In vivo biodistribution study of TAT-L-Sco2 fusion protein, developed as protein therapeutic for mitochondrial disorders attributed to SCO2 mutations

Georgios C Kaiafas; Dionysia Papagiannopoulou; Αndroulla N Miliotou; Anastasia S Tsingotjidou; Parthenopi C Chalkidou; Aikaterini C Tsika; George A Spyroulias; Asterios S Tsiftsoglou; Lefkothea C Papadopoulou

doi:10.1016/j.ymgmr.2020.100683

In vivo biodistribution study of TAT-L-Sco2 fusion protein, developed as protein therapeutic for mitochondrial disorders attributed to SCO2 mutations

Mol Genet Metab Rep. 2020 Dec 8:25:100683. doi: 10.1016/j.ymgmr.2020.100683. eCollection 2020 Dec.

Authors

Georgios C Kaiafas¹, Dionysia Papagiannopoulou², Αndroulla N Miliotou¹, Anastasia S Tsingotjidou³, Parthenopi C Chalkidou¹, Aikaterini C Tsika⁴, George A Spyroulias⁴, Asterios S Tsiftsoglou¹, Lefkothea C Papadopoulou¹

Affiliations

¹ Laboratory of Pharmacology, School of Pharmacy, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki 54124, Macedonia, Greece.
² Laboratory of Medicinal Chemistry, School of Pharmacy, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki 54124, Macedonia, Greece.
³ Laboratory of Anatomy, Histology and Embryology, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece.
⁴ Department of Pharmacy, University of Patras, 26504 Patras, Greece.

Abstract

The rapid progress achieved in the development of many biopharmaceuticals had a tremendous impact on the therapy of many metabolic/genetic disorders. This type of fruitful approach, called protein replacement therapy (PRT), aimed to either replace the deficient or malfunctional protein in human tissues that act either in plasma membrane or via a specific cell surface receptor. However, there are also many metabolic/genetic disorders attributed to either deficient or malfunctional proteins acting intracellularly. The recent developments of Protein Transduction Domain (PTD) technology offer new opportunities by allowing the intracellular delivery of recombinant proteins of a given therapeutic interest into different subcellular sites and organelles, such as mitochondria and other entities. Towards this pathway, we applied successfully PTD Technology as a protein therapeutic approach, in vitro, in SCO2 deficient primary fibroblasts, derived from patient with mutations in human SCO2 gene, responsible for fatal, infantile cardioencephalomyopathy and cytochrome c oxidase deficiency. In this work, we radiolabeled the recombinant TAT-L-Sco2 fusion protein with technetium-99 m to assess its in vivo biodistribution and fate, by increasing the sensitivity of detection of even low levels of the transduced recombinant protein. The biodistribution pattern of [^99mTc]Tc-TAT-L-Sco2 in mice demonstrated fast blood clearance, significant hepatobiliary and renal clearance. In addition, western blot analysis detected the recombinant TAT-L-Sco2 protein in the isolated mitochondria of several mouse tissues, including heart, muscle and brain. These results pave the way to further consider this PTD-mediated Protein Therapy Approach as a potentially alternative treatment of genetic/metabolic disorders.

Keywords: 99mTc, Technetium-99 m.; BSA, Bovine Serum Albumin;; COX, Cytochrome c oxidase;; FBS, Fetal bovine serum;; IBs, Inclusion bodies;; ID, Injected dose;; PBS, Phosphate buffered saline;; PRT, Protein Replacement Therapy;; PTD, Protein Transduction Domain;; RA, Radioactivity;; RT, Room Temperature;; SD, Standard Deviation;; SEC, Size Exclusion Chromatography;; TAT-L-Sco2, 10xHis-XaSITE-TAT-L-Sco2-HA;; i.p., Intraperitoneal;; i.v., Intravenous;; l-Arg, l-Arginine;; p.i., Post-injection;.