Update: June 2010
Our PKU research efforts continue to move forward at a pleasing pace here at the Children’s Hospital at Westmead. There are currently three directions we are taking, all aimed at either improving our understanding of PKU or working towards the development of new therapies.
1. How do the PKU gene mistakes (mutations) affect the phenylalanine hydroxylase (PAH) enzyme?
There are now over 550 different mutations in the PAH gene that can give rise to PKU. Some mutations completely destroy the function of the PAH enzymes, and individuals with these types of mutations usually have the lowest phenylalanine tolerance, requiring very tight dietary control to maintain their blood levels in the target range. Other mutations are less disruptive on the enzyme, and people with these mutations generally can tolerate more phenylalanine in their diet.
Most individuals with PKU have two different PAH mutations, one inherited from each of that individual’s parents, and the two mutations acting together determine the phenylalanine tolerance level in the individual. Sometimes however, two individuals can have the same two mutations but appear to have different levels of phenylalanine tolerance. The mechanism for this is not clear. Also, there are quite a number of mutations in the PAH gene where it is difficult to predict the severity of that mutation.
Gladys Ho, PhD student in the laboratory, is working on these two questions. Using sophisticated gene manipulation studies in cells in culture, she hopes to be able to unravel at least some of the unknowns relating to the interactions of two PAH mutations in the same individual. She has now developed and optimized most of the molecular tools she will need to be able to start teasing out these questions.
2. Can we induce the production of functional PAH enzyme by correcting the mutation in some people with PKU?
In about 10% of people with PKU one of the mutations is a nonsense mutation, ie the mistake in the genetic sequence tells the liver cells to stop making the PAH protein beyond the site of the mutation, resulting in a shorter form of the PAH protein that no longer works at all, and sometimes is so unstable it falls apart altogether. There is an experimental drug available, which in other diseases where nonsense mutations affect the gene has been able to trick the cell’s machinery into making a full length copy of the protein, restoring some of the protein’s function. This drug has proved promising in clinical trials of cystic fibrosis, and studies seem to indicate that it is safe and nontoxic.
No one has tried this type of drug in PKU yet. Based on our experience with other inborn errors of metabolism, we estimate that improvement of PAH activity by as little as 10 – 15% could have a dramatic impact on dietary phenylalanine tolerance in patients. We have been able to obtain a small amount of this experimental drug, and Gladys will soon be undertaking studies, again using cells in culture in the first instance, to see if this drug is potentially beneficial.
3. Can we provide an alternate enzyme to do the job of poorly functioning PAH enzyme in PKU patients?
A potential form of therapy for PKU would be to provide a different simpler enzyme than PAH that can potentially do the job of the faulty PAH enzyme. There is an enzyme called phenylalanine-ammonia lyase (PAL), which is not present in humans, but which comes from yeast and certain plants. It is able to break down phenylalanine to a nontoxic chemical called transcinnamic acid. Early studies giving the purified PAL enzyme to a mouse with PKU showed that PAL was able to reduce blood phenylalanine levels in the mouse by up to 50%. The trick is to be able to deliver PAL in a form what will retain its effectiveness. The purified PAL enzyme cannot be given in an oral capsule form because digestive enzymes in the gut break it down pretty quickly. Also, because it is a foreign protein, an injectable form has the potential to induce immune responses in humans.
We are taking a different approach to delivering the PAL enzyme, namely by developing a genetically modified probiotic. Probiotics are “health-promoting” bacteria, and most people will probably be familiar with the product Yakhult™. For some time now XingZhang Tong, a talented postdoctoral scientist in our research group, has been working on this project. He has been able to successfully introduce a PAL gene from parsley into a probiotic organism called Lactococcus, and has shown that the Lactococcus organism is able to make functional PAL. He has been fine-tuning the system with the aim being to make the system as efficient as possible.
We are not close to taking this to the next level, ie testing it out in the PKU mouse. We have begun the process of importing the PKU mouse from the US, and once the colony is established in our research facility (will take about 6 months) we will be ready to start preliminary testing. If this works it will be a brand new therapy that we hope will lead to improved phenylalanine tolerance in patients with PKU.
It goes without saying that performing this kind of research is time consuming and costly. Fortunately our small but dedicated team of PKU researchers is enthusiastically tackling the questions. We have been very fortunate in being to obtain a third year of funding from the Rotary Australian Health Research Fund to maintain the momentum of our research. More recently the Rotary Club of Pennant Hills and the NSW PKU Association have both contributed further funds in support of our research. As a result of this, we are able to continue our research efforts for a further 12 months, and take them all to the next level. We are very grateful to all the people working behind the scenes supporting the fundraising efforts. Without them we would not have progressed to the point where we are now ready to consider trying the new PKU probiotic therapy in the mouse model for PKU. This time next year I hope to be able to share preliminary results of this study.
Finally, for those PKU patients waiting to gain access to tetrahydrobiopterin (BH4; you know who you are!), hang in there! An application to have BH4 approved for individuals with PKU is currently under review by the Therapeutic Goods Authority in Canberra, and we understand the recommendations will be handed down by the end of this year. We will continue to advocate for its use at every opportunity.