Tat-mediated delivery of human alanine:glyoxylate aminotransferase in a cellular model of Primary Hyperoxaluria Type I

Defects in liver peroxisomal alanine:glyoxylate aminotransferase (AGT), as a consequence of inherited mutations on the AGXT gene, lead to primary hyperoxaluria type I (PH1), a rare metabolic disorder characterized by the formation of calcium oxalate stones at first in the urinary tract and then in the whole body. The curative treatments currently available for PH1 are pyridoxine therapy, effective in only 10–30 % of the patients, and liver transplantation, an invasive procedure with potentially serious complications. A valid therapeutic option for PH1 patients would be the development of an enzyme administration therapy. However, the exogenous administration of the missing AGT would require the crossing of the plasma membrane to deliver the protein to liver peroxisomes. In this study, we constructed, purified and characterized the fusion protein of AGT with the membrane-penetrating Tat peptide (Tat-AGT). Although Tat-AGT shows subtle active site conformational changes as compared with untagged AGT, it retains a significant transaminase activity. Western-blot analyses, enzymatic assays and immunofluorescence studies show that active Tat-AGT can be successfully delivered to a mammalian cellular model of PH1 consisting of chinese hamster ovary cells expressing glycolate oxidase (CHO-GO), whereas untagged AGT cannot. Moreover, the intracellular transduced Tat-AGT makes CHO-GO cells able to detoxify endogenously produced glyoxylate to an extent similar to that of CHO-GO cells stably expressing AGT. Altogether, these results show that the Tat peptide is capable of delivering a functional AGT to mammalian cells, thus paving the way for the possibility to use Tat-AGT as an enzyme replacement therapy to counteract PH1.