To assess the efficacy of thymidine phosphorylase (TP) intracellular enzyme replacement therapy (ERT) in experimental models of TP enzyme deficiency.

MNGIE is a slowly progressive, rare autosomal recessive disease caused by mutations in TYMP, which encodes TP. Patients manifest a combination of gastrointestinal dysmotility, severe cachexia, extraocular muscle weakness, peripheral neuropathy, and leukoencephalopathy. MNGIE is characterized by elevated concentrations of the TP substrates thymidine (Thd) and deoxyuridine (dUrd). Elevated Thd/dUrd result in imbalanced deoxynucleoside triphosphate pools which, in turn, cause mitochondrial DNA instability and depletion, and ultimately the MNGIE phenotype. Since current experimental interventions result in excess morbidity and mortality, there is a need for improved therapeutic options for MNGIE patients.

We developed a series of novel intracellular ERTs consisting of 3 covalently-linked components: 1) an engineered form of recombinant human TP, 2) a cell-penetrating peptide (CPP) to facilitate enzyme delivery into the cell, and 3) a polyethylene glycol chain to optimize the pharmacokinetic properties of the molecules. The activities of multiple TP-ERT constructs were evaluated in 1) a TP-deficient mammalian cell line and 2) the MNGIE murine Tymp^-/-Upp1^-/- model, in which plasma Thd levels exceed 10 μM, in contrast to 1.5 μM in wild type (WT) mice.

CPP-TP conjugates delivered active TP into TP-deficient cells in vitro in a dose-dependent manner. In the MNGIE Tymp^-/-Upp1^-/- mouse model, the lead CPP-TP construct, ENTR-501, rapidly reduced plasma Thd/dUrd to WT or lower levels that were sustained for 72 hours after a single dose of 5 mg/kg. Reduced Thd/dUrd levels were repeatedly achieved following weekly doses of 5 mg/kg ENTR-501.

CPP-conjugated-TP corrected metabolic abnormalities in the TP-deficient MNGIE mouse. These data support the development of CPP-TP intracellular enzyme replacement therapy for MNGIE.