Taking on the world’s first genetic disease

Identified over a century ago, Alkaptonuria still has no cure. But with several possible approaches, this is something the AKU society think can change...

Alkaptonuria (AKU) was the first genetic disease ever identified, by Sir Archibald Garrod in 1901¹. More than 100 years later, AKU still has no cure, nor treatment.

AKU is a serious, autosomal recessive, multisystem disorder of peak adulthood affecting approximately one in every 250,000 people, although this figure can rise to one in 19,000 in certain countries such as Slovakia². A recent study of 44 AKU patients, showed 76 joint replacements, 28 ruptures (tendons/ligaments/muscle), 10 fractures, four with renal stones, four with prostatic stones and seven with aortic valve disease³. This morbidity is caused by increased homogentisic acid (HGA) due to a deficient enzyme, homogentisate 1,2-dioxygenase (HGD), despite efficient urinary excretion. The absence of HGD results in patients being unable to fully metabolise the amino acid tyrosine. Tyrosine is broken down via HGA, which in unaffected individuals is then converted to acetoacetate and fumarate, via maleylacetoacetate, in the liver⁴.

[caption id="attachment_43143" align="alignright" width="302"] Tetrad of diagnostic features of AKU. Note the dark urine, eye and ear pigment and joint ochronosis. The diagnosis of AKU relies on recognising the black urine present from birth, blue/black ear pigment, black scleral pigment and clinical arthritis. These features usually alert a physician into considering AKU as the diagnosis.[/caption]

Despite efficient and marked urinary excretion of much of the HGA, some of it is oxidised to a melanin-like polymeric pigment via benzoquinone acetic acid (BQA), in a process termed ochronosis. The pigment polymer is deposited in connective tissues, particularly cartilage⁵, leading to early onset, severe arthritis^{3, 4}. Progressive arthritic pain, affecting all synovial joints, can start around age 30. There are few symptoms or signs before the late 20s or early 30s, apart from constant dark urine and sometimes renal stones³.

Visible external ochronosis in ears and eyes begins at the same time as serious and painful spinal disease, which accompanies progressive kyphoscoliosis, impaired spinal and thoracic mobility. This causes poor pulmonary inflation/decreased respiratory reserve and disc disease/prolapse (resulting in spinal stenosis, cord compression and myelopathy) and vertebral and long bone fractures. Development of spinal disease is inevitable over time. A recent patient survey by the AKU Society showed constant pain, difficulty with daily activities, poor sleep, depression, poor quality of life, unemployment and isolation. If current assumptions are correct that ochronosis is irreversible⁶, early treatment is required to modify the disease. Later treatment may stop progression, but will lead to greater residual disease.

[caption id="attachment_43144" align="alignleft" width="320"] Metabolic pathway of tyrosine catabolism. Names in brown boxes are enzymes. Names in bold are two disorders on the pathway (Alkaptonuria and Tyrosinaemia Type I). Other names are metabolic products.[/caption]

Several therapeutic approaches have been tried in AKU patients. These palliative treatments include ascorbic acid, low-protein diets, physiotherapy, pain control and joint replacement. As they do not treat the intrinsic cause of AKU, success is very limited.

A better treatment may exist in a drug called nitisinone. Nitisinone is a competitive inhibitor of 4-hydroxy-phenyl-pyruvate-dioxygenase and decreases formation of HGA. The US National Institutes of Health began research in nitisinone for the treatment of AKU in 2005⁷. Their research found that nitisinone works biochemically, by reducing the acid which causes AKU. Due to an imprecise endpoint and too few patients, their trials unfortunately ended without conclusive proof of efficacy.

The AKU Society is the UK patient group for AKU patients. As a direct result of the unsuccessful trials in the USA, the AKU Society led the development of AKU research in the UK and Europe. Over the next decade, the group funded research into the understanding of AKU. Highlights of the research follow and include the development of models of disease that can be used to further investigate the effect of nitisinone in AKU.

In vitro ochronosis in monolayer and organotypic cultures. The deposition of HGA pigment in tissues leads to structural, biochemical and remodelling changes that cause severe musculoskeletal problems as well as associated cardiac, renal and prostate problems. Our partners, at the universities of Liverpool and Siena have collaborated on the development of an in vitro model of ochronosis⁸. Using a high throughput screen in which SaoS-2 osteosarcoma cells are cultured in medium containing HGA, ochronotic pigment is detected with Schmorl’s stain.

ENU-induced mouse AKU model. The University of Liverpool established an experimental colony of AKU mice in 2010, based on a Balb/cAnNcr strain. They have high urinary and plasma HGA and show progressive ochronotic pigmentation⁹. The model seems otherwise healthy with near normal growth and activity. In this model, nitisinone reduces circulating HGA by about 90% and completely inhibits ochronosis^{6, 9}.

[caption id="attachment_43148" align="alignright" width="280"] AKU in ENU-mouse[9]. Ochronosis in the tibio-femoral joint of a 65 week old AKU mouse stained with Schmorl’s. Individual ochronotic chondrons are stained blue/green and can easily be identified and counted.[/caption]

AKUSSI. Our focus also included clinical research. Our partners at the Royal Liverpool University Hospital performed an observational study of 40 AKU patients^{3, 10}. The results of which led them to develop a systematic assessment system called the AKUSSI (Alkaptonuria Severity Score Index). The AKUSSI incorporates multiple clinically meaningful outcomes that can be described in a single score. It includes kidney and prostate stones, aortic stenosis, bone fractures, tendon/ligament/muscle ruptures, kyphosis, scoliosis, joint replacements and all other clinical features of AKU. This score is sensitive to all morbidity features of AKU and not any one feature.

Anecdotal evidence strongly suggests that nitisinone should reduce the disease burden in AKU. The basic work provides additional evidence in disease models, and provides a custom assessment system to measure effects of the drug in patients. However, reliable evidence of a working treatment can only be found through clinical research. Therefore, the AKU Society founded the DevelopAKUre consortium, including Sobi (the pharmaceutical company with the licence for nitisinone), three clinical sites (Royal Liverpool University Hospital in the UK, National Institute of Rheumatic Disease in Slovakia, and Hôpital Necker and Institute Necker in France), a CRO and Consultancy (PSR Group and Cudos), several labs (University of Liverpool, Nordic Bioscience, University of Siena and the Institute of Molecular Physiology and Genetics in Slovakia), as well as our French sister group, Association pour la Lutte Contre l'Alcaptonurie (ALCAP). With funding from the European Commission’s Seventh Framework Programme, DevelopAKUre began clinical trials of nitisinone in human AKU patients in 2012.

[caption id="attachment_43145" align="alignleft" width="400"] Metabolic pathway of tyrosine catabolism. Names in brown boxes are enzymes. Names in bold are two disorders on the pathway (Alkaptonuria and Tyrosinaemia Type I). Other names are metabolic products.[/caption]

The first published clinical trial results from the DevelopAKUre consortium has shown that nitisinone decreases urinary HGA excretion in a dose-dependent manner in AKU patients and is well tolerated¹¹. A phase III trial of nitisinone is underway to determine if nitisinone can prevent ochronosis and osteoarthopathy in AKU patients.

However, nitisinone is an imperfect treatment. The major side effect of nitisinone is severe hypertyrosinaemia and marked increases in urine tyrosine, 4-hydroxyphenylpyruvice acid and 4-hydroxyphenyllactic acid. This is associated with potentially serious side effects including corneal keratopathy¹². As a result of the potential nitisinone toxicity in adults with AKU, and even more so in children and young adults with AKU, it would be desirable to develop treatments that ‘normalise’ the pathophysiology of AKU, i.e. decrease HGA without increasing tyrosine. This would allow us to target and treat patients with AKU from birth or as soon as the diagnosis is made without concerns of treatment-related on-target toxicity.

[caption id="attachment_43146" align="alignright" width="320"] Regression of AKUSSI on age. To a good approximation, AKUSSI increases linearly with age at an average rate of two units per year for both male and female patients. However, females score, on average, seven units less than males of the same age (M – male, regression line 1; F – female, regression line 2).[/caption]

The AKU Society is now focused on the search for treatments beyond nitisinone, which would have the same therapeutic results, but without the problems of induced tyrosinaemia. We hope to achieve this through the creation of a newly formed AKU consortium called GARROD AKU (Genetic Approaches for Repair & Replacement of Disease Mutations in Alkaptonuria). This new approach to the treatment of AKU would take the quest for a cure into five new therapeutic areas:

Small molecules. Using high-throughput screening of libraries of FDA-approved biological compounds, we plan to create a large-scale drug-repurposing project to identify any compounds that interact with AKU models to reduce HGA without the effect of increasing tyrosine.

HGD gene therapy. Using S/MAR DNA vectors, we plan to research the possibility of gene therapy in AKU, which would result in patients producing the HGD enzyme and, if it works, should result in a complete cure of the disease.

CRISPR-Cas9 correction of mutant HGD. By using an innovative approach to CRISR-Cas9-mediated genome editing, we plan to investigate this method of gene therapy. This approach would be plasmid-free and has the potential to lead to a more refined therapy. The end result would be the ability of patients to produce a functional HGD enzyme.

HGD replacement. We plan to create wild-type analogue HGD enzymes which can replace the malformed enzyme present in AKU patients. This approach would return patients to a biological norm; correcting the enzyme at fault in AKU.

[caption id="attachment_43159" align="aligncenter" width="560"] The scientific approach for GARROD AKU.[/caption]

Cell therapy. Our final approach would develop a cell encapsulation technique, allowing us to inject microcapsules containing healthy liver cells into the body of patients. Encapsulation would prevent any immune response and healthy liver cells would produce HGD naturally to prevent AKU.

All of the above techniques are in early stages of development, and following successful funding applications would require several years of study in in vitro and animal models of disease before we could gather sufficient data to warrant use in human patients. However, the AKU Society believes that the future of AKU treatment beyond nitisinone could result from GARROD AKU.

The author:

Oliver Timmis of the AKU Society.

References

AKU Society: www.akusociety.org  / DevelopAKUre: www.developAKUre.eu

1. Garrod AE (1902) The incidence of alkaptonuria: a study in chemical individuality.  Lancet, vol. ii, 1902, pp. 1616-1620.

2. Zatková A, de Bernabé DB, Poláková H et al. (2000) High frequency of alkaptonuria in Slovakia: evidence for the appearance of multiple mutations in HGO involving different mutational hot spots. Am J Hum Genet 67:1333-9.

3. Ranganath LR, Cox TF (2011)  Natural history of alkaptonuria revisited: analyses based on scoring systems. J Inherit Metab Dis 34:1141-51.

4. La Du BN, Zannoni VG, Laster L et al (1958) The nature of the defect in tyrosine metabolism in alcaptonuria. J Biol Chem.1958;230:251-260.

5. Helliwell TR, Gallagher JA, Ranganath L (2008) Alkaptonuria - a review of surgical and autopsy pathology. Histopathol 53:503-512.

6. Keenan CM, Preston AJ, Sutherland H et al (2015)  Nitisinone Arrests but Does Not Reverse Ochronosis in Alkaptonuric Mice. J Inherit Metab  in press.

7. Introne WJ, Perry MB, Troendle J et al. (2011) A 3-year randomised therapeutic trial of nitisinone in alkaptonuria. Mol Genet Metab 103: 307-14

8. Tinti L, Taylor AM, Santucci A et al. (2011) Development of an in vitro model to investigate joint ochronosis in alkaptonuria. Rheumatology 50: 271-7

9. Preston AJ, Keenan CM, Sutherland H et al. (2014) Ochronotic osteoarthropathy in a mouse model of alkaptonuria, and its inhibition by nitisinone.  Ann Rheum Dis 73:284-9.

10. Cox TF, Ranganath L. (2011) A quantitative assessment of alkaptonuria: testing the reliability of two disease severity scoring systems. J Inherit Metab Dis 34:1141-51.

11. Ranganath LR, Milan AM, Hughes AT  et al (2014) Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment. Ann Rheum Dis. [Epub ahead of print]

12. Stewart RM, Briggs MC, Jarvis JC, et al (2014) Reversible Keratopathy Due to Hypertyrosinaemia Following Intermittent Low-Dose Nitisinone in Alkaptonuria: A Case Report.  JIMD Rep. 17:1-6.