO) humans, if told they are taking a medication that might have side effects, are most likely to will themselves into having those three. Perhaps I'm cynical?

headache, nausea, and dizziness" Doesn't EVERY drug have this as possible side effects?

Actually, when you look at the placebo group at baseline, then during treatment, you usually do see an increase in minor symptoms like this. That is why you have a control group in a well designed study.

Dr. McCoy's miracle pill can't be that far away after all.

This is very interesting information, thanks for posting it.

I have cystic fibrosis, I'm 35 years old and do not use a treatment center, my doctors scratch their heads and wonder why I'm not sicker (is that a word?). CF patients are expert pill swallowers. I do comparatively little for treating my CF, I swallow 6 pills every meal, four pills with any snack and another 4 pills which are herbs I've found helpful in managing symptoms. If I get a cold add one or two antibiotic pills, and possibly two more Prednisone pills. I find the possibility of being able to take a pill in exchange for not coughing up a lung encouraging.