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

Marjolein Meinders,Debbie Shoemark,Johannes G.G Dobbe,Geert J Streekstra,Jan Frayne,Ashley M Toye

doi:10.1016/j.omtm.2020.03.029

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.

Highlights

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive metabolic disorder[1] that usually manifests during early teens, with an average life expectancy of 37.5 years.[2,3] The disease is very rare, with an estimated occurrence in the population of 1:1,000,000 but this may be an underestimate[4] due to the varied clinical presentation, which includes severe gastrointestinal dysmotility, cachexia, peripheral neuropathy, diffuse leukoencephalopathy, and mitochondrial abnormalities.[3]MNGIE is caused by mutations in the TYMP gene, which encodes the thymidine phosphorylase (TP) enzyme
This study has demonstrated for the first time that exogenous human TP can be overexpressed using lentivirus transduction of the TP cDNA in CD34+ hematopoietic cells and in an erythroid line, BEL-A
We have demonstrated that it is possible to express TP in in vitro-cultured reticulocytes, the enzyme is degraded by ubiquitination during differentiation, limiting the abundance of TP per reticulocyte

Summary

Introduction

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive metabolic disorder[1] that usually manifests during early teens, with an average life expectancy of 37.5 years.[2,3] The disease is very rare, with an estimated occurrence in the population of 1:1,000,000 but this may be an underestimate[4] due to the varied clinical presentation, which includes severe gastrointestinal dysmotility, cachexia, peripheral neuropathy, diffuse leukoencephalopathy, and mitochondrial abnormalities.[3]. MNGIE is caused by mutations in the TYMP gene, which encodes the thymidine phosphorylase (TP) enzyme. TP catalyzes the phosphorolysis of thymidine (dThd) and deoxyuridine (dUrd) to thymine or uridine, and 2-deoxy ribose 1-phosphate (2DR1P) in the cytosol. Homozygous or compound heterozygous mutations in the TYMP gene cause a drastic reduction in protein expression or activity, which results in thymidine accumulation, and subsequently leads to an imbalanced intramitochondrial deoxynucleotide pool.[4,6,7,8] This is thought to destabilize mitochondrial DNA by affecting mitochondrial DNA repair and replication, resulting in the broad variety of symptoms.[9]

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Conclusion