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Expression and Retention of Thymidine Phosphorylase in Cultured Reticulocytes as a Novel Treatment for MNGIE.

Authors :
Meinders M
Shoemark D
Dobbe JGG
Streekstra GJ
Frayne J
Toye AM
Source :
Molecular therapy. Methods & clinical development [Mol Ther Methods Clin Dev] 2020 Apr 01; Vol. 17, pp. 822-830. Date of Electronic Publication: 2020 Apr 01 (Print Publication: 2020).
Publication Year :
2020

Abstract

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal metabolic disorder caused by thymidine phosphorylase (TP) deficiency. Successful therapeutic interventions for this disease rely on a means for efficient and long-lasting circulation of the TP enzyme. In this study we exploit lentiviral transduction of hematopoietic stem cells and an erythroid cell line (BEL-A) to generate reticulocytes that contain active TP. Significant loss of overexpressed TP during erythroid differentiation can be reduced by addition of the ubiquitination inhibitor MG132. However, the ubiquitination sites are located in the substrate binding site in human TP, and their removal abolished enzyme activity. Examination of the TP structure and mechanism suggested that these sites are only exposed in the absence of substrate. We show that supplementation of culture media with thymidine during differentiation reduces enzyme degradation, doubling the amount of TP retained in reticulocytes. This study provides proof of principle that therapeutic reticulocytes expressing TP can be generated in vitro and that ubiquitin-mediated degradation can be subverted through masking ubiquitination sites to ensure retention of human TP in reticulocytes following erythroid differentiation.<br /> (© 2020 The Authors.)

Details

Language :
English
ISSN :
2329-0501
Volume :
17
Database :
MEDLINE
Journal :
Molecular therapy. Methods & clinical development
Publication Type :
Academic Journal
Accession number :
32368563
Full Text :
https://doi.org/10.1016/j.omtm.2020.03.029