THE DEFICIT OF ALANINE:GLYOXYLATE AMINOTRANSFERASE LEADS TO PRIMARY HYPEROXALURIA TYPE I: A BIOCHEMICAL STUDY TO UNDERSTAND THE ROLE OF INTERALLELIC COMPLEMENTATION IN COMPOUND HETEROZYGOUS PATIENTS AND TO PROJECT THE DEVELOPMENT OF AN ENZYME ADMINISTRATION THERAPY.

Primary Hyperoxaluria Type I (PH1) is a rare autosomal recessive disorder characterized by a high level of oxalate in the urine, which in turn results in the formation of insoluble calcium oxalate crystals at first in the kidneys and urinary tract and then, in absence of an appropriate treatment, in the whole body. PH1 is caused by the deficiency of human liver alanine:glyoxylate aminotransferase (AGT), a peroxisomal pyridoxal 5'-phosphate (PLP)-dependent enzyme. AGT detoxifies glyoxylate to glycine, thus preventing glyoxylate oxidation to oxalate and the subsequent calcium oxalate formation. AGT is encoded by the AGXT gene, which presents, in humans, two polymorphic forms: the major allele (encoding AGT-Ma) and the minor allele (encoding AGT-Mi). At the time of writing, more than 150 mutations associated with PH1 have been reported and several studies allowed for interesting progresses in the understanding of the molecular mechanisms by which each mutation leads to AGT deficiency. However, quite often patients affected by PH1 are compound heterozygous and their enzymatic phenotype could depend on interallelic complementation (IC) effects. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, while the genotype-enzymatic phenotype-clinical phenotype relationship of compound heterozygous patients is completely unknown. During my PhD, we elucidated the enzymatic phenotype linked to the S81L mutation on AGT-Ma, concerning a PLP binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functional properties, is present in the second allele. By using a bicistronic eukaryotic expression vector we demonstrated that (i) S81L-Ma has a significant peroxisomal localization, and (ii) the interaction of the S81L and G170R monomers occurs in the cell yielding the G170R-Mi/S81L-Ma heterodimer, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the recombinant purified heterodimer compared with those of the homodimeric counterparts obtained by a dual vector prokaryotic expression strategy, provided evidence for a positive IC between the S81L and G170R monomers. This study represents the first investigation of the pathogenesis of PH1 in compound heterozygous patients at molecular level. PH1 is a very difficult-to-treat disease. Only two curative therapeutic approaches are currently available: the administration of pyridoxine, a precursor of PLP that is only effective in a minority of patients, and liver transplantation, a very invasive procedure. It follows that the development of new treatment strategies, less invasive and effective for all the patients, would be highly desirable. In this regard, since PH1 originates from the deficit of a single enzyme, the opportunity to restore the catalytic pool of the hepatocytes by administering exogenous enzyme is an intriguing perspective. One of the major issues for the development of an enzyme administration therapy is the intracellular delivery of the exogenous protein. During my PhD, to obtain an AGT form able to cross the plasma membrane, a dual approach was used: (i) the construction of a fusion protein between AGT and the Tat peptide exploiting the membrane crossing capabilities of the Tat moiety, and (ii) the conjugation of AGT with a polymeric nanocarrier able to deliver the functional enzyme across the plasma membrane. Both strategies did not significantly alter the structural and functional properties of AGT and proved to be effective in transducing active AGT into a cellular disease model and in restoring their glyoxylate detoxification ability. These results can be considered an encouraging starting point for the development of an enzyme administration therapy for PH1.