Expression and retention of thymidine phosphorylase in cultured reticulocytes as a novel treatment for MNGIE

Marjolein Meinders, Deborah K Shoemark, Johannes G. G. Dobbe, Geert J. Streekstra, Jan Frayne, Ash M Toye*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)

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Abstract

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal metabolic disorder caused by thymidine phosphorylase (TP) deficiency. Successful therapeutic interventions for this disease rely upon a means for efficient and long-lasting circulation of TP enzyme. In this study we exploit lentiviral transduction of haematopoietic 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.
Original languageEnglish
Pages (from-to)822-830
Number of pages9
JournalMolecular Therapy - Methods and Clinical Development
Volume17
Early online date31 Mar 2020
DOIs
Publication statusPublished - 12 Jun 2020

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