Understanding the role of the kidney in alkaptonuria (AKU) and development of cell lines for the in vitro study of HGD gene therapies

Alkaptonuria (AKU) is an ultra-rare, autosomal recessive metabolic disease cause by a deficiency of homogentisate 1,2- dioxygenase (HGD) activity and characterised by elevated levels of urinary and circulatory homogentisic acid (HGA). HGD expression is normally localised in the hepatocytes and kidney proximal tubule cells. As the disease progresses, the elevated HGA in the circulation, results in the deposition of dark, HGA-derived ochronotic pigment in the connective tissues causing a severe, early onset osteoarthropathy, often requiring surgical intervention. The main aim of this thesis was to understand the role of the kidney in alkaptonuria, with a primary focus on the contribution of kidney HGD to the elevated levels of HGA observed in the AKU phenotype. This was achieved with the development of kidney-specific Hgd conditional knockout models, using Pax8-CreERT2 and Six2GCiP Cre recombinases, to explore the effect of eliminating kidney HGD expression on physiological HGA levels. Additionally, the wider role of the kidney in tyrosine handling was investigated by comparison of tyrosine metabolic enzyme expression in the liver versus the kidney. The final aim was to develop AKU cell models, using human immortalised liver and kidney cells, to aid the in vitro development of HGD replacement through gene/mRNA therapies. The kidney Hgd conditional knockout mouse models, showed that deletion of kidney HGD expression, has no effect on circulating HGA, but caused an elevation in urinary HGA concentration. The increase in urinary HGA observed in the kidney Hgd conditional knockout mouse models was 100-fold less than the urine HGA concentrations observed in AKU mouse models. The data suggests that the loss of kidney HGD expression in AKU only minimally contributes to the HGA levels detected in AKU, with the loss of liver HGD expression being the main contributor to the characteristically high HGA seen in AKU patients. Therefore, any future HGD gene/mRNA therapies should be focussed on restoration of liver, rather than kidney HGD expression. Analysis of tyrosine metabolic enzyme expression in the liver versus the kidney, showed that the levels of expression are much higher in the liver compared to the kidney, with tyrosine aminotransferase (TAT) not detected in the kidney. This suggest the primary role of the kidney in tyrosine handling is to reabsorb tyrosine from the renal filtrate into the circulation, with no need to metabolise tyrosine intracellularly to produce other essential compounds. Finally, CRISPR-Cas9 was used to knockout endogenous HGD expression in liver cell lines, HepG2 and Huh7, to produce novel AKU liver cells models for the in vitro development of HGD gene therapies and allowing a reduction in the number of mice required for in vivo testing of constructs. Since 2020, nitisinone has been approved by EMA for treatment of AKU, by inhibiting 4-hydroxyophenylpyruvate dioxygenase (HPPD) activity resulting in lowering of HGA production. Although, nitisinone is an effective treatment, the inhibition of HPPD, leads to the accumulation of upstream metabolites of HGA, including tyrosine, which have their own detrimental effects, with suggested links to cataracts, vitiligo and Parkinson’s disease. As a result, nitisinone-treated AKU patients have to restrict their dietary protein intake, which can be challenging. Therefore, the development of HGD gene therapies would provide a more perfect treatment of AKU, by repairing the genetic defect.