Erythrocytes as carriers of oxalate decarboxylase from Bacillus subtilis: an innovative approach for the treatment of hyperoxaluria

Hyperoxaluria is a pathologic condition due to genetic and non-genetic causes that leads to the deposition of calcium oxalate (CaOx) crystals at first in the urinary system and, in the most severe forms, in the whole body. The disease can be due to either an increased endogenous oxalate production (primary hyperoxaluria or PH) or an increased intestinal oxalate absorption (secondary hyperoxaluria or SH). Three forms of PH are known. They are inherited disorders caused by the deficit of enzymes involved in liver glyoxylate metabolism. The most common and most severe form is PH1. Two curative therapies are currently available for the treatment of PH1: pyridoxine administration (PN) and liver transplantation. However, The first is only effective in 10-30% of the patients, while the second is a very invasive and risky procedure. Thus, the development of new therapeutic strategies represents an urgent need. In this regard, we hypothesized that a possible approach could be the use of an oxalate-degrading enzyme, which would reduce plasma oxalate concentration thus counteracting the formation of CaOx. Oxalate Decarboxylase (OxDC) from B. subtilis is an hexameric Mn-dependent enzyme belonging to the bicupin family that catalyses the cleavage of the oxalate C-C bond to give carbon dioxide and formate. A mutated form of the enzyme, called OxDC-DSSN, shows a reduced decarboxylase specific activity, but is endowed with the ability to catalyse an oxalate oxidation reaction. It should be underlined that OxDC displays an optimum pH around 4 and a deep characterization of the enzyme at neutral pH is still lacking. Based on these considerations, the aim of my PhD was the study of the biochemical features of OxDC at neutral pH and their possible improvement by protein engineering techniques. Moreover, since the direct administration of a non-human protein would elicit a remarkable immune reaction, we thought to encapsulate OxDC in red blood cells (RBCs) and use loaded RBCs as oxalate-degrading bioreactors. The data obtained indicate that: 1) OxDC and OxDC-DSSN (i) display optimal activity at pH 4.2 but retain a detectable residual activity at pH 7.2, the intracellular pH of RBCs, (ii) do not undergo major structural changes at neutral pH, (iii) are able to detoxify oxalate endogenously produced in a cellular model of PH1. 2) OxDC can be efficiently encapsulated in human and murine RBCs and does not loose catalytic activity during the encapsulation process. 3) by using directed evolution approaches, a mutated form of OxDC could be engineered that is more resistant to thermal stress and aggregation under physiological conditions as compared with wild-type OxDC. Overall these data provide the proof-of-principle for the feasibility of a therapy for PH based on the administration of RBCs-loaded with an oxalate-degrading enzyme. Future studies will be focused on the testing of the ability of wild-type and engineered OxDC to detoxify oxalate in a mouse model of PH1.