Therapy (2011) 18, 795–806 & 2011 Macmillan Publishers Limited All rights reserved 0969-7128/11 www.nature.com/gt

ORIGINAL ARTICLE Hematopoietic gene therapy restores thymidine activity in a cell culture and a murine model of MNGIE

J Torres-Torronteras1,2,AGo´mez3, H Eixarch3,6, L Palenzuela1,2,6, G Pizzorno4, M Hirano5, AL Andreu1,2, J Barquinero3 and R Martı´1,2

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the TYMP gene, which encodes (TP). TP dysfunction results in systemic thymidine (dThd) and deoxyuridine (dUrd) overload, which selectively impair mitochondrial DNA replication. Allogeneic hematopoietic transplantation has been used to treat MNGIE patients; however, this approach has serious adverse effects, including the toxicity of myeloablative conditioning, graft rejection and graft-versus-host disease. With the aim of testing the feasibility of gene therapy for MNGIE, we transduced TP-deficient B-lymphoblastoid cells from two MNGIE patients, with lentiviral vectors carrying a functional copy of the human TYMP DNA coding sequence. This restored TP activity in the cells, which reduced the excretion of dThd and dUrd and their concentrations when added in excess. Additionally, lentiviral-mediated hematopoietic gene therapy was used in partially myeloablated double Tymp/Upp1 knockout mice. In spite of the relatively low levels of molecular chimerism achieved, high levels of TP activity were observed in the peripheral blood of the transplanted mice, with a concomitant reduction of nucleoside concentrations. Our results suggest that hematopoietic gene therapy could be an alternative treatment for this devastating disorder in the future. Gene Therapy (2011) 18, 795–806; doi:10.1038/gt.2011.24; published online 31 March 2011

Keywords: MNGIE; lentiviral vector; thymidine phosphorylase; TYMP; mitochondria

INTRODUCTION micromolar concentrations in MNGIE patients.6–8 This is relevant Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is from a pathogenic point of view as in vitro and in vivo studies a multisystemic disease caused by autosomal recessive mutations in have demonstrated that an excess of dThd induces an increase of the TYMP gene encoding thymidine phosphorylase (TP).1 The main thymidine triphosphate (dTTP), which interferes with the clinical features of MNGIE are progressive external ophthalmoplegia, correct replication of mitochondrial DNA, causing mitochondrial severe gastrointestinal dysmotility, cachexia, peripheral neuropathy, dysfunction.9,10 diffuse leukoencephalopathy on brain magnetic resonance imaging Therapeutic approaches have so far focused on reducing the and evidence of mitochondrial dysfunction (histological, biochemical systemic overload of these nucleosides.7,11–14 In recent years, several and genetic abnormalities of mitochondria).2 Patients usually die from MNGIE patients have been treated with allogeneic hematopoietic stem complications of their gastrointestinal problems and their critical cell transplantation (HSCT).4,12,15,16 After successful HSCT, donor- nutritional status, with an average life expectancy of 37 years.2,3 derived nucleated blood cells and platelets provided enough TP MNGIE is a rare disease, with fewer than 200 patients known to be activity to reduce the dThd and dUrd concentrations to undetectable affected worldwide, but the true incidence of the disease is unknown or nearly undetectable levels, and subsequent slowing of disease and calculations based on known cases may lead to underestimations progression or mild clinical improvement.4,12,15,16 However, this because MNGIE patients are often misdiagnosed.4 approach has several limitations, which include the high morbidity TP catalyzes the phosphorolysis of thymidine (dThd) or and mortality of allogeneic HSCT, and the difficulty of obtaining deoxyuridine (dUrd) nucleosides to the corresponding bases, thymine suitable donors for a high proportion of patients. In MNGIE patients, or uracil, and deoxyribose-1-phosphate.5 In MNGIE patients, muta- HSCT implies additional difficulties. MNGIE patients are generally in tions in TYMP markedly reduce TP activity, resulting in a systemic poor medical condition, with limited capacity to tolerate transplant- accumulation of dThd and dUrd. Plasma and tissue dThd and dUrd, related complications. The gastrointestinal function is disturbed which are undetectable (o0.05 mM) in normal individuals, reach in most cases, with potential impairment of absorption, so that

1Laboratori of Neuromuscular and Mitochondrial Disorders, Institut de Recerca Hospital Universitari Vall d’Hebron, Universitat Auto` noma de Barcelona, Barcelona, Spain; 2Biomedical Network Research Centre on Rare Diseases (CIBERER) ISCIII, Spain; 3Banc de Sang i Teixits, Institut de Recerca Hospital Universitari Vall d’Hebron, Universitat Auto` noma de Barcelona, Barcelona, Spain; 4Department of Drug Development, Nevada Cancer Institute, Las Vegas, NV, USA and 5Department of Neurology, Columbia University Medical Center, New York, NY, USA Correspondence: Dr R Martı´, Laboratori de patologia mitocondrial, Institut de Recerca Hospital Universitari Vall d’Hebron, Pg. Vall d’Hebron 119, Barcelona 08035, Spain. E-mail: [email protected] 6Current address: Centre d’Esclerosi Mu´ ltiple de Catalunya, Unitat de Neuroimmunologia Clı´nica (HE) and Servei de Medicina Interna-Hepatologia (LP), Institut de Recerca Hospital Universitari Vall d’Hebron, Barcelona, Spain. Received 29 August 2010; revised 28 December 2010; accepted 29 January 2011; published online 31 March 2011 Gene therapy corrects TP deficiency J Torres-Torronteras et al 796

parenteral application of drugs is needed. In addition, drugs with (Figure 2a). Transduction with p305-TP increased three- to sixfold possible mitochondrial toxicity must be avoided.4 the levels of TYMP mRNA in controls and P1. Surprisingly, B-LCLs of As occurs with many monogenic disorders that can benefit from an P2 transduced with p305-TP showed reduced TYMP mRNA levels allogeneic HSCT, MNGIE is probably a very good candidate for relative to untransduced and sham-transduced cells. Levels of TP hematopoietic stem cell gene therapy (HSCGT). Robust and stable protein were similar in non-transduced or sham-transduced B-LCLs TP expression in hematopoietic cells should clear the systemic from controls, and were undetectable or barely detectable in patients’ accumulation of dThd and dUrd, as observed in MNGIE patients cells (Figures 2b and c). p305-TP transduction significantly increased undergoing an allogeneic HSCT.12 Here, we show that gene transfer TP protein levels, which paralleled increases observed in TYMP mRNA using lentiviral vectors results in stable TP activity in cell lines from in controls and in P1. In P2, p305-TP induced low, but clearly MNGIE patients and in a double knockout (Tymp À/À; Upp1À/À) measurable levels of TP protein, which were undetectable in their murine model of the disease,10 reverting dThd and dUrd overload to non-transduced and sham-transduced lines. normal levels. Our results suggest that gene therapy is a promising therapeutic approach for MNGIE. p305-TP restored TP activity in TP-deficient cells Figure 3 shows the results for TP activity in cell lines from patients and RESULTS controls. activity was undetectable or barely detectable in Transduction of TP-deficient B-lymphoblastoid cell lines (B-LCLs) B-LCLs from MNGIE patients (below 5 nmol of thymine formed We generated B-LCLs through Epstein–Barr virus (EBV) infection of hÀ1 per mg protein), and between 550 and 1100 nmol hÀ1 per mg lymphocytes from two MNGIE patients (P1 and P2) and two normal protein in B-LCLs from controls. TP activity was strongly increased in controls (C1 and C2). These B-LCLs were transduced with a lentiviral B-LCLs transduced with p305-TP. A strong correlation was observed vector containing the TYMP DNA coding sequence (cDNA) between TP protein levels and TP activity, and the linear distribution of (p305-TP) or the same vector without the TYMP cDNA (p305- the data in this representation suggests that both transgenic and sham) (Figure 1a). Both vectors contained human phosphoglycerate endogenous TP proteins have similar enzymatic Vmax,asourenzyme kinase (hPGK) promoter and also included the sequence encoding the assay is performed at substrate saturation. TP activity gained after TP enhanced green fluorescent protein (EGFP) as a marker gene (see transduction in B-LCLs (TP activity in TP-transduced cellsÀTP activity Materials and Methods). in sham-transduced cells) was directly proportional to the average Flow cytometry analyses revealed variable levels of transduction in transgenic TYMP DNA copy number per cell (r¼0.978; P¼0.022, B-LCLs derived from MNGIE patients and controls (Figure 1b). Cell Pearson correlation). We also assessed the effects of TP transduction sorting was performed to enrich cell lines in EGFP+ cells. TP- and on HEK293T cells. Although non-transduced and sham-transduced sham-transduced cells from P2 did not survive after sorting, which HEK293T cells had undetectable TP activity, after transduction with prompted us to use unsorted transduced B-LCLs from patients and p305-TP the activity reached 6345 nmol hÀ1 per mg protein. EGFP+-sorted cell population from control B-LCLs for further experi- Both endogenous and transgenic TP activity prevented or limited ments. Transduction efficiencies were studied by measuring the copy dThd and dUrd accumulation in the culture medium (Figure 4). number of TYMP and EGFP sequences in transduced cells, using Extracellular concentrations of dThd and dUrd increased with cul- quantitative real-time PCR (qRT-PCR) (Table 1). For p305-TP-trans- tured TP-deficient cells over time, whereas control and p305-TP- duced cell lines, post-sorted C1, C2 and pre-sorted P1 showed values transduced cells (patients and controls) showed a very low excretion between 1.1 and 1.7 average TYMP copies per cell, and only 0.12 or catabolic reduction of these nucleosides in the culture medium copies per cell for pre-sorted P2. Similar results were obtained for the (Figures 4a–c). The addition of 10 and 20 mM of exogenous dThd and copy number of the EGFP gene, consistent with the fact that the p305- dUrd to the medium (mimicking extracellular concentrations TP vector carries one TYMP copy per EGFP copy. Higher averages observed in MNGIE patients17) showed that TP-deficient cells are of EGFP copy numbers per cell were observed, indicating higher unable to catabolize an excess of nucleosides, but this catabolic ability efficiencies of transduction in sham-transduced cells relative to was restored after transduction with p305-TP (Figures 4d–f). In the TP-transduced B-LCLs. These differences were not observed when case of B-LCLs from patient 2, which achieved only moderate TP transduction was assessed by flow cytometry (Figure 1b), probably activity after p305-TP transduction (Figure 3), the catabolic reduction because of a low level of EGFP expression of the p305-sham vector was less pronounced, but it was still noticeable compared with the when compared with that of p305-TP. This notion was supported by non-transduced or sham-transduced counterparts. the observation of lower EGFP mRNA levels per EGFP DNA copy in sham-transduced cells as compared with TP-transduced cells p305-TP-mediated TYMP expression did not induce cytotoxicity (Table 1). As p305-TP transduction in C1, C2 and P1 B-LCLs resulted in high TP activities, well above those observed in non-transduced B-LCLs TYMP mRNA and TP protein levels were increased in from controls, we investigated whether this increase could result in TP-transduced cell lines dTTP depletion. Thus, we determined deoxyribonucleoside tripho- To determine whether the integrated p305-TP vector can efficiently sphate amounts in cellular extracts, including the HEK293T cell line. drive TYMP expression in B-LCLs, levels of TYMP mRNA and TP Although we did not observe dTTP depletion in B-LCLs from patients protein in cell homogenates were quantified. Analyses were performed or controls overexpressing TYMP after p305-TP transduction, TP- at least 2 weeks after transduction to avoid possible interference from transduced HEK293T cells had a significant reduction (B40%) of the non-integrated vector expression. TYMP mRNA levels were measured dTTP percentage (Figure 5a). To determine whether this biochemical by qRT-PCR and normalized to cyclophilin A (PPIA) mRNA, which imbalance was associated with alterations in the cell cycle status, we was homogenously expressed (data not shown). Untransduced and analyzed cell cycle by flow cytometry of Hoechst-stained non-trans- p305-sham-transduced B-LCLs showed similar levels of TYMP mRNA duced, sham-transduced and TP-transduced cells (Figure 5b). No in both control and patients’ cell lines, except for non-transduced significant changes in the percentage of proliferating cells (S+M B-LCLs from P1, which had lower amounts of TYMP mRNA phases) after TP transduction or sham transduction were detected,

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Figure 1 Transduction efficiency in B-LCLs. (a) Schematic representation of the lentiviral vectors. p305-TP, p305-sham and p-sham vectors and their elements. RRE, rev-responsive element; cPPT, central polypurine tract; hPGK, human phosphoglycerate kinase promoter; TYMP, coding sequence of the TYMP gene; IRES, internal ribosome entry site; eGFP, enhanced green fluorescent protein; WPRE, woodchuck hepatitis post-transcriptional regulatory element; LTR, long terminal repeat. Both circular vectors are schematically represented as linear forms. (b) Flow cytometry analysis of B-LCLs obtained from two controls (C1, C2) and two MNGIE patients (P1, P2) transduced at 15 MOI and cultured for at least 10 days before analysis (pre-sorted) and 10 days after sorting of EGFP+ cells (post-sorted). The x axis shows green fluorescence intensity, y axis shows cell counts. Open histograms: non-transduced cell lines used as negative controls. Filled histograms: transduced cell lines. either in B-LCLs or in HEK293T cells. However, slightly higher Stability of transgenic TYMP percentages of proliferating cells were observed in EGFP+ subpopula- Figure 6 shows the stability of TP activity provided by p305-TP tions (cells effectively transduced) than in EGFPÀ subpopulations, vector in B-LCLs from P1. At early passages after transduction, although this difference was not statistically significant. This trend was cells increased TP activity to 1490 nmol thymine hÀ1 per mg protein, observed in both TP-transduced and sham-transduced cells, indicating compared with undetectable TP in sham and untransduced B-LCLs that this effect was TP independent. (Figures 3a and 6a). Although cell counts were not performed,

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Table 1 Transduction efficiencies and EGFP expression in B-LCLs

+ Cell line Vector EGFP cells (%) EGFP DNA copy number TYMP DNA copy number EGFP mRNA

per cell per transduced cell per cell per transduced cell referred to PPIA mRNA referred to EGFP DNA copy number

C1 p305-TP 96 1.43 1.49 1.46 1.52 2.37 1.66 p305-sh 97 2.86 2.95 UND — 0.83 0.29 C2 p305-TP 98 1.43 1.46 1.74 1.78 1.21 0.85 p305-sh 33 3.15 9.55 UND — 0.32 0.10 P1 p305-TP 31 0.89 2.87 1.10 3.55 1.00 1.12 p305-sh 31 2.23 7.19 UND — 0.71 0.32 P2 p305-TP 6 0.10 1.67 0.12 2.00 0.08 0.80 p305-sh 3 0.17 5.67 UND — 0.06 0.38

Abbreviations: B-LCLs, B-lymphoblastoid cell lines; EGFP, enhanced green fluorescent protein; sh, sham; TP, thymidine phosphorylase; UND, undetectable. Percentages of EGFP+ cells were determined by flow cytometry. EGFP and TYMP DNA copies were assessed by quantitative real-time PCR (qRT-PCR), referred to the single-copy gene RNase P, and expressed as average copy number per cell, or per transduced cell. EGFP mRNA levels were determined by qRT-PCR, referred to endogenous levels of PPIA mRNA (cyclophilin A) and shown as fold increase referred to the result obtained for the P1 transduced with p305-TP. In addition (last column), this value was referred to the EGFP DNA copy number.

Figure 2 Expression of TP. (a) TYMP mRNA levels in B-LCLs. Values are normalized to the endogenous PPIA gene mRNA (cyclophilin A) levels and shown as fold increase with respect to the result obtained for the P1 transduced with p305-TP. Data are expressed as mean of two independent determinations on the same cDNA. (b) Representative western blot showing the amounts of TP protein in B-LCL extracts. nt, non-transduced cell lines; sh and tp, cell lines transduced with p305-sham and p305-TP, respectively. (c) Quantitative levels of TP protein obtained by densitometry; all values are normalized to b-actin and shown as fold increase referred to the result obtained for a calibrator protein extract (cal). Results are expressed as the mean (±s.d.) of three independent experiments (three different protein extracts). Asterisks indicate statistically significant differences between TP transduced as compared with both non-transduced and sham transduced (*Po0.05; **Po0.001; one-way analysis of variance with Bonferroni correction for multiple comparisons).

microscopic observation of the culture and stability of the medium We additionally used the immortalized HEK293T cell line to study color indicated that growth rate suddenly decreased between 4 and 16 long-term transgene expression in p305-TP-transduced cells. As shown weeks, and TP activity almost disappeared. Growth rate and in Figure 6c, non-transduced and sham-transduced HEK293T cells had TP activity recovered after week 16, reaching stable values of around barely detectable TP activity (around or below 5 nmol thymine hÀ1 per 5000 nmol thymine hÀ1 per mg protein at week 28 and over mg protein), whereas p305-TP-transduced HEK293T cells achieved 17 subsequent weeks (Figure 6a). In parallel, the percentages of activities of 5000 nmol hÀ1 per mg protein or higher, which were EGFP+ cells initially declined and, after week 16, increased maintained over at least 15 weeks in culture. The percentage of EGFP+ (Figure 6b). In this experiment, sham-transduced cells did not cells was also stable (Figure 6d), around 70%, suggesting that there was survive beyond week 20, probably because true immortalization by not any selective advantage for p305-TP-transduced cells due to EBV was not achieved. differential survival or differential rates of growth in culture.

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that show higher TP activities.10 Circulating dThd and dUrd concen- trations in untreated and sham-treated double KO mice were between two- and fivefold higher than those observed in the wt mice. After treatment with the p305-TP vector, double KO mice reduced their plasma dThd and dUrd concentrations to levels in the range of wt mice (Figures 7b and c). In fact, plasma dThd levels in TP-treated double KO mice were lower than those of wt mice (median 3.3, range 2.0–4.2 mM for wt, versus 1.6 and 0.7–2.9 for TP-treated double KO). In order to assess the stability of TP expression, TP activity was monitored over 29 weeks after transplantation in blood samples of the mice. Figure 7d shows that, after a moderate decline of TP activity between the fourth and eighth weeks, all eight TP-treated mice maintained high TP activities up to 29 weeks after the transplantation, more than 20-fold higher than those of wt animals. At week 25, gene- marking levels based on EGFP fluorescence ranged between 1.0 and 7.5%, (Figure 7e) indicating that low levels of molecular chimerism are able to provide high TP activity in vivo. At this point, TP activity did not significantly correlate with molecular chimerism levels (percentage of EGFP+ cells) in PB, but it did with bone marrow chimerism (P¼0.038; Supplementary Figure 1). The sham group (Figure 7f) showed higher percentages of trans- duction in vivo, and three out of eight mice showed a progressive increase of the %EGFP+ cells between weeks 8 and 26. These increases and those observed in the TP-treated group were not associated with apparent hematopoietic malignant transformation in these mice. Absolute and differential blood cell counts performed in all the animals 29 weeks after treatment were within the normal range for C57BL/6J mice (Peters LL. Aging study: Blood hematology in 31 Figure 3 TP activity. (a) TP activity in B-LCLs from controls and MNGIE inbred strains of mice. MPD: Peters4. Mouse Phenome Database web patients. Results are expressed as mean (±s.d.) of four different protein extracts. Asterisks indicate statistically significant differences between TP site, The Jackson Laboratory, Bar Harbor, ME, USA. http://pheno- transduced and either non-transduced or sham transduced (**Po0.001; me.jax.org, December 2010) and did not reveal different distributions one-way analysis of variance with Bonferroni correction for multiple of lineages between TP-treated (lymphocytes 84.4±3.1%; granulo- comparisons). (b) Linear correlation (Spearman) between relative TP protein cytes 12.5±1.9%; monocytes 3.2±1.6%; N¼8), sham-treated amount (data from Figure 2c) and TP activity (data from a). (lymphocytes 82.6±6.6%; granulocytes 14.6±6.2%; monocytes 2.8±0.7%; N¼10), KO (lymphocytes 85.3±1.4%; granulocytes 12.1±1.4%; monocytes 2.7±0.5%; N¼8) and wt animals (lympho- p305-TP provided high levels of TP activity and reduced nucleoside cytes 83.1±3.1%; granulocytes 14.0±2.2%; monocytes 2.9±1.1%; overload in vivo N¼9). In addition, increased TP activity did not affect the ability of To test the efficacy of our lentiviral vector in vivo,weusedadouble the hematopoietic stem cells to differentiate into multiple lineages. Tymp/Upp1 knockout murine model.10 As the in vitro study had Flow cytometric analysis of two TP-treated mice’s PB 33 weeks after shown that EGFP was poorly expressed by the p305-sham vector treatment revealed that all lineages analyzed (CD11b+,Gr-1+,CD19+ (Figure 1b and Table 1), we decided to use a modified version of the and CD4/8a+) contributed to the EGFP+ cell subpopulation (data not sham vector in the animal model (p-sham), identical to p305-sham shown). but lacking the internal ribosomal entry site (IRES) (Figure 1a). Previous work in our laboratory showed that this vector resulted in DISCUSSION higher EGFP expression levels than p305-sham. Immunoselected In this work, we show that TP can be restored in human TP-deficient hematopoietic lineage negative (LinÀ) cells from double KO mice cells and that HSCGT using lentiviral vectors can ameliorate the were transduced with our p305-TP or p-sham vector. Flow cytometric metabolic abnormalities in a murine model of MNGIE. All therapeu- analysis revealed p305-TP transduction efficiencies of 26–28% in two tic strategies have been aimed at normalizing the accumulation of the different extractions of donor cells, and 16–18% for the corresponding toxic metabolites dThd and dUrd in patients.7,13,14 The observation p-sham transductions. Transduced cells were infused into partially that platelet transfusions transiently reduced the levels of dThd and myeloablated syngenic double KO mice. Four weeks after transplanta- dUrd in MNGIE patients,18 suggested that HSCT from healthy donors tion, the level of gene marking in the peripheral blood (PB) of could provide a permanent source of TP at levels, producing a TP-transplanted mice (n¼8) ranged between 2.0 and 10.5%, with a significant clinical benefit. Patients undergoing a successful allogeneic mean copy number per transduced cell ranging from 0.5 to 1.5. As HSCT show permanent restoration of TP activity with correction of shown in Figure 7a, high TP activities (median: 11.3 nmol thymine biochemical abnormalities and progressive improvements in clinical hÀ1 per mg protein, range: 7.6–13.3) were achieved in PB cells of parameters.4,12,15,16 However, allogeneic HSCT is not an ideal therapy treated mice, as compared with undetectable or negligible values in because of toxicity of the conditioning regimen and the risk of severe untreated and sham-treated double KO. Interestingly, wild-type (wt) complications, such as graft failure and graft-versus-host disease.19 mice had detectable but very low TP activity in the blood cell fraction We hypothesized that HSCGT using autologous cells could (median: 0.04; range: undetectable to 0.10), in contrast to other tissues overcome some of these limitations, and represents a reasonable

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Figure 4 Metabolism of dThd and dUrd. B-LCLs were seeded in medium with dialyzed fetal bovine serum and maintained for 4 days in culture without any medium exchange; culture medium was then recovered to measure dThd and dUrd excretion (shown as nucleoside concentration in the medium) in non- transduced (a), p305-sham-transduced (b) and p305-TP-transduced (c) cultures. dThd and dUrd catabolism was studied for non-transduced (d), p305- sham-transduced (e) and p305-TP-transduced (f) B-LCLs in a similar experiment in which 10 mM dThd and 20 mM dUrd were added to the medium at day 0. Results are expressed as mean of two independent experiments, which are represented as vertical bars (not s.d.).

therapeutic alternative for MNGIE patients. To this aim, we have used transduction by the p305-sham vector, this difference could be lentiviral-mediated gene transfer to demonstrate the feasibility of explained if we take in account that EGFP was expressed at lower restoring TP activity in cellular models, and in vivo in a murine levels in the p305-sham vector, as compared with the p305-TP vector. model of the disease. HSCGT has already been proven successful in a Therefore, the titrations to obtain batches of p305-sham vectors and number of human disorders,20–22 but these successes were partially the multiplicity of infection (MOI) used for the p305-sham transduc- overshadowed by the observation of severe adverse effects (insertional tions were most likely underestimated. To avoid this problem, we used oncogenesis) in some of the patients.23,24 In order to minimize such a modified version of the sham vector lacking the IRES for the animal risks, novel integrative vectors, which show increased safety, have been studies. developed and some of them have already been used in clinical trials.25 We used B-LCLs from two patients. P1 was a compound hetero- Two main strategies are currently under investigation to minimize zygote for a mutation involving the splicing of intron 2 (c.215-1G4C) potential risks and increase the biosafety profile of vectors. One is the and a missense mutation in the exon 7 (c.886A4C),18 and P2 was use of lentiviral instead of g-retroviral vectors, as they were shown to homozygote for an in-frame 18-bp duplication in the exon 8 be less prone to integrate near oncogenes and near their origin of (c.994_1011dup).28 In both cases, negligible or undetectable levels of transcription.26,27 A second strategy is the use of vectors with self- TP protein were observed in cell homogenates from non-transduced inactivating (SIN) configuration, which greatly reduces the transacti- and sham-transduced lines, even though they showed similar TYMP vating potential of the viral long terminal repeat sequences.26 mRNA levels as controls (except for non-transduced B-LCLs from P1, We used EBV transformation to obtain B-LCLs as cellular models which had lower mRNA levels). The presence of mRNAs and absence for our study. We transduced B-LCLs from two MNGIE patients and of both TP protein and enzyme activity suggest that either mutant two controls, and HEK293T cells with our TP vector, and observed mRNAs are not translated or that aberrant TP protein is rapidly significant transduction efficiencies and good expression levels of degraded. Transduction with TP resulted in significant increases in functional TP. Only in one case (P2), we observed low levels of TYMP mRNA levels in C1, C2 and P1, whereas lower transcript levels transduction, due to unknown reasons. However, this low efficiency were observed in P2. The reason for this unexpected reduction is was sufficient to provide a TP activity of a third of the normal value unknown, but it could be partly explained by a decrease in the for B-LCLs from healthy controls. Overall, the TP activities obtained transcription of the endogenous TYMP gene in response to the were directly proportional to the transduction efficiency. Higher copy transcription of the transgene. This putative repression would be numbers were detected in the p305-sham-transduced lines as com- masked in C1, C2 and P1 by the higher levels of transgenic TYMP pared with those TP transduced. Rather than a more efficient mRNA, which is indistinguishable from the endogenous mRNA.

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Figure 5 Deoxyribonucleoside triphosphates (dNTPs) in cell homogenates and cytotoxicity. (a) Percentages of dTTP in cell homogenates. Cultured cells were lysed and dNTPs extracted from the homogenates. dATP, dGTP, dCTP and dTTP concentrations were determined. The results are expressed as the percentage of dTTP on the total dNTP pool (dTTPÂ100/(dATP+dGTP+dCTP+dTTP)). The mean (±s.d.) of three independent experiments is depicted. Asterisk indicates statistically significant differences between TP transduced and either non-transduced and sham transduced (*Po0.01; one-way analysis of variance with Bonferroni correction for multiple comparisons). (b) Percentages of proliferating cells (S+M phases) as assessed by flow cytometry of Hoechst 33342-stained cells. Bars represent the percentages of proliferating cells in non-transduced cultures (open bars), and those observed in the EGFP+ (gray bars) and EGFPÀ (black bars) cell populations. Numbers under the cell line label indicate the percentage of EGFP+ cells out of the total cells in the transduced cell line. Owing to the reduced number of EGFPÀ events in sham-transduced 293T and TP-transduced C2, no reliable results for this fraction could be obtained in these cell lines (asterisks).

Figure 6 Stability of transgenic TP expression. Monitoring of transgenic TP activity and the percentage of EGFP+ cells in B-LCLs from P1 (a, b), and HEK293T cells (c, d) after transduction. Cells were maintained in culture and samples were collected for TP activity assay and flow cytometry analysis. TP activity is expressed as 103 nmol of thymine formed hÀ1 per mg protein. When present, error bars indicate duplicate results obtained from the same homogenate (not s.d.). Note different scales for different series in plots a and c. TP activities close or below 5 nmol thymine hÀ1 per mg protein are detectable but barely quantifiable. Note that sham-transduced B-LCL from P1 stopped proliferating and did not survive beyond week 20.

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Figure 7 Effect of p305-TP in vivo.(a, b and c): Effect of HSCGT in a Tymp/Upp1 double KO mice, 1 month after transplantation. Box plots represent the median (horizontal line), inter-quartile range (box) and minimum and maximum (whiskers), except outliers, which are depicted as dots. WT, untreatedwild- type mice; KO, untreated TympÀ/À/Upp1À/À mice; SHAM, p-sham-treated TympÀ/À/Upp1À/À mice; TP, p305-TP-treated TympÀ/À/Upp1À/À mice. (a)TP activity in the PB cell fraction. (b) plasma dThd, and (c) plasma dUrd. Open triangles indicate significant differences with respect to wt mice (Po0.01) and open stars indicate significant differences with respect to KO and sham-treated mice (Po0.01). Note the discontinuity of the y axis scale of a.(d, e and f): Long-term monitoring in vivo obtained from peripheral mice’s blood (weeks 4 to 26) or from intracardiac puncture blood (dashed lines, week 29). Values for each animal are depicted in individual lines with individual symbols. Some symbols are slightly displaced at the last time points to facilitate identification. (d) TP activity in p305-TP treated mice. The gray area at the bottom indicates the range of TP values observed in n¼15 untreated wt mice (undetectable for untreated and p-sham-treated TympÀ/À/Upp1À/À mice). (e) Percentage of EGFP+ cells in p305-TP-treated mice. (f) Percentage of EGFP+ cells in p-sham-treated mice.

In fact, reduced TYMP mRNA observed after p305-TP transduction in Transgenic TP activity was stable in HEK293T cells over at least 15 P2 was accompanied by the expression of TP protein and TP activity, weeks. Our long-term TP expression studies in the B-LCL model were both undetectable in the corresponding sham-transduced or non- partially affected by the special features of EBV transformation of transduced B-LCL from P2. B-lymphocytes. Several reports have demonstrated that true immor- The ratios between TP protein amounts and TP activities in B-LCL talization only occurs in some B-LCL clones that survive after long homogenates were similar for endogenous and transgenic TP, indicat- periods of proliferation in pre-immortal phases.29 Our experiments to ing that both forms of TP have similar Vmax. Transgenic TYMP assess lentiviral transduction and TP expression were performed in induced higher levels of protein than those observed in non-trans- B-LCLs with less than 100 culture passages; therefore, it is unlikely that duced control cells, probably due to a higher potency of the hPGK cell lines achieved true immortalization. TP-transduced B-LCLs from promoter as compared with the endogenous human TYMP promoter. P1 survived after a growth crisis and reached stable levels of TP activity This prompted us to test whether this excess of TP activity could lead for at least 17 weeks. In fact, the results depicted in Figures 6a and b to intracellular dTTP depletion, which would be a potentially deleter- indicate a clonal selection of p305-TP-transduced cells, probably ious effect. Significant dTTP depletion was observed only in the related with the EBV-induced immortalization process discussed HEK293T cells, probably because this cell line, with a virtually above,29 although we cannot rule out the possibility that this pro- absolute lack of TP activity, achieved the highest enzyme activity liferative advantage was induced by the increased TP activity or by a among all TP-transduced cell lines. On the other hand, flow cytometry vector insertion enhancing cell proliferation. Alternatively, a biased analysis of Hoechst-stained cells did not reveal detectable cytotoxicity transduction of rapidly proliferative cells that eventually predominate or abnormal proliferation associated to this partial dTTP depletion. in the cultures could also account for this observation. Although the Interestingly, for almost all cell lines, EGFP+ cell subpopulations molecular reasons for this clonal selection should be further investi- showed slightly higher percentages of proliferating cells than EGFPÀ gated, our results show that, at late passages, TP activity was high and subpopulations for both TP-transduced and sham-transduced cells, stable in this transduced B-LCL and HEK293T cells. Therefore, we can which could be reflecting the fact that cells or clones with higher conclude that our p305-TP vector was able to provide long-term proliferative potential were preferentially transduced. At the same expression of active TP to two different TP-deficient cell lines. time, TP-deficient lines recovered the capacity to catabolize an excess We used double Tymp/Upp1 KO mice to evaluate our therapeutic of exogenous dThd and dUrd after TP transduction, which is an tool in vivo because this model recapitulates the biochemical imbal- important goal from the therapeutic point of view. ances and some other features observed in MNGIE patients.10

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However, several differences in the pyrimidine deoxynucleoside ment of the gene-corrected hematopoietic cells. In this regard, regimes metabolism between human and mice have to be taken into account. based on chemotherapeutic agents as those successfully used in human First, both TP and contribute to the catabolism clinical trials can make the therapy safer. Our results suggest that the of dThd and dUrd in mice, whereas this role is played entirely (or level of correction required to obtain a therapeutic benefit is probably almost entirely) by TP in humans.30 Second, white blood cells and very low, and that, for therapeutic purposes, transgene expression platelets are among the richest tissues in TP activity in humans,8 does not seem to require a strict regulation, at least in the murine whereas in mice, blood cells have the lowest TP activity among all model tested. tissues analyzed.10 Third, normal dThd and dUrd plasma levels are In summary, we have generated a lentiviral vector capable of higher in mice (1–4 mM) than in humans (below 0.05 mM). Therefore, introducing a fully functional version of the TYMP gene in vitro HSCGT may constitute a more rational approach in MNGIE patients and in vivo. Transduction of human TP-deficient cells with this vector than in the murine model because restoration in humans would take provided stable expression of functional TP that catabolizes an excess place in a more relevant target cell type. of dThd and dUrd present in the extracellular medium. Overexpres- Our preliminary in vivo results demonstrate that p305-TP is fully sion of TYMP did not produce cytotoxicity in our conditions, and the functional as a potential therapeutic vector. We provided allotopic allotopic targeting of the gene in a Tymp/Upp1 double KO model restoration of TP activity to the mice and reached an average TP restored the correct balanced dThd and dUrd concentrations. Our activity around 11 nmol thymine hÀ1 per mg protein in blood cellular results strongly suggest that treatment of MNGIE with HSCGT using fraction, 1 month after transplantation, and high activities (between lentiviral vectors is feasible, and support carrying out long-term 2–30 nmol hÀ1 per mg protein) were maintained at least 29 weeks after studies in the animal model to ensure full correction of the phenotype transplantation. TP activity of blood cells significantly correlated with and the safety of the procedure. %EGFP+ bone marrow cells, but did not with %EGFP+ blood cells. The reasons for this discrepancy are unknown, but could be related MATERIALS AND METHODS with factors masking the correlation in PB but not in bone marrow. This study was approved by our institutional review board and animal care and For example, high white cell and platelet counts should be associated use committee. to higher TP activity, and our TP activity test does not control this variable because most of the protein homogenate comes from ery- Cell culture conditions and lymphocyte transformation throcytes, which presumably contain very low TP, and would mask the Peripheral blood mononuclear cells were obtained from two patients previously protein of TP-expressing cells. We measured TP in total blood cells diagnosed with MNGIE by clinical, biochemical and genetic criteria, after instead of buffy coat because of the limited volumes of blood available. informed consent. P1 was compound heterozygote for a missense mutation 18 Although reference values for circulating TP activity in humans have (c.886A4C), and a mutation in the splice site of intron 2 (c.215-1G4C). 28 been reported only for buffy coat,7,17 we have measured activities in P2 harbored a homozygous 18-bp duplication in exon 8 (c.994_1011dup). Peripheral blood mononuclear cells were obtained from both patients and two PB cells of healthy controls, which ranged between 4–8 nmol hÀ1 per healthy controls through Ficoll gradient centrifugation, and cultured in 3.2 ml mg protein (Torres-Torronteras and Martı´, unpublished results), of complete medium (RPMI-1640, Invitrogen, Carlsbad, CA, USA; 15% indicating that our vector has potential to restore circulating TP dialyzed heat-inactivated fetal bovine serum, Invitrogen; 10 mM 4-(2-hydro- 1 activity to normal levels in humans. This activity was sufficient to xyethyl)-1-piperazineethanesulfonic acid; 2 mML-glutamine; 1 U lÀ penicillin; maintain plasma dThd and dUrd concentrations within the normal 1UlÀ1 streptomycin; 50 mgmlÀ1 gentamicin), supplemented with 1.8 ml of range. dThd (but not dUrd) concentrations were moderately over- EBV-containing supernatant from cultured B95-8 marmoset cells. In all, reduced, but it is unlikely that this difference would produce detri- 1 mgmlÀ1 of anti-CD3 OKT3 (eBioscience, San Diego, CA, USA) was added mental effects, given the broad range of dThd levels in the wt mice, to avoid T-cell response. Cells were maintained in culture for 15–21 days and which partially overlaps that observed for the TP-treated double KO. subcultured in complete medium until cell clumps were visible. Flow cytometry This point will have to be analyzed further in the future. As the main analysis revealed that in all EBV-transformed lines, more than 90% of the cells were CD45+ and CD19+ (data not shown). HEK293T cells were cultured objective of a potential treatment for MNGIE is to reduce the systemic in Dulbecco’s modified Eagle’s medium containing 4.5 g lÀ1 of glucose overload of dThd and dUrd, these results strongly support further (Invitrogen), supplemented with 10% heat-inactivated fetal bovine serum, À1 À1 studies of this therapy in this animal model. This will allow us to 2mML-glutamine, 1 U l penicillin and 1 U l streptomycin. address several questions raised by our results. For example, pro- + nounced increases of blood TP activity and/or %EGFP cells were Animal model observed in some mice 26–29 weeks after the treatment, although A Tymp1/Upp1 double KO murine model in C57BL/6J genetic background these increases were not associated to apparent hematopoietic malig- previously characterized 10 was used for in vivo tests. Two separated reproduc- nant transformation. This and other important issues will have to be tive groups were segregated: homozygous TympÀ/À/Upp1À/À (KO group) and clarified with more specific tests in an extended follow-up. homozygous Tymp+/+/Upp1+/+ (wt group). Both inbred groups had isogenic Unlike most hereditary diseases, TP deficiency constitutes a unique background, except for the Tymp/Upp1 genotype. situation for several reasons. The molecular defect can be corrected, theoretically, in any tissue or cell type, and the enzyme does not need Vector construction to be secreted because its substrates are small diffusible molecules. The IRAT p970B1069D6 clone from IRAT MGC Human-verified full-length Moreover, the correction of a limited number of cells providing cDNA library, containing the human TYMP full cDNA (NCBI accession enough TP activity can be sufficient to clear the systemic overload BC052211) was purchased from RZPD (Berlin, Germany). The coding frag- ment was amplified by PCR, excluding the polyA tract (forward primer: 5¢-GA of nucleosides. We used bone marrow cells as target and sublethal total ACCCTGAACCCTACGGTC-3¢ and reverse: 5¢-CGCGGCAAAGGAGCTTTA body irradiation as a conditioning regimen before transplantation. As TT-3¢). A high fidelity polymerase (Platinum Taq DNA polymerase, Invitrogen) transduced cells expressing TP are not expected to have a selective was used to reduce the rate of amplification errors. PCR was carried out as advantage, as opposed to what occurs in patients with adenosine follows: one denaturation step at 94 1C 2 min; 25 cycles at 94 1C30s,551C30s, deaminase-deficient severe combined immunodeficiency,20 we think 681C 100 s; and one final extension step at 68 1C 7 min. The amplified cDNA that some amount of myeloablation is required to facilitate engraft- was cloned into pCR4-TOPO using TOPO TA Cloning kit for sequencing

Gene Therapy Gene therapy corrects TP deficiency J Torres-Torronteras et al 804

(Invitrogen), and sequenced with BigDye and ABI-3100 Genetic Analyzer Brea, CA, USA). Data was analyzed using the FCS Express version 3 software (Applied Biosystems, Foster City, CA, USA). We flanked the TYMP cDNA (De Novo Software, Los Angeles, CA, USA). with the appropriate restriction targets for further directional cloning into the PB cell differential counts were performed using a BC-2800 Auto lentiviral vector (EcoRV at 5¢ and XbaIat3¢) by subcloning into pBluescript SK Hematology analyzer (Mindray, Mahwah, NJ, USA). plasmid. A clone with the insert in the correct orientation was selected and digested with EcoRV (blunt ends) and XbaI (New England Biolabs, Ipswich, Assessment of DNA copy number and mRNA levels by qRT-PCR MA, USA) to obtain the TYMP cDNA fragment ready to be cloned into the qRT-PCR determinations were performed using pre-designed or custom- vector. The self-inactivated lentiviral vector pCCL.sin.PPT.hPGK.deltaNGFR. designed TaqMan assays in the ABI PRISM 7500 sequence detection system IRESemvcv.wt.eGFP.Wpre (p305) was kindly provided by Dr L Naldini (San (Applied Biosystems). For copy number quantification of specific DNA Raffaele Telethon Institute for Gene Therapy, Milano, Italy). We removed the sequences, total DNA was obtained by phenol–chloroform extraction from deltaNGFR fragment by SmaI (blunt ends) and XbaI digestion, and introduced 107 cells. Transgenic TYMP cDNA copy number (integrated in the genome our TYMP cDNA fragment to obtain our active vector (p305-TP). We obtained from p305-TP transduction) was quantified using the pre-designed TaqMan a sham vector (p305-sham) by simply removing the deltaNGFR fragment MGB assay Hs00157317_m1 (Applied Biosystems), which through BamHI digestion and re-ligation. The p305-sham vector was used for amplifies a 95-nucleotide amplicon covering the exon 4–5 junction. Transgenic the in vitro study in cultured cells. A different sham vector, identical to p305- TYMP cDNA is detected by this assay because it does not contain intronic sham but lacking the IRES (p-sham) was kindly provided by L Naldini, and sequences. EGFP gene copy number (integrated in the genome from p305-TP, used for the animal studies. Previous work in our laboratory showed that this p305-sham or p-sham transduction) was quantified using custom-designed vector resulted in higher EGFP expression levels than p305-sham. Figure 1a specific primers and TaqMan MGB probe, as previously described.31 Both shows the representation of p305-TP (containing the lentiviral backbone, the TYMP cDNA and EGFP copy numbers were normalized to the copy number of human phosphoglycerate kinase, hPGK, promoter to drive the bicistronic human RNase P gene (TaqMan RNase P detection reagents kit) in B-LCLs, and expression of TYMP and EGFP), the p305-sham and the p-sham. No mutations to the copy number of murine Ang1 gene (pre-designed TaqMan MGB gene were detected in the TP-coding region, and correct orientation of integration expression assay, Id assay Mm00833184_s1) for murine samples. As both RNase was confirmed by direct sequencing, as compared with the wt sequence number P and Ang1 are single copy (two copies per diploid cell), we could express NM_001953.3 in the NCBI (data not shown). the results as copy number per cell. In all experiments, the quantification was based on a standard curve prepared with different dilutions of DNA from a control transduced with p305-TP (which contains the same number of copies Vector production, titration and transduction of TYMP and EGFP) or with a plasmid containing the RNAse P or Ang1 Lentiviral vectors were produced by transient co-transfection of HEK293T cells fragments amplified in the assay. The ratios between TYMP, EGFP and RNAse P with p305-TP (or p305-sham, or p-sham) and the packaging plasmids pRSV or Ang1 were calculated and a correction factor obtained from different 1 REV, pMDLg RRE and pMD VSVG at 37 Cand5%CO2. The supernatant amplification efficiencies was applied. medium containing transductant particles was recovered 48, 56 and 72 h after For mRNA quantification, total RNA was extracted from 5Â106 cells using transfection, filtered through a 0.45-mm membrane and enriched by at least Trizol reagent (Invitrogen). The amount and integrity of the RNA obtained was 1 100-fold through ultracentrifugation at 50 000 g, for 2 h at 4 C. Titration was assessed by capillary electrophoresis using the RNA 6000 Nano Chip kit carried out by treating HEK293T cells with serial dilutions of the enriched (Agilent Technologies, Santa Clara, CA, USA). The levels of RNA degradation supernatants. After 4 days in culture, HEK293T cells were trypsinized and were always negligible (data not shown). In all, 2 mg of RNA was treated with EGFP fluorescence was analyzed by flow cytometry to estimate transduction DNase I (Invitrogen). Reverse transcription was performed on DNA-free RNA 31 rates, and viral titers were calculated. using the High-capacity cDNA Reverse Transcription Kit (Applied Biosystems). À2 B-LCL transductions were performed at 15 MOI in 14 mgcm RetroNectin The resulting cDNA was used for qRT-PCR assays using the probes indicated À1 (Takara, Otsu, Japan)-coated 24 multi-well plates in the presence of 4 mgml above for TYMP and EGFP. Results of TYMP and EGFP mRNA levels were protamine sulfate. HEK293T cells were transduced at 10 MOI without Retro- referred to PPIA mRNA (pre-designed TaqMan MGB gene expression assay, Id 1 Nectin. After addition of vectors, the plates were centrifuged at 300 g,32 Cfor assay Hs99999904_m1). Amplifications were performed as follows: one step at 90 min, and transductions were allowed to proceed for an additional period of 50 1C 2 min; one step at 95 1C10min;40cyclesat951C15s,601C 1 min. For 1 48 h at 37 C, 5% CO2. Then, cells were washed once with phosphate-buffered each experiment, all reactions were run in triplicates. saline (PBS) and fresh medium was added to the cultures. For transduction of murine hematopoietic progenitors, 5Â105 LinÀ cells per well, obtained as detailed below, were seeded in 24-well plates at 5Â105 cells per Western blot analysis ml in Iscove’s modified Dulbecco’s medium (Invitrogen) supplemented with Protein extracts were obtained as described below for TP activity determina- 1 tion. In all, 3.5 mg of protein extracts were electrophoresed in a 10% SDS- 10% heat-inactivated fetal bovine serum, 2 mML-glutamine, 1 U lÀ penicillin, 1UlÀ1 streptomycin and recombinant murine growth factors (rmSCF polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride 10 ng mlÀ1,rmTPO10ngmlÀ1,rmIL310ngmlÀ1 and rmFlt3 10 ng mlÀ1) membrane, and probed with an anti-TP mouse monoclonal antibody (ImmunoTools, Friesoythe, Germany). Lentiviruses were added at 100 MOI (Calbiochem, Darmstadt, Germany) and an anti-b-actin mouse monoclonal and cells were centrifuged at 300 g,321C for 90 min. Transductions were antibody (Sigma-Aldrich, St Louis, MO, USA), and then with peroxidase- conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West allowed to proceed overnight at 37 1C, 5% CO2.Then,cellswerewashedwith PBS and re-suspended (2.5Â106 cells per ml) in PBS immediately before the Grove, PA). Bands were visualized by treating the membranes with ECL infusion to the recipient mice. chemiluminescent kit (GE Healthcare, Buckinghamshire, England), and quan- tified using Quantity One software (Bio-Rad, Hercules, CA, USA). All the TP-to-b-actin ratios were referred to the corresponding ratio obtained from a Flow cytometry analysis control extract, used as a calibrator that was run in all gels. EGFP fluorescence was analyzed by flow cytometry to assess the percentage of transduced cells. Cultured cells, or buffy coat and bone marrow cells from mice, Deoxyribonucleoside triphosphate determination were re-suspended in fresh medium and fluorescence was measured through a Deoxyribonucleoside triphosphate determinations were performed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The polymerase assay based on the incorporation of 3H-dATP or 3H-dTTP in fluorescence cutoff was chosen at that value that rendered a 1% or less positive synthetic oligonucleotides,32 in total cell homogenates obtained as previously events in a negative control for EGFP or the fluorescent antibody indicated in described.33 Briefly, cells were lysed by mechanical disruption through a the specific experiment. For cell cycle analyses, cultured cells were stained with 27-gaugeÂ1-inch needle in 2 ml of isolation buffer (220 mM mannitol, 70 mM Hoechst 33342, then EGFP- and Hoechst-derived fluorescence was simulta- sucrose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2, neously measured using a MoFlo Legacy cell sorter device (Beckman Coulter, 1mM ethylene glycol tetraacetic acid, 0.5% bovine serum albumin), and

Gene Therapy Gene therapy corrects TP deficiency J Torres-Torronteras et al 805 proteins were precipitated through addition of 60% methanol (final concen- ACKNOWLEDGEMENTS tration). Supernatants containing total cell deoxyribonucleoside triphosphates We would like to thank Luigi Naldini for kindly providing the lentiviral vectors were dried under speed vacuum and stored at À80 1C. Dry pellets were p305 and p-sham, and Michael Terry for his English Language assistance. re-suspended in 400 ml of water and 10 ml were added to each polymerase This work was supported by the Spanish Instituto de Salud Carlos III reaction.32 [PI 06/0735, PS09/01591, Miguel Servet grants to JB and RM] and the United Mitochondrial Disease Foundation [UMDF 04/042]. Transplantation of mice with transduced bone marrow LinÀ Double KO donors (8- to 12-week-old) were killed by CO2 inhalation, and bone marrow was obtained by crushing femorae, tibiae and iliac crests with mortar and pestle. LinÀ bone marrow cells were immunoselected by lineage 1 Nishino I, Spinazzola A, Hirano M. Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder. Science 1999; 283: 689–692. depletion (Lineage Cell Depletion Kit for Mouse, Miltenyi Biotech, Bergisch 2 Hirano M, Nishigaki Y, Marti R. Mitochondrial neurogastrointestinal encephalomyo- Gladbach, Germany). After enrichment, cells were transduced with p305-TP or pathy (MNGIE): a disease of two genomes. Neurologist 2004; 10:8–17. p-sham vectors, as detailed above. Between 2Â105 and 5Â105 transduced cells 3 Lara MC, Valentino ML, Torres-Torronteras J, Hirano M, Martı´ R. Mitochondrial were injected in the tail vein of 8- to 12-week-old double KO recipient mice, neurogastrointestinal encephalomyopathy (MNGIE): biochemical features and thera- peutic approaches. Biosci Rep 2007; 27: 151–163. after sublethal myeloablation using total body irradiation (6 Gy, in two doses of 4 Halter J, Schu¨pbach WM, Casali C, Elhasid R, Fay K, Hammans S et al. Allogeneic À1 3 Gy at a dose rate of 2.24 Gy min given at a 2-h interval). Mice were housed hematopoietic SCT as treatment option for patients with mitochondrial neurogastroin- in filter-isolated ventilated cages for 15 days after transplantation to reduce the testinal encephalomyopathy (MNGIE): a consensus conference proposal for a standar- risk of infection, and then housed in conventional cages. PB was collected at dized approach. Bone Marrow Transplant 2011; 46:330–337. 5 Desgranges C, Razaka G, Rabaud M, Bricaud H. Catabolism of thymidine in human different times from the saphenous vein for different determinations. At week blood platelets: purification and properties of thymidine phosphorylase. Biochim 29, p305-TP- and p-sham-treated mice were killed by CO2 inhalation, and Biophys Acta 1981; 654: 211–218. blood was collected immediately by intracardiac puncture, as well as bone 6 Marti R, Nishigaki Y, Hirano M. Elevated plasma deoxyuridine in patients with marrow, as detailed above. thymidine phosphorylase deficiency. Biochem Biophys Res Commun 2003; 303: 14–18. 7 Spinazzola A, Marti R, Nishino I, Andreu AL, Naini A, Tadesse S et al. Altered thymidine Nucleoside levels and TP activity determination metabolism due to defects of thymidine phosphorylase. JBiolChem2002; 277: dThd and dUrd were measured in cell culture media and mice plasma by a 4128–4133. 8 Valentino ML, Martı´ R, Tadesse S, Lo´pez LC, Manes JL, Lyzak J et al. Thymidine and binary gradient-elution ultra performance liquid chromatography (UPLC) deoxyuridine accumulate in tissues of patients with mitochondrial neurogastrointestinal method. Cultured cells were centrifuged at 300 g for 5 min, and the supernatant encephalomyopathy (MNGIE). FEBS Lett 2007; 581: 3410–3414. was recovered and deproteinized through ultrafiltration (Microcon YM-10, 9 Ferraro P, Pontarin G, Crocco L, Fabris S, Reichard P, Bianchi V et al. Mitochondrial Millipore, Billerica, MA, USA) at 14 000 g,30min,251C. The cellular pellet was deoxynucleotide pools in quiescent fibroblasts: a possible model for mitochondrial 1 neurogastrointestinal encephalomyopathy (MNGIE). JBiolChem2005; 280: PBS washed and kept at À80 C for TP assay. In all, 50 ml of mouse antic- 24472–24480. oagulated (EDTA) PB was collected from the saphenous vein and diluted in 10 Lopez LC, Akman HO, Garcı´a-Cazorla A, Dorado B, Martı´ R, Nishino I et al. Unbalanced 400 ml PBS, centrifuged at 3000 g for 5 min at 4 1C and the supernatant (diluted deoxynucleotide pools cause mitochondrial DNA instability in thymidine phosphory- lase-deficient mice. Hum Mol Genet 2009; 18:714–722. plasma) was deproteinized through addition of 0.5 M (final concentration) 11 De Vocht C, Ranquin A, Willaert R, Van Ginderachter JA, Vanhaecke T, Rogiers V et al. 1 m perchloric acid. Blood cell fraction was kept at À80 C for TP assay. In all, 5 l Assessment of stability, toxicity and immunogenicity of new polymeric nanoreactors of medium or diluted plasma, both deproteinized, were injected into an for use in enzyme replacement therapy of MNGIE. J Control Release 2009; 137: Acquity UPLC apparatus (Waters, Milford, MA, USA) and eluted at 246–254. 1 0.5 ml minÀ with a saline buffer eluent A (20 mM ammonium acetate, pH 12 Hirano M, Martı´ R, Casali C, Tadesse S, Uldrick T, Fine B et al. Allogeneic stem cell 5.6) and an organic eluent B (methanol gradient grade), according to the transplantation corrects biochemical derangements in MNGIE. Neurology 2006; 67: 1458–1460. following gradient: 0 to 1.1 min, 100% eluent A; 1.1 to 5 min, 100 to 86.4% 13 Moran NF, Bain MD, Muqit MM, Bax BE. Carrier erythrocyte entrapped thymidine eluent A; 5 to 6.1 min, 0% eluent A; 6.1 to 7.2 min, 100% eluent A. The column phosphorylase therapy for MNGIE. Neurology 2008; 71: 686–688. used was an Acquity UPLC BEH C18 column 100Â2.1 mm, 130 A˚ pore size, 14 Yavuz H, Ozel A, Christensen M, Christensen E, Schwartz M, Elmaci M et al. Treatment 1.7 mm particle size (Waters). Optical absorbance of the eluate was monitored of mitochondrial neurogastrointestinal encephalomyopathy with dialysis. Arch Neurol 2007; 64: 435–438. at 267 nm, and definitive identification of the dThd and dUrd peaks was based 15 Hirano M, Casali C, Tadesse S, Stanzani M, Savage DG. Sustained biochemical and upon retention time and by treatment of a second aliquot of the sample with a clinical improvements two years post-allogeneic stem cell transplantation in a patient large excess of purified Escherichia coli TP (Sigma-Aldrich) to eliminate the with MNGIE. Neurology 2008; 70 (Suppl 1): A406–A407. dThd and dUrd. The quantitation of the nucleosides was based on peak areas 16 Schupbach M, Benoist JF, Casali C, Elhasid R, Fay K, Hahn D et al. Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for Mitochondrial Neurogastrointest- using external aqueous standards. inal Encephalomyopathy (MNGIE). Neurology 2009; 73: 332–332. TP activity was determined in cultured cells and PB cell fraction. Cultured 17 Marti R, Spinazzola A, Tadesse S, Nishino I, Nishigaki Y, Hirano M. Definitive diagnosis cells (5Â106) or PB cell fraction obtained as detailed above were homogenized of mitochondrial neurogastrointestinal encephalomyopathy by biochemical assays. Clin À1 Chem 2004; 50: 120–124. in 200 ml of lysis buffer (50 mM Tris-HCl, pH 7.2; 10 ml l Triton X-100; 2 mM À1 18 Lara MC, Weiss B, Illa I, Madoz P, Massuet L, Andreu AL et al. Infusion of platelets phenylmethylsulfonyl fluoride; 0.2 ml l 2-mercaptoethanol) by passing 12 transiently reduces nucleoside overload in MNGIE. Neurology 2006; 67: 1461–1463. times through a 27-gaugeÂ1-inch needle. TP activity was determined as detailed 19 Copelan EA. Hematopoietic stem-cell transplantation. NEnglJMed2006; 354: elsewhere.8 Briefly, 74 ml of homogenate containing 10 mg (cultured cells) or 2 mg 1813–1826. (blood cell fraction) of total protein was incubated at 37 1Cfor1hinthe 20 Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science M 8 presence of 10 m dThd in the reaction buffer, and the thymine formed was 2002; 296: 2410–2413. measured through the UPLC method described above for nucleoside determina- 21 Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G, Schmidt M, Kutschera I et al. tion. The protein content was determined through the Bradford method.34 Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleuko- dystrophy. Science 2009; 326: 818–823. 22 Ott MG, Schmidt M, Schwarzwaelder K, Stein S, Siler U, Koehl U et al. Correction of X- Statistical analysis linked chronic granulomatous disease by gene therapy, augmented by insertional Statistical analysis was performed with the SPSS 15.0 software. Tests used are activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 2006; 12: 401–409. indicated in the Results section and/or the figure legends. For statistical 23 Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for purposes, undetectable values were considered as zero. SCID-X1. Science 2003; 302: 415–419. 24 Stein S, Ott MG, Schultze-Strasser S, Jauch A, Burwinkel B, kinner A et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 CONFLICT OF INTEREST activation after gene therapy for chronic granulomatous disease. Nat Med 2010; 16: The authors declare no conflict of interest. 198–204.

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