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Thymidine Phosphorylase Is Both a Therapeutic and a Suicide Gene in a Murine Model of Mitochondrial Neurogastrointestinal Encephalomyopathy

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Thymidine Phosphorylase Is Both a Therapeutic and a Suicide Gene in a Murine Model of Mitochondrial Neurogastrointestinal Encephalomyopathy Gene Therapy (2014) 21, 673–681 © 2014 Macmillan Publishers Limited All rights reserved 0969-7128/14 www.nature.com/gt ORIGINAL ARTICLE Thymidine phosphorylase is both a therapeutic and a suicide gene in a murine model of mitochondrial neurogastrointestinal encephalomyopathy S López-Estévez1,5, G Ferrer1,5, J Torres-Torronteras2,3, MJ Mansilla1, S Casacuberta-Serra1, L Martorell1, M Hirano4, R Martí2,3 and J Barquinero1 Suicide gene therapy (SGT) is a promising strategy for treating cancer. In this work, we show that thymidine phosphorylase (TP) deficiency, the underlying genetic defect in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), presents an opportunity to apply SGT using capecitabine, a commonly used prodrug that is converted into 5-fluorouracil by TP. Using an immortalised B-lymphoblastoid cell line from a patient with MNGIE, the tumourigenic EL-4 cell line, lentiviral vectors encoding TP and a double knockout (TympÀ/ÀUpp1À/À) murine model, we found that EL-4 cell-derived TP+ tumours were exquisitely sensitive to capecitabine and generated a significant local bystander effect. In addition, we detected a spontaneous cytolytic immune response in a significant fraction of the animals surviving more than 20 days after termination of the therapy. These data indicate that, in individuals lacking TP expression, TP is a highly specific suicide gene, which can be used to treat tumours that could hypothetically arise in MNGIE patients undergoing gene therapy, as these tumours will likely originate from the gene-modified cells and will be selectively targeted by capecitabine. These observations have important implications for gene therapy for MNGIE. Gene Therapy (2014) 21, 673–681; doi:10.1038/gt.2014.41; published online 8 May 2014 INTRODUCTION haematopoietic stem cell gene therapy is an effective long-term Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) treatment even at levels of correction below 10% of the bone 3 is a rare metabolic disease caused by autosomal recessive marrow cells and we are currently investigating novel preclinical mutations in TYMP, which encodes the enzyme thymidine gene therapy strategies that can be easily translated into humans. phosphorylase (TP). This enzyme catalyses the first step in the Depending on the target, long-term correction of genetic catabolism of thymidine (dThd) and deoxyuridine (dUrd); there- defects typically requires vectors that integrate into the genome fore, in patients with TP deficiency, these two nucleosides of target cells therapeutic genes that can be transmitted to accumulate to reach abnormally high systemic and plasma levels, progeny cells. Retroviral vectors were initially used in clinical gene which can be used diagnostically.1 This imbalance selectively therapy trials and, two decades later, are still the most widely used impairs mitochondrial DNA synthesis, which results in mitochon- vectors in stem cell-based applications and have been successfully drial DNA depletion, multiple deletions and site-specific point applied to treat inherited diseases including several primary mutations, without detectable changes in the nuclear DNA. In immunodeficiencies.4 Although these successes have brought healthy individuals and in heterozygous carriers of pathogenic renewed hope to the discipline, the development of T-cell TYMP mutations (who are otherwise asymptomatic), dThd and leukaemias in some of the patients treated unveiled the risk of dUrd produced during normal metabolism cross cell membranes insertional oncogenesis by integrative vectors. Although those via specific transporters, diffuse into the extracellular fluid and are first-generation vectors have been progressively replaced by more cleared from the circulation by tissues expressing the enzyme; efficient and safer vectors, like self-inactivating lentiviral vectors, thus, their systemic concentrations are maintained below a non- the potential oncogenic risks still represent a major concern to the 1 toxic threshold (approximately o0.05 μM in plasma). Over the clinical application of gene therapy. last years, we have studied different gene therapy strategies to Suicide gene therapy (SGT; also known as Gene Directed restore TP activity and to correct the biochemical alterations in Enzyme/Prodrug therapy or Gene Prodrug Activation Therapy) can TympÀ/ÀUpp1À/À double knockout (dKO) mice (the only be defined as the therapeutic introduction, into target cells or available animal model of MNGIE). These mice have no apparent tissues, of heterologous genes encoding enzymes able to trans- clinical abnormalities but display a biochemical phenotype form an inactive prodrug into an active drug. Most suicide genes (with increased systemic levels of dThd and dUrd) and it has encode enzymes involved in the nucleotide metabolism, for been reported that elderly animals also have mDNA depletion in example, herpes virus thymidine kinase or cytosine deaminase, the brain.2 We previously reported that sub-myeloablative which confer sensitivity in the transduced cells to ganciclovir or 1Gene and Cell Therapy Laboratory, Vall d'Hebron Research Institute (VHIR)/Universitat Autònoma de Barcelona, Barcelona, Spain; 2Mitochondrial Pathology Unit, VHIR/Universitat Autònoma de Barcelona, Barcelona, Spain; 3Biomedical Network Research, Centre on Rare Diseases (CIBERER) ISCIII, Madrid, Spain and 4Department of Neurology, Columbia University, New York, NY, USA. Correspondence: Dr J Barquinero, Gene and Cell Therapy Laboratory, Vall d'Hebron Research Institute (VHIR)/Universitat Autònoma de Barcelona, Passeig Vall d'Hebron 119-129, 08035 Barcelona, Spain. E-mail: [email protected] 5These authors contributed equally to this work. Received 6 September 2013; revised 7 March 2014; accepted 26 March 2014; published online 8 May 2014 TP is both a therapeutic and a suicide gene in MNGIE S López-Estévez et al 674 5-fluorocytosine, respectively. This strategy has been used in combination of exogenous TP with 5-FU prodrugs has been used clinical trials to treat cancers and other conditions such as graft- experimentally as a form of SGT,13–15 with a documented versus-host disease.5–7 Capecitabine is an orally administered bystander effect.16 With this background, we hypothesised that À À chemotherapeutic agent used in the treatment of colorectal and the absence of TP activity in MNGIE patients (or in Tymp / À À metastatic breast cancers, as well as other cancer types. It is a Upp1 / dKO mice) would render virtually all their cells resistant TP-activated oral fluoropyrimidine that was rationally designed to to capecitabine because of inability to convert the prodrug into generate 5-fluorouracil (5-FU) preferentially within tumours. Upon 5-FU. One implication of this phenomenon is that capecitabine oral administration, this prodrug is rapidly absorbed in the will not be effective in treating tumours in MNGIE patients. On gastrointestinal tract as an inactive molecule. In the liver, it is the other hand, if a MNGIE patient is eventually treated with gene converted to 5′-deoxy-5-fluorocytidine by the action of a hepatic therapy and develops a tumour associated with the procedure carboxyl-esterase, which is subsequently metabolised to 5′-deoxy- (because of insertional mutagenesis), the therapeutic gene will 5-fluorouridine (doxifluridine, 5′-DFUR) by cytidine deaminase, also behave as a suicide gene, as the tumour will most likely derive and then to 5-FU by the TP expressed in the tumour, the liver or from transduced cells expressing TP (as opposed to the rest of the other normal tissues (Figure 1).8,9 When present, intracellular tissues in the organism), a unique scenario that will make tumour TP can convert 5-FU to 2'-deoxy-5-fluorouridine (2'-DFUR), which is cells selectively sensitive to the prodrug. Herein, we provide further phosphorylated by thymidine kinase 1 to the active evidence that both in vitro and in the murine model of MNGIE, the molecule fluorodeoxyuridine monophosphate, which inhibits therapeutic gene can also behave as a suicide gene. These results thymidylate synthase and disrupts DNA synthesis.10 On the other can most likely be extrapolated to MNGIE patients undergoing hand, 5-FU can undergo a series of enzymatic steps that result in gene therapy in the hypothetical scenario that they develop the generation of fluorouridine triphosphate, a false metabolite for tumours in association with the therapy. Thus, this form of SGT RNA synthesis. 5-FU catabolism is mainly due to the action of may represent a safeguard in cases of oncogenicity, although at dihydropyrimidine dehydrogenase, which is abundant in the liver. the potential expense of elimination of all gene-corrected cells, The relative selectivity of capecitabine in cancer partially derives thereby neutralising the therapeutic effect. However, this would from the fact that most tumours naturally overexpress TP, which probably be an acceptable side effect if the priority is to save the acts as an angiogenic factor, primarily through the production of patient's life, as gene therapy can be performed again later on. 2-deoxy-D-ribose.11 In most tumours, TP has a dual role. Although Moreover, we also show that the use of this murine model its angiogenic activity (mainly due to the release of 2-deoxy-D- provides an optimal tool to investigate various aspects of SGT with capecitabine, such as local and distant bystander effects or ribose) favours tumour growth, its overexpression sensitises the potential synergies between chemotherapy and the anti-tumour cells to the toxic effects
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