Phenotyping of a new conditional mouse model of alkaptonuria and investigation of nitisinone-induced tyrosinaemia

Alkaptonuria (AKU) is an ultra-rare, autosomal recessive metabolic bone disease characterised by increased urinary and circulating homogentisic acid (HGA) caused by homogentisate 1,2-dioxygenase (HGD) enzyme deficiency. The deposition of a dark, HGA-derived ochronotic pigment in cartilage and other connective tissues causes a severe and early-onset osteoarthropathy, due to ochronotic tissue being stiff and brittle. In addition to joint manifestations in AKU, eye pigmentation, tendon rupture, kidney and prostate stones, and heart valve stenosis are common features. An existing mutagenesis AKU mouse model has previously been used to investigate initial pigmentation of cartilage and to evaluate the effectiveness of the HGA-lowering drug nitisinone. Nitisinone therapy was proven to halt ochronosis in this mouse model and has been the subject of human clinical trials (DevelopAKUre), where it has been shown to reduce HGA and slow disease progression in AKU patients. Nitisinone-induced tyrosinaemia however is an upstream effect of 4-hydroxyphenylpyruvate dioxygenase inhibition that can cause keratopathy. The major aims of this thesis were to phenotype a new Hgd-targeted AKU mouse model, obtained from the KOMP repository, to use the LacZ gene which is expressed as a fusion transcript with the Hgd gene to determine the tissue expression of Hgd via ex vivo LacZ staining, to explore the effect of conditional liver-specific Hgd deletion on the AKU phenotype using double transgenic Hgd tm1d (fl/fl) MxCre mice, and finally to explore the effectiveness of dietary restriction and the potential application of an exogenous tyrosine-degrading enzyme at reducing tyrosine. The level of HGA and the tissue distribution of ochronotic pigmentation were evaluated using the knockout-first Hgd tm1a -/- model. HGA was shown to be increased in the plasma and urine, with the first signs of knee joint pigmentation found at 9 weeks in the knee joint, increasing linearly with age. Pigmentation was only found associated with chondrocytes within the calcified articular cartilage of joints, the calcified endplates of vertebrae, and the calcified knee joint meniscus. Hgd tm1a -/- mice exhibit the early stages of human ochronosis, with pigmentation confined to calcified tissues. Positive LacZ staining was identified in the cytoplasm of hepatocytes and kidney proximal convoluted tubule cells, beginning at E12.5 and E15.5 respectively, in addition to the developing male germ cells within the testis and epididymis, with qPCR showing liver Hgd mRNA expression to be approximately 2000-7,500-fold greater than the Hgd mRNA expression in the testis and epididymis. Germ cell expression was deemed insignificant and unlikely to contribute towards HGA metabolism. Liver-specific Hgd deletion via polyinosinic: polycytidylic acid induction of MxCre in Hgd tm1d (fl/fl) MxCre +/- mice demonstrated that 20% residual liver Hgd mRNA does not reduce circulating HGA leading to joint pigmentation and that kidney Hgd mRNA cannot rescue AKU. It was determined that the minimum level of liver Hgd mRNA required is 26-43% to reduce circulating HGA. The detection of downstream metabolites of intravenously injected 13C6-HGA in Hgd tm1a -/+ plasma revealed that hepatocytes can take up and metabolise HGA. The liver should therefore be the target of future gene therapy in AKU. Nitisinone was shown to reduce HGA and increase tyrosine, phenylalanine, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid in the plasma of AKU mice. Dietary restriction of tyrosine/phenylalanine significantly reduced nitisinone-induced tyrosinaemia, with phenylalanine-only restriction not effective. Evidence of AKU patients reducing tyrosine was observed, although the reductions were not comparable with that of mice. Due to the proof-of-principle provided by the dietary studies, preliminary experiments then demonstrated tyrosine degradation by the bacterial enzyme tyrosine ammonia lyase, which has the potential to be developed into an oral therapeutic for reducing nitisinone-induced tyrosinaemia