Research Blog
Prepared by: Professor John Christodoulou, Head, PKU Clinic
Contact phone number: 02 9845 3452
PKU Research at the Children’s Hospital at Westmead
Progress Report - 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.
PKU Research at the Children’s Hospital at Westmead
Update: January 2010
We had a busy year in 2009, and continue to make steady progress in our PKU research activities.
PKU Gene Studies:
Wecontinue to offer screening of the PKU (phenylalanine hydroxylase; PAH)gene to any of the patients we see in our clinic. PhD student, GladysHo, is performing this work. We anticipate that by identifying thecombination of mistakes (mutations) in the PAH gene, we will be betterable to predict the level of dietary restriction that will be needed. In addition, there are some mutations that allow us to predict thatsome individuals (perhaps up to a third of PKU patients currently ondiet) will be responsive to the essential cofactor, tetrahydrobiopterin(BH4). This is important information, because once BH4 becomes moreeasily available in Australia, we will be in a position to identifythose individuals who are likely to be BH4 responsive for immediateaccess to it.
On that front, we understand that the companytrying to get BH4 approved and licensed in Australia submitted a formalapplication to the Federal agency responsible for reviewing suchapplications in December, and we hope that it will approved in thefirst quarter of this year, although there is no guarantee about thisat present.
So, if you or your PKU child has not had genetictesting done yet and would like this to be done, please discuss thiswith your PKU doctor. The genetic testing is available as part of aresearch project based at the Children’s Hospital at Westmead. We haveinformation sheets and consent forms to become involved in thisresearch, and we are happy to make these available to you through yourPKU doctor.
Progress in the Development of a PAL Probiotic Therapy:
Readerswill recall from previous newsletters that we have been working on anew therapy for PKU, namely the provision of an alternate enzyme,phenylalanine-ammonia lyase (PAL) that is able to break down excessamounts of phenylalanine. A combined research effort in Canada and theUS has developed a chemically modified form of PAL that is delivered topatients by regular injection. Whilst this is a great step forward inthe development of a novel treatment approach to PKU, our view is thatfor many individuals regular injections (it is not clear at this stagehow frequently the injections will need to be given) of the PAL enzymewould not be acceptable.
Our approach is to create agenetically modified (GM) probiotic to produce PAL. This PAL couldthen be delivered through a regular oral drink of a Yakhult type ofpreparation. If it works, phenylalanine released from dietary proteinin the intestine would be broken down before it is even absorbed intothe bloodstream, and this should translate to improved proteintolerance with better control of blood phenylalanine levels.
Withthe generous support of a research grant from the Australian RotaryHealth Fund (sponsored by the Rotary Club of Pennant Hills), andfinancial support from the Rotary Club of Liverpool (Greenaway), wehave been able to develop this idea. We are pleased to report that wenow have working PAL being made by a probiotic called Lactococcus. Weare currently making adjustments to the genetic regulatory machinery sothat we can maximize the efficiency with which PAL is produced by theLactococcus probiotic.
The next step will be to test out our newPKU therapy in a PKU mouse. There is a commercially available mousethat has a severe mutation in the mouse version of the PKU gene, whichin turn results in very high blood phenylalanine levels. There aresignificant costs involved in importing the mouse to Australia,establishing a PKU mouse colony and then performing the trial of the GMLactococcus. Unfortunately, at present we do not have the funds toestablish the mouse colony and perform the therapeutic trial, and so weare currently unable to progress to the next stage. We are currentlyin the process of trying to obtain additional funding from a number ofsources, and if successful in our fundraising activities, we will beable to perform the initial mouse studies over the next 12 months.
We will keep you informed of progress on all these research fronts, so stay tuned for the next installment!
PKU Research at the Children's Hospital at Westmead
Update: April 2009
Thanks to the financial support of the Rotary Club of Pennant Hills, the Rotary Club of Liverpool-Greenway and Australian Rotary Health, we have been able to initiate research into PKU at the Children's Hospital at Westmead. Our research currently has two main directions.
Genetic and Functional Studies of the Phenylalanine Hydroxylase (PAH) Gene: As we all know, individuals with PKU are unable to clear excessive amounts of phenylalanine from the blood because the liver enzyme phenylalanine hydroxylase (PAH) does not work properly. This in turn is a result of mistakes (mutations) in the gene that encodes PAH. To date well over 500 different mutations have been identified in the PAH gene.
There is value in knowing which PAH mutations have caused PKU in individuals for several reasons. Firstly, knowing the mutations may give us some idea as to the level of phenylalanine tolerance in that individual. Secondly, we now know that for perhaps up to a third of people with PKU, treatment with the cofactor tetrahydrobiopterin (BH4), which comes in tablet form, may lead to an improvement in phenylalanine tolerance, ie these people would be able to have a higher protein intake and yet keep their blood phenylalanine levels in the satisfactory range. We can predict with some confidence whether someone is likely to respond to BH4 treatment if we know what their PAH mutations are.
Gladys Ho, who started her PhD studies into PKU in mid 2008, has established an efficient process for screening the PAH gene for mutations. The aim is to provide us with information that will allow us to predict whether someone with PKU is likely to have more severe or milder PKU, and whether they are likely to be responsive to BH4. She has already screened DNA from over 80 individuals with PKU, and has indeed found that a significant number are likely to be responsive to BH4. This will be very important information when BH4 eventually becomes available in Australia (hopefully in the next 12 months). A form of BH4 called Kuvan, has already been approved for use in PKU patients in the US and Europe.
Gladys has also discovered a number of mutations which have not been previously reported. She hopes to be able to study the effect of these mutations on PAH function using a range of test tube studies.
If you have not yet had mutation testing done and would like to, please discuss this with the doctor who you see for your PKU management. Your doctor could then contact us to arrange for testing. There are no charges for this for patients residing in NSW.
Development of Genetically Modified Probiotics for the Treatment of PKU: Whilst there have been pleasing developments in the range of more palatable low-phenylalanine products available for patients with PKU, it would be fair to say that there have been no major advances in the dietary treatment of PKU in the last two decades. Dietary treatment of PKU becomes more onerous for everyone concerned as children mature into adolescence and adulthood. The development of new treatments that might overcome the need for diet would be a great advance in the treatment of PKU. Once such approach, gene therapy, where a correctly functioning copy of the PAH gene is delivered to the liver, where it needs to be active, would be one such approach. However, gene therapy is has a number of potential problems associated with it, and for now at least does not appear to be an option in the immediately foreseeable future.
We have decided to take a different approach, and see if it is possible to overcome the dysfunctional PAH enzyme with an alternative enzyme that might do the job of clearing phenylalanine. One such enzyme is phenylalanine-ammonia lyase (PAL). PAL is not normally found in humans (it is in yeast and plants). Research performed in Canada a number of years ago showed that if purified PAL was injected into PKU mice (yes, there is a mouse with PKU!), the blood phenylalanine levels could be reduced by up to 50%. However, purification of PAL is a time-consuming and very expensive process, and so does not seem a practical option. We have taken a different approach, that being to see if we can genetically modify probiotic organisms (eg Lactobacillus and Lactococcus, which are harmless bacteria that have been found to have many health benefits when regularly consumed), so that they can make PAL. The idea is that the genetically modified PAL-producing probiotics could taken be taken on a regular basis, and break down dietary phenylalanine in the gut before it is absorbed into the bloodstream. If this approach worked then it should be possible for the diet to be relaxed, allowing a higher natural protein intake.
It has proven quite tricky working with the probiotic bugs because they are finicky little critters! However, XingZhang Tong, a postdoctoral research scientist in our laboratory, has been working hard at this, and we now believe he has been able to trick a Lactococcus bug to make PAL. There is much work to be done to make this process as efficient as possible, but we hope to be able to commence studies to assess the safety and effectiveness of our GM Lactococcus in one of the PKU mouse models in the next 12-18 months. If these studies are promising, then we would hope to move into human trials after that.
There is still much work to be done, but we are excited about our results so far. We hope that with ongoing support we will be able to progress these two research themes to the benefit of everyone touched by PKU.