Self-care for Rare – Research Update Summaries

At this year’s Self Care 4 Rare event, Dr Juliette Hughes and Dr Brendan Norman gave an update on some of the alkaptonuria (AKU) research currently taking place at the University of Liverpool.

For those who were unable to attend – or would simply like a reminder of what we discussed – here are some of the highlights.

Our AKU research group at Liverpool is led by Dr Juliette Hughes and Dr Brendan Norman, working alongside a fantastic team of researchers, PhD and Master’s students and collaborators. Between us, we are looking at AKU from several different angles, from understanding why joints become damaged to exploring gene therapy and some of the less well-understood features of the condition.

A major focus remains ochronosis – the build-up of dark pigment in tissues caused by homogentisic acid (HGA). Understanding exactly how this pigment forms, and how it contributes to tissue damage, could help us better understand how AKU progresses.

One particularly exciting development comes from PhD student Daisy Quinn’s work on cartilage.

We know from previous research that pigmentation in AKU joints begins deep within a region called calcified cartilage, where cartilage meets the underlying bone. However, our AKU mouse models develop only very mild pigmentation in their joints, which makes some aspects of the disease difficult to study.

Daisy has therefore developed a new laboratory model. Cartilage cells are grown to form cartilage-like tissue and mineral, and HGA is then added. Encouragingly, we can actually watch pigmentation begin to develop in the laboratory.

Interestingly, the pigment appears alongside the mineralised cartilage – similar to what we see in AKU joints. This gives us a useful new way of asking why pigmentation starts in this particular part of the joint and, importantly, what HGA and pigment do to the health of cartilage.

We are also interested in the relationship between AKU and osteoarthritis.

AKU mice do not naturally develop osteoarthritis in the same way that people can, so we are using a non-invasive method to produce osteoarthritis-like changes in their knee joints. We can then compare AKU and non-AKU mice and look at the severity of osteoarthritis, pigmentation, bone health and changes in molecules found in the blood and urine. This should help us tease apart the relationship between the biochemical changes caused by AKU and the processes that ultimately lead to joint degeneration.

Another new area for us is oxidative stress. Our cells naturally experience oxidative stress and have built-in antioxidant systems that protect them. One important controller of this protective response is a gene called Nrf2.

We have been studying a new AKU mouse model in which this antioxidant protection is reduced. These mice showed increased pigmentation. In separate laboratory experiments, cartilage cells exposed to HGA also increased the activity of genes involved in responding to oxidative stress.

Together, these early findings suggest that HGA is causing oxidative stress in joint cells, and that the body’s antioxidant response may be relevant to pigmentation and joint disease in AKU. There is much more work to do before we understand exactly what this means, but it gives us an important new avenue to investigate.

Although joint disease is a major feature of AKU, our research increasingly looks at the condition more widely.

For example, we are investigating whether HGA encourages cartilage to become more calcified. We are also studying tendons and ligaments, where our work suggests that AKU tendons are mechanically different and can be prone to calcification in areas with dense pigmentation.

We have also examined ear biopsies from eight children with AKU under the age of 16, in collaboration with Dr Marius Kujawa in Poland. We did not identify pigmentation in these samples. This is useful information as we continue trying to understand when and where pigmentation begins.

Other projects are asking some quite different questions. Why is hypothyroidism more common in people with AKU? Our work in mice suggests that the relevant tyrosine pathway is not present within the thyroid itself, meaning that HGA must reach the thyroid through the bloodstream. At present, however, we do not know the mechanism linking this to thyroid problems.

We are also beginning to explore the gut microbiome – the community of bacteria living in the gut. We are analysing samples from AKU and non-AKU mice, including animals with different levels of tyrosine, to see whether AKU or altered tyrosine levels are associated with changes in gut bacteria. This work is ongoing.

A major focus of our research is on finding better ways to treat AKU, including managing the effects of nitisinone and exploring future treatments that address the underlying cause of the disease.

Managing high tyrosine during nitisinone treatment:

Nitisinone is an effective treatment because it greatly reduces the production of HGA, the substance that builds up in AKU. However, it also causes tyrosine levels in the blood to rise, which can sometimes lead to symptoms including eye problems (corneal keratopathy) and other unwanted effects. We are investigating whether inhibiting a protein called SLC6A19, which helps the body absorb and retain amino acids such as tyrosine, could help remove more tyrosine through the urine. This would potentially support the use of a SLC6A19 inhibitor drug to be used in conjunction with nitisinone. If successful, this approach would effectively provide a way to keep the benefits of nitisinone while also reducing the rise in tyrosine.

In our mouse studies, experimental SLC6A19 inhibitors reduced blood tyrosine by around 40–50% in the short term and also lowered tyrosine in tissues including the brain, muscle and heart. A longer study showed that this reduction could be maintained over a longer period of several weeks. These are encouraging early results, but this approach still needs further research before it could be considered for people with AKU.

Exploring mRNA therapy for AKU

Nitisinone is the only known disease-modifying therapy for AKU. However, it does not represent a cure for the condition. The only way to definitively cure AKU is to directly restore activity of the HGD enzyme. We are investigating whether we can temporarily give cells the instructions they need to produce a working HGD enzyme. In AKU, changes in the HGD gene mean that this enzyme does not work properly, leading to the build-up of HGA.

In our laboratory, we have been carrying out studies on delivery of messenger RNA (so-called mRNA) for HGD in the AKU mouse model. HGD mRNA can be thought of the molecular instructions required for cells to produce a functional version of the HGD enzyme. In the mouse model, we saw HGA levels fall dramatically for several days after treatment before beginning to rise again. This suggests that mRNA therapy could one day offer a new way of treating the underlying biochemical problem in AKU, although it is still at an early, preclinical stage. 

Overall, our aim is to develop treatments that not only reduce HGA, but also make existing therapies easier to manage and, in the longer term, address the underlying cause of AKU itself.

One of the most exciting things about AKU research in Liverpool at the moment is the number of different questions we can now investigate.

Some of our projects are aimed at understanding the fundamentals of AKU: how HGA turns into pigment, why pigmentation begins where it does, and how this eventually damages tissues. Others are exploring oxidative stress, calcification and symptoms beyond the joints. Alongside these projects, our longer-term work on future therapies/cures for AKU also continues.

Many of these studies are still at an early stage, so they do not yet translate into changes in treatment or self-care. But each one adds another piece to our understanding of what happens in AKU – and, we hope, brings us closer to finding new ways of preventing or reducing the damage caused by the condition.

It was a pleasure to share our progress at Self Care for Rare, and we would like to thank the AKU Society, the patients and families who support AKU research, and all of our researchers and collaborators who make this work possible.