Widespread biochemical correction of murine ... - Nature

Gene Therapy (2009) 16, 746–756 & 2009 Macmillan Publishers Limited All rights reserved 0969-7128/09 $32.00 www.nature.com/gt

ORIGINAL ARTICLE

Widespread biochemical correction of murine mucopolysaccharidosis type VII pathology by liver hydrodynamic plasmid delivery M Richard1,2,3,4,5, A Arfi1,2,3,4,5, J Seguin1,2,3,4, C Gandolphe1,2,3,4 and D Scherman1,2,3,4 Institut de Sciences et Techniques du Me´dicament, Inserm, U640, Paris, France; 2Institut de Chimie, CNRS, UMR8151, Paris, France; Chemical and Genetic Pharmacology Laboratory, Department of Therapeutic and Diagnostic Innovation, Faculte´ de Pharmacie, Universite´ Paris Descartes, Paris, France and 4Friedel Department of Molecular Chemistry, Ecole Nationale Supe´rieure de Chimie de Paris, Paris, France 1 3

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by a deficiency of the acid hydrolase b-glucuronidase. MPS VII mice develop progressive lysosomal accumulation of glycosaminoglycans (GAGs) within multiple organs, including the brain. Using this animal model, we compared two plasmid gene administration techniques: muscle electrotransfer and liver-directed transfer using hydrodynamic injection. We have evaluated both the expression kinetics and the biodistribution of b-glucuronidase activity after gene transfer, as well as the correction of biochemical abnormalities in various organs. This study shows that MPS VII mice treated with a plasmid-bearing mouse b-glucuronidase cDNA, acquire the ability to produce the b-glucuronidase enzyme for an extended period of time.

The liver seemed to be more appropriate than the muscle as a target organ to enable enzyme secretion into the systemic circulation. A beneficial effect on the MPS VII pathology was also observed, as liver-directed gene transfer led to the correction of secondary enzymatic elevations and to the reduction of GAGs storage in peripheral tissues and brain, as well as to histological correction in many tissues. This work is one of the first examples showing that non-viral plasmid DNA delivery can lead to improvements in both peripheral and brain manifestations of MPS VII disease. It confirms the potential of non-viral systemic gene transfer strategy in neurological lysosomal disorders. Gene Therapy (2009) 16, 746–756; doi:10.1038/gt.2009.36; published online 9 April 2009

Keywords: non-viral gene transfer; lysosomal disease; hydrodynamic; CNS

Introduction Mucopolysaccharidoses are a group of lysosomal storage diseases caused by a deficiency in specific acid hydrolases, which are responsible for the degradation of complex glycosaminoglycans (GAGs).1 This deficiency causes substrate accumulation in the lysosomes of the cells of the central nervous system and of other organs. Patients with mucopolysaccharidosis (MPS) share several clinical signs, including a severe neurodeterioration and systemic symptoms.1 To date, no treatment provides clinical benefit for patients with neurological form of a MPS, except allogeneic bone marrow transplantation that shows limited results. The MPS type VII (Sly disease) is caused by a severe deficiency of the lysosomal enzyme b-glucuronidase (GUSB; EC3.2.1.31), which is one of the enzymes responsible for the sequential degradation of chondroitin sulfate, dermatan sulfate and heparan sulfate.2 Affected patients Correspondence: Professor D Scherman, Institut de Sciences et Techniques du Me´dicament, Inserm, U640; Institut de Chimie, CNRS, UMR8151; Chemical and Genetic Pharmacology Laboratory, Department of Therapeutic and Diagnostic Innovation, Faculte´ de Pharmacie, Universite´ Paris Descartes, 4, avenue d’observatoire, 75006 Paris, France. E-mail: [email protected] 5 The two authors contributed equally to this work. Received 28 September 2008; revised 19 February 2009; accepted 21 February 2009; published online 9 April 2009

have a variable phenotype that usually includes coarse facies, growth and mental retardation, corneal clouding, hepatosplenomegaly and skeletal deformities. The severe form of Sly disease is rarely seen in humans, as affected individuals probably die perinatally. However, the MPS VII murine model is a very convenient disease model for the study of different therapeutic approaches to this and similar genetic deficiencies. Indeed, extensive data is available about GUSB, and animal models display features very close to the human MPS VII phenotype, including many biochemical and histopathological similarities.2–4 Lysosomal storage diseases are the good candidates for gene therapy because of two main properties. First, lysosomal enzymes, such as GUSB, are secreted from producing cells and taken up by the neighboring or distant cells in a process mediated by the mannose-6phosphate receptors.5 Second, it is also known that 1–5% only of the normal level of GUSB activity are sufficient to reduce biochemical and histopathological abnormalities of the MPS VII disease in some organs.6–11 Therefore, the gene transfer localized to a depot organ such as the liver or the muscle could enable the secretion of the affected enzyme into circulation at a therapeutic level, leading to the global correction of lysosomal storage. Gene therapy of MPS has been pursued primarily using viral vectors.12,13 However, several characteristics could make non-viral gene therapy more attractive: lower cost of plasmid large-scale production and storage, possibility to

Plasmid-mediated gene therapy in MPS VII mouse model M Richard et al

readminister the treatment, absence of immune response to vector and no risk of insertional mutagenesis. In this study, we focused on two non-viral gene administration techniques: muscle electroporation (also called electrotransfer) and liver-directed transfer using hydrodynamic intravenous injection. Skeletal muscle is the most widely targeted tissue for electrotransfer, as muscle fibers offer numerous advantages such as accessibility, efficient DNA cellular uptake and long-term transgene expression.14,15 Concerning hydrodynamic gene delivery method, it has proven to be simple, efficient and versatile.16,17 We have evaluated both kinetics and biodistribution of GUSB activity after each of these gene transfer methods. We have also evaluated the correction of biochemical and histological abnormalities in various organs. This study shows that MPS VII mice treated with a plasmid encoding the mouse GUS cDNA (p-GUSb) acquired the ability to produce GUSB enzyme for an extended period of time. Hydrodynamic-based gene transfer to liver of p-GUSb allowed enzyme secretion and uptake by most organs. In addition, this non-viral approach had a beneficial effect on the disease as it corrected secondary enzymatic abnormalities and reduced GAG storage, not only in several peripheral tissues but also in brain. To our knowledge, this is one of the first demonstration18 of a non-viral strategy providing successful replacement gene therapy for Sly disease and more generally for a neurological form of MPS.

Results GUSB expression and biodistribution in MPS VII mice after plasmid electrotransfer into muscle Mice receiving 25–100 mg of either pCMV-GUSb or pCAG-GUSb at 6–8 weeks of age were killed at several time points from 1 to 8 weeks after intramuscular electrotransfer. The GUSB expression in injected muscles was exceptionally high; when administering 100 mg of plasmid, it represented about 1000% of normal level in wild-type mice (Figure 1a). In contrast, GUSB level in all other tested tissues such as liver, spleen, or brain and in sera was below the detection level. Similar results were obtained with more than twenty mice at different times after electrotransfer (data not shown). This activity level seemed to be dose-dependent (Figure 1b). We observed very high and sustained GUSB activity in injected muscles for 60 days, suggesting that high expression continued for an extended period of time. This GUSB production at a level, which was far in excess to that of normal tissue did not seem to induce any negative side effects, such as decrease in weight or increased mortality. Sections of injected muscle were stained for b-glucuronidase activity (Figure 1c). We observed two types of positive cells. Several muscle fibers with intense GUSB staining were presumably transfected expressing cells. In addition, the neighboring cells presenting a weaker staining were presumably fibers, which had been taken up by the receptor-mediated endocytosis GUSB molecules secreted by the transfected fibers. GUSB expression and biodistribution in MPS VII mice after hydrodynamic tail-vein plasmid injection Mice receiving 100 mg of pCAG-GUSb at 6–8 weeks of age were killed at day 8, day 30 and day 60 after

hydrodynamic tail-vein injection. The CAG promoter was used preferentially to prevent the rapid silencing of the CMV promoter in the liver.19,20 Before killing, mice were perfused with saline to reduce the enzyme activity because of circulating blood. GUSB activity was measured in serum at various time points after DNA injection (Figure 2). In treated MPS VII mice, the secretion by the liver resulted in high-serum GUSB activity, corresponding to about 100–500% of normal level at days 2–8, and 15% of normal value at day 30, versus less than 1% of normal level for non-treated MPS VII mice (Figure 2). However, circulating GUSB activity at day 60 decreased to less than 5% of normal level. The GUSB activity was also measured in a variety of tissues at days 8, 30 and 60 after injection (Figure 3). As expected, the liver seemed to be the organ with the highest GUSB activity level (about 240 units per mg protein at day 8) corresponding to 110% of normal level. At day 8 after injection, the GUSB activity was also high in the spleen (65% of normal), the kidney (25% of normal), the heart (300% of normal), the lung (45% of normal), the muscle (20% of normal) and significantly detectable in the brain (3% of normal). In contrast, the untreated MPS VII mice exhibited less than 0.5% of normal activity in all tissues. The GUSB activity was decreased at day 30 and day 60, but remained exceptionally high in the liver (50% at day 30 and 15% at day 60 of normal level) and significantly higher than in non-treated mice for most of the tested organs (Figure 3). Intense GUSB staining was observed in multiple liver cells after hydrodynamic injection (Figure 4). The morphology was typical of a regenerating liver parenchyma, as observed earlier in corrected MPS VII animals.8,21 This is consistent with the hypothesis that liver may be the major site of GUSB production after hydrodynamic injection. This was confirmed by a quantification of plasmid amounts in various tissues of treated MPS VII mice compared with those of nontreated animals 8 days after hydrodynamic injection. It showed that the liver was the major site for gene transfer, as it exhibited much higher amount of plasmid than any other tested organs, such as heart, spleen or brain (Table 1). The liver presented also a significantly higher GUSB mRNA level in treated MPS VII mice than in non-treated MPS VII mice. In contrast, in all other tested tissues, such as kidney, spleen and heart, no increase of GUSB mRNA level could be detected (Table 1). This confirmed that the liver was the major site for the transgene expression. The lack of correspondence we observed between plasmid content and GUSB activity in kidney, spleen and heart also confirmed that in these organs, the GUSB activity was mainly because of the cross-correction phenomenon.

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Secondary enzymatic elevations and GAG accumulation after hydrodynamic tail-vein plasmid injection Lysosomal hydrolases, such as b-hexosaminidase A and B (b-Hex) and a-galactosidase (a-Gal), are secondarily elevated in the tissues of MPS VII mice,21 and are usually normalized after effective therapeutic intervention.22,23 A reduction in secondary elevations has been used as a sensitive biochemical surrogate for therapeutic Gene Therapy


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Figure 1 b-Glucuronidase (GUSB) activity in muscle of mucopolysaccharidosis VII (MPS VII) mice treated by pCMV-GUSb electrotransfer. Muscle extracts were collected (a) at day 10 (n ¼ 4), day 14 (n ¼ 4), day 20 (n ¼ 3), day 30 (n ¼ 3) and day 60 (n ¼ 3) after electrotransfer of 100 mg of plasmid encoding GUSB, (b) at day 8 after electrotransfer of 10 mg (n ¼ 3), 50 mg (n ¼ 3) and 100 mg (n ¼ 3) of plasmid-encoding GUSB. GUSB activity was measured and expressed as the percentage of level in wild-type muscle extracts. The error bars indicate the s.d. (c) Histochemical staining for GUSB activity on frozen sections of electrotransfered MPS VII muscles, wild-type muscles and untreated MPS VII muscles. Deeply stained foci seen in panels a and b (indicated by black arrows) are GUSB-expressing cells. Weakly stained foci seen in panels a and b panels (indicated by white arrows) are cells GUSB taking up cells. Bars ¼ 1 mm.

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Figure 2 Circulating levels of b-glucuronidase (GUSB) activity after liver-targeted hydrodynamic injection of plasmid pCAG-GUSb (100 mg). Sera were colleted at the times indicated and assayed for bglucuronidase in treated (white circles, n ¼ 5) MPS VII mice. GUSB activity was expressed as nmol/ml/h. Ranges for this parameter were determined in wild type (wt, n ¼ 4) and non-treated MPS VII (nt, n ¼ 4) mice, and are shown as mean values (solid line)±s.d. (dashed lines). The error bars indicated the s.d.

response. More specifically, we measured the level of b-Hex (Figure 5a) in several tissues and that of a-Gal (Figure 5b) in the liver, the heart and the brain of wildGene Therapy

type mice, and of untreated or pCAG-GUSb-treated MPS VII mice at days 8 and 30. All examined tissues in treated MPS VII mice exhibited a consistent trend toward normalization of secondary elevations. For some tissues, such as kidney, heart and lung, the difference between the injected and non-injected MPS VII mice was statistically significant (Po0.005, Student’s t-test), and even comparable to wild-type mice levels in some organs (Figures 5a and b). Furthermore, GAG contents were significantly decreased in several organs of treated MPS VII mice as compared with those in non-treated MPS VII mice at days 8, 30 and 60 after plasmid injection (Figure 6). The GAG-reduction rate ranged between 50% in spleen, kidney and brain, and 75% in liver. We did not observe any further GAG accumulation between day 30 and day 60, except in the spleen.

Correction of the pathology in several organs We performed histological evaluation for lysosomal storage abnormalities using bright-field microscopy on the liver, the spleen, the bone marrow, the joints and the brain from MPS VII mice killed 4 weeks post injection, compared with tissues from age-matched control MPS VII and normal mice (Figure 7). Severe histological abnormalities such as vacuolation and macrophage


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Figure 3 b-Glucuronidase (GUSB) activity in various tissues of mucopolysaccharidosis VII (MPS VII) mice treated by liver-targeted hydrodynamic procedure. Liver, spleen, kidney, heart, lung, muscle and brain were collected at day 8, day 30 and day 60 after hydrodynamic injection of plasmid pCAG-GUSb (100 mg). GUSB activity was measured (expressed as the percentage of wild-type enzymatic levels), in nontreated (nt, n ¼ 3), and treated MPS VII mice at day 8 (D8, n ¼ 3), day 30 (D30, n ¼ 6) and day 60 (D60, n ¼ 3) after hydrodynamic procedure. Data points represent results from individual mice. The error bars indicate the s.d. Statistical significance was assessed by Student t-test, *Po0.005, **Po0.001.

infiltration were observed in tissues from MPS VII mice (Figures 7b, e, h, k and n), in comparison with normal mice (Figures 7a, d, g, j and m). However, we observed a complete resolution of vacuolated cells in the liver, the spleen and the bone marrow of treated MPS VII mice, showing a global reversion of the pathology in these tissues. Such a global correction of pathological changes was not seen in cartilage and joints, as the hyperplastic synovium and the abnormalities in chondrocytes and articular cartilage matrix were still present in treated MPS VII mice (Figure 7l). Nevertheless, we noticed a slight decrease of the lesion in articular tissue of treated MPS VII mice (Figure 7l) as compared with non-treated MPS VII mice (Figure 7k). Finally, an extensive observation of different brain regions in normal, treated and nontreated MPS VII mice, failed to show any significant correction of the neuropathology at the optical microscopic level (Figures 7m–o). Nonetheless, minor improvements were observed in cortical region such as the reduction of vacuoles number in glial cells (Figures 7n and o).

Discussion Gene therapy of lysosomal storage diseases, including MPS, requires sustained expression of the therapeutic

gene. Among non-viral gene transfer strategies, electrotransfer into skeletal muscle can enable high and sustained transgene expression, as well as protein secretion.24 However, in this study, we observed that although we obtained a very high and sustained GUSB activity level in electrotransfered muscle, skeletal muscle was not able to secrete this enzyme into the blood circulation. This phenomenon has already been described after AAV-GUSB intramuscular injection6 in MPS VII mice; the muscle was also significantly less efficient than the liver to secrete the lysosomal enzyme a-glucosidase in Pompe disease,25 as well as the arysulfatase B in MPS VI mouse and cat models,26 whereas in other studies on Pompe disease27 or Fabry disease,28 skeletal muscle was able to secrete efficiently lysosomal enzyme. Regarding b-glucuronidase, Naffakh et al.29 showed that genetically modified myoblasts engrafted into muscle could secrete small amount of human GUSB, subsequently taken up by cells in other organs. We can hypothesize that the size of GUSB tetrameric protein (332 kDa) could be a hindrance for enzyme delivery in systemic circulation by muscle because of the basal lamina, whereas hepatocytes are directly in contact with blood circulation through the fenestrae of the thin endothelium of sinusoides. The staining of GUSB activity in sections of electrotransfered Gene Therapy


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muscle showed that several muscular cells expressed GUSB. It also suggested that the neighboring muscular cells were able to take up the enzyme. However, the enzyme diffusion was limited to the very close neighborhood of expressing fibers, and no enzyme could be detected in the serum. Thus, massive GUSB uptake by neighboring muscle fibers might be responsible for the lack of blood secretion. This phenomenon does not occur with protein such as factor IX or Epo, which have been shown to be secreted readily by muscle tissue.24,30 hydrodynamically treated MPS VII liver

wild type liver

non treated MPS VII liver

Figure 4 Histological evaluation of plasmid-mediated b-glucuronidase (GUSB) activity in liver of mucopolysaccharidosis VII (MPS VII) mice treated by liver-targeted hydrodynamic procedure. Histochemical staining for GUSB activity on liver frozen sections of injected MPS VII, wild type and untreated MPS VII mice. Deeply stained foci seen in liver sections of MPS VII treated mice (indicated by black arrows) are GUSB-expressing cells. Bars ¼ 1 mm.

Table 1

Taking into account this high GUSB activity level obtained in eletrotransfered muscle, we can hypothesize that with another lysosomal enzyme exhibiting a better ability to be secreted by muscle, such as a-glucosidase27 or a-galactosidase,28 electrotransfer into muscle could be a very interesting therapeutic approach. This has been tested earlier in a murine MPS II model, showing limited enzyme secretion, but the electrotransfer conditions were probably not optimized.31 Unlike muscle-targeted approach, liver-directed nonviral gene transfer based on hydrodynamic procedures resulted in several therapeutical effects in MPS VII murine model, which were comparable to those observed using viral vectors.32 First, a high GUSB activity has been obtained, not only in the liver, but also in most of the tested organs, including the brain, as well as in the serum. The serum level of GUSB obtained 30 days after injection was higher than those described earlier with Sleeping Beauty transposon-mediated gene delivery.18 Furthermore, GUSB activity was still detected 30 and 60 days after gene transfer in almost all the tissues. This expression maintenance could be attribuated to the lack of a strong immune response directed toward bglucuronidase, as confirmed by ELISA assay performed on several injected MPS VII mice at different times post injection (data not shown). Unlike the earlier study of Aronovich et al.18 in which the human GUSB gene was used, in this study we used the murine GUSB cDNA. First, adult MPS VII mice have a known defect in immunity33 and might not generate a strong immune response to a foreign protein in the liver. It is more likely that the residual production of partial length inactive b-glucuronidase in this murine model could be sufficient to avoid a strong immune response against the restored murine protein. A long-lasting GUSB expression has been observed earlier in another study using murine GUSB for gene transfer in the liver,32 confirming the advantage of using murine cDNA. The decrease of GUSB activity observed in this study might more likely result from a silencing of the gene expression as it has been widely described earlier after hydrodynamic injection of plasmid.34,35

Plasmid biodistribution and relative GUSB mRNA levels in various organs after hydrodynamic injection of pCAG-GUSb GUSB mRNA level (fold change)

GUSB activity (nmol per mg protein per h)

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Treated MPS VII mice (n ¼ 3) Liver 24.1 Spleen 0.056 Heart 0.044 Brain 0.069

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Non-treated MPS VII mice (n ¼ 3) Liver 0.048 Spleen 0.054 Heart 0.045 Brain 0.064

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Abbreviations: GUSB, b-glucuronidase; MPS VII, mucopolysaccharidosis VII. Plasmid pCAG-GUSb amount was evaluated in several organs at day 8 after hydrodynamic injection in MPS VII mice (n ¼ 3) by quantifying kanamycin-resistant bacterial colonies after transformation with 1 mg of DNA extracted from organs. Relative GUSB mRNA level in heart, kidney, liver and spleen of non-treated (n ¼ 3) and treated MPS VII mice at day 8 after hydrodynamic injection (n ¼ 3) was determined using quantitative PCR. Data are expressed as means of fold-change in MPS VII mice, as compared with normal mice (n ¼ 3). 18S rRNA was used for normalization. GUSB activity was also measured in the same organs and expressed as means of specific activity (nmol/h per mg proteins, n ¼ 3). Gene Therapy


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Figure 5 b-hexosaminidase A and B (b-Hex) (a) and a-galactosidase (a-Gal) (b) activities in various tissues of mucopolysaccharidosis VII (MPS VII) mice treated by liver-targeted hydrodynamic procedure. Liver, spleen, kidney, heart, lung, muscle and brain were collected at day 8 and day 30 after hydrodynamic injection of plasmid pCAG-GUSb (100 mg). b-Hex (a) and a-Gal (b) activities were measured in non-treated (white bars, nt, n ¼ 3) and in treated MPS VII mice at day 8 (spotted bars, D8, n ¼ 3) and day 30 (hatched bars, D30, n ¼ 6) after hydrodynamic injection in MPS VII mice, and in wild-type mice (black bars, wt, n ¼ 3). Enzymatic activities were expressed as nmol/h per mg proteins. The error bars indicate the s.d.. Statistical significance was assessed by Student t-test, *Po0.005, **Po0.001.

In addition to GUSB activity restoration, a normalization of the secondary enzymatic elevations of b-Hex and a-Gal was observed. Most importantly, the liver-directed treatment enabled to dramatically decrease GAG content in several tissues of MPS VII mice, such as liver, spleen, kidney and brain. The effect on secondary elevations and GAG content was maintained or improved 30 days after gene transfer, whereas GUSB activity level decreased between days 8 and 30. This

suggests that GUSB activity level at day 30 could be sufficient in most organs including brain, to correct biochemical pathology. These GUSB activity levels, comprised between 5 and 10% of normal level for all organs except brain, were comparable to those obtained in earlier studies showing long-term clinical improvements,36 confirming the potential of our approach. This enzyme restoration led also to a drastic vacuolated-cells clearance in several tissues such as liver, spleen, bone Gene Therapy


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Figure 6 Effects of hydrodynamic treatment on glycosaminoglycans (GAG) levels in liver, spleen, kidney and brain. The liver, spleen, kidney and brain were collected at day 8, day 30 and day 60 after hydrodynamic injection of plasmid pCAG-GUSb (100 mg). Sulfated GAG levels were measured in non-treated (white bars, nt, n ¼ 3), and treated MPS VII mice at day 8 (gray bars, D8, n ¼ 3), day 30 (gray bars, D30, n ¼ 6) and day 60 (gray bars, D60, n ¼ 3) after hydrodynamic injection in MPS VII mice, and in wild-type mice (black bars, wt, n ¼ 3). Values were expressed as mg of GAG per mg of dried tissue. Heparan sulfate from bovine testis was used as standard. The error bars indicate the s.d. Statistical significance was assessed by Student t-test, *Po0.005, **Po0.001.

marrow and in a lesser extent in joints. The microscopal aspect of these tissues was undistinguishable in treated MPS VII mice and in normal mice (Figures 7a, c, d, f, g and i). Nevertheless, the circulating GUSB activity after 60 days (below 5% of normal activity) could be unsufficient to achieve substantial clinical improvement in this work, as it has been shown earlier with 2.5% of circulating GUSB activity.11 More specially, higher enzyme restoration may be required to completely reverse some pathological changes, such as brain or articular abormalities, as we observed poor improvements of morphological abnormalities after plasmid injection. Interestingly, despite the blood–brain barrier, the brain exhibited low but significant GUSB activity, presumably because of the high and sustained presence of the enzyme in the blood circulation. This putative permeation phenomenon has been described recently after enzyme replacement therapy in neonates37,38 or in adult mice using high doses of enzyme,39–41 but also following gene therapy in adult MPS VII mice.32 Several hypotheses were proposed by Vogler et al.39 to explain enzyme uptake and brain correction after systemic enzyme replacement therapy, such as saturation of residual mannose-6-phosphate receptors on the blood–brain barrier, phagocytosis phenomenon or enzyme uptake by large molecules. Unlike proteotherapy, characterized by irregular protein circulation, our gene therapy protocol provides constant enzyme production and circulation. Therefore, the same putative mechanisms could be proposed to explain enzyme restoration in the brain after the hydrodynamic injection of plasmid. Still, this GUSB activity in the brain could reflect residual GUSB from the circulation, in spite of the intense perfusion procedure used. Therefore, we considered as a much experimental result the study of secondary enzymatic elevations and GAG content in brain parenchyma. Indeed, we observed in our study a significant correction of these biochemical abnormalities in the brains of treated MPS VII mice. As we verified that there was no direct plasmid transfection of the brain in Gene Therapy

treated MPS VII mice, we could conclude that GUSB activity detected in brain did not result from plasmid expression by brain cells. Nevertheless, we observed a rather poor correction of the cellular damages in the brains of treated MPS VII mice, suggesting that enzyme restoration in the brain could not be sufficient to enable complete histological correction of the central nervous system. In conclusion, as compared with other non-viral methods, hydrodynamic gene delivery seems to be a promising procedure, which provides remarkable efficiency and convenience, as shown earlier in numerous disease models.17 Regional approaches (through inferior vena cava for instance) have been shown to be clinically applicable.42,43 In our case, the GUSB enzyme was measured at therapeutic level in most organs of injected MPS VII animals. The only limitation to this technique remains the transient nature of the transgene expression. As shown recently,34,44 this could be overcome by the use of new non-viral tools such as minicircles.45 A longer gene expression could also be achieved with a liver-specific promoter combined with regulation sequences.46,47 On the basis of these data, we will next focus on nonviral vector optimization to achieve long-lasting enzyme secretion by the liver and enhanced uptake by the organs, including the brain. This could thus lead not only to biochemical and histological corrections, but also to clinical improvement of MPS VII disease state. This therapeutic strategy should also be extended to treat other neurological lysosomal storage disorders.

Materials and methods Plasmids construction and production pVAX-CAG-GUSb and pVAX-CMV-GUSb. Murine cDNA sequence of GUSb gene was obtained by reverse transciptase-PCR with total cDNA from C57Bl/6 liver. The primers used were 50 -ATGTCCCTAAAATG and 50 -CCAAATAGAAACAC GAGTGCGTGTT-30


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Figure 7 Histological evaluation of lysosomal storage abnormalities after plasmid administration. Tissue section was imaged by bright field microscopy. Representative micrographs of liver (a–c), spleen (d–f), bone marrow (g–i), joints (j–l) and brain (m–o) from aged-matched normal (a, d, g, j and m), aged-matched non-treated MPS VII (b, e, h, k and n) and treated MPS VII mice (4 weeks post plasmid-injection) (c, f, i, l and o) were shown. The degree of lysosomal storage, manifested by foci of vacuolated cells, was reduced in tissues from injected MPS VII mice. Black arrows indicate vacuoles and clarification of the cytoplasm, observed mainly in macrophage-like cells. In brain cortex (n and o), cells with distended cytoplasm and cytoplasmic vacuoles were likely oligodendrodytes. White arrrrows indicate synovium in articular tissue, either normal (j) or hyperplastic in MPS VII tissue (l). Arrowheads indicate cartilage in articular tissue, either normal (j) or damaged in MPS VII tissue (k and l). Original magnification: 200 (a–c, g–i, m–o) and 100 (d–f, j–l).

TAGTGTCAGGAA-30 . This cDNA was cloned in pVAX2 backbone (derived from pVAX1 from Promega, Charbonnie`res-Les-Bains, France) under CMV and CAG promoters, respectively.

Experimental animals MPS VII mice and normal controls C57Bl/6 were generated from the carrier strain gusmps/gus+4 obtained from B Soper and the Jackson Laboratory (Bar Harbor, Gene Therapy


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ME, USA). This strain has been generated by backcrossing the original B.CH2bm1-gusmps/gusmps to B6+/+ mice to move gusmps onto the B6 background. The mice were genotyped by PCR at 3 weeks from birth. The studies were conducted following the recommendations of both the European Convention for the Protection of Vertebrates Animals used for Experimentation and the local Ethics Committee on Animal Care and Experimentation.

Muscle electrotransfer Plasmid DNA in saline (final volume 20 ml) was injected into the tibial cranial muscle of anaesthetized mouse. After 20 s, eight square-wave electric pulses (200 V/cm, 20 ms, 2 Hz) were delivered through two stainless steel plate electrodes placed on each side of the shaved leg, using a electropulsator (Sphergen, Evry, France).14 Electrical contact with the skin was ensured by application of a conductive gel. Hydrodynamic injections Mice were injected with plasmid DNA in saline through tail vein using a hydrodynamic-based procedure as described earlier.48,49 Briefly, DNA injection was completed in less than 5 s, using a total volume representing 10% of mouse weight (v/w). Preparation and analysis of tissues Blood was collected by retro-orbital puncture. Mice were perfused with saline, killed by cervical dislocation, and selected organs were flash frozen and stored at 80 1C for biochemical and histochemical analyses. Enzymatic assays The samples of liver, spleen, kidney, heart, lung, brain and tibial cranial muscle were mechanically homogenized in an appropriate volume of lysis buffer (25 mM Tris pH 7.8, 2 mM EDTA, 2 mM dithiothreitol, Triton X-100 1%, 10 ml mg1 tissue). Measurement of GUSB activity was performed using 4-methylumbelliferyl b-D-glucuronide (Sigma, St Louis, MO, USA) as the substrate as described earlier.50,51 Enzyme activity is reported within the text as the percentage of wild-type levels to allow for comparison across studies. The wildtype levels were from tissues obtained from homozygous normal mice (C57Bl/6) in our breeding colony. In a similar manner, a-galactosidase and b-hexosaminidase activity levels were measured using 4-methylumbelliferyl a-Dgalactopyranoside and 4-methylumbelliferyl-N-acetyl-b-Dglucosaminide (Sigma, St Louis, MO, USA), respectively, as the substrates. One unit is defined as the amount of enzyme activity required to release 1 nmol of 4-methylumbelliferone per hour at 37 1C. Each sample was assayed for total protein content by using BCA Protein Assay kit (Pierce, Brebie`res, France). Glycosaminoglycan assay Delipidated tissue homogenates were dried, weighed and suspended in 3 ml of 100 mM sodium acetate buffer pH 5.5 containing 5 mM cysteine, 5 mM EDTA and papain (0.3% wt/vol), then left overnight at 65 1C for digestion. GAG concentration was determined in supernatants (200 ml) by measuring absorbance at 535 nm after the addition of dimethylmethylene blue dye solution (330 mM in format buffer 0.2 M pH 3.3, 2.5 ml). Heparan Gene Therapy

sulfate from bovine testis (Sigma, St Louis, MO, USA) was used as the standard. Data (mean of duplicates) were expressed per mg of dried tissue.

Histochemical detection of GUSB activity To detect GUSB activity by histochemical staining, 10mm-thick sections were obtained from frozen tissues, fixed with paraformaldehyde, and incubated with 25 mM naphtol AS-BI-N-acetyl-b-D-glucuronide (Sigma, St Louis, MO, USA) in 50 mM citrate buffer, pH 4.5, for 3 h at 4 1C. Subsequently, sections were incubated with 0.25 mM naphtol AS-BI-N-acetyl-b-D-glucuronide, 0.2 mg ml1 of Fast Garnet GBC salt (Sigma, St Louis, MO, USA) in 50 mM citrate buffer, pH 5.2, for 2 h at 20 1C. Quantitative reverse transcriptase-PCR Total RNA was isolated from the tissue using RNeasy kit (QIAGEN, Courtaboeuf, France) following the manufacturer’s protocol. The RNA concentrations were determined by using Ribogreen RNA quantification kit (Invitrogen, Cergy Pontoise, France). One microgram of total RNA was reverse transcribed in a final volume of 20 ml containing 4 ml of 5 RT buffer, 20 units of RNasin RNase inhibitor (Invitrogen), 10 mM DDT, 100 units of Superscript II RNase H- reverse transcriptase (Invitrogen), 3 mM random hexamers (Invitrogen) and 0.5 mM dNTP (NEB, Ipswich, MA, USA). The samples were incubated at 20 1C for 10 min, 42 1C for 30 min and 99 1C for 5 min. PCR primers for target genes were designed from GenBank sequences using Oligo6. Real-time PCR reactions were carried out using ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) in a 384-well, clear optical reaction plate with optical adhesive covers (Applied Biosystems). Reactions were run in a 5 ml volume in duplicate, with 2 ml of cDNA solution and 3 ml of a homemade targetspecific mix composed of 5/6 2 Power SYBR Green Master Mix (Applied Biosystems) and 1/6 of 10 mM primers solution. The PCR program was: 95 1C for 10 min, followed by 45 cycles of 15 s at 95 1C and 1 min at 60 1C. Cycle threshold values were obtained from the ABI PRISM 7900 SDS software (Applied Biosystems). Relative expression of mRNA for the target genes was determined using 18S rRNA as an internal standard. Plasmid quantification Total DNA was isolated from the tissue using QIAamp kit (QIAGEN) following the manufacturer’s protocol. The DNA concentrations were determined by measuring optical density at 260 nm. A total of 1 mg of DNA was used to transform 100 ml of competent bacteria (DH5a) according to the classical molecular biology protocols. We expressed the results as kanamycin-resistant colonies number, obtained using 1 mg of DNA. Subsequently, plasmid DNA was extracted and, as expected, its restriction profile was similar to pVAX-CAG-GUS profile. ELISA assay Blood was collected by eye bleed to measure antibodies to murine GUS by ELISA 8, 15, 30 or 45 days after plasmid injection. Analysis of sera from MPS VII injected mice was done by ELISA assay on microtiter aliquots. The wells of 96-well microtiter plates were coated


overnight at 4 1C with 4 mg ml1 purified recombinant murine b-glucuronidase in 15 mM Na2CO3, 35 mM NaHCO3, 0.02% NaN3, pH 9.6. The wells were washed three times with Tris-Buffered Saline Tween-20 (TBST) (10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20), then blocked for 1 h at room temperature with 3% casein in phosphate buffered saline (pH 7.2). After washing three times with TBST, 100 ml of serial 10-fold dilutions of mouse plasma (102–106) in TBST were added to the wells and incubated at 37 1C for 2.5 h. The wells were washed four times with TBST, then 100 ml of TBST containing a 1:1000 dilution of peroxidase-conjugated goat anti-mouse IgG was added to the wells and incubated at room temperature for 1 h. The wells were washed three times with TBST and two times with Tris buffered saline (10 mM Tris, pH 7.5, 150 mM NaCl). Peroxidase substrate (TMB solution, Sigma, St Louis, MO, USA) was added (200 ml per well) and plates were incubated at room temperature for 30 min. The reaction was stopped with the addition of 100 ml of 2N H2SO4 and the plates read at OD 450 nm on an automatic ELISA plate reader.

Histopathological analysis of the tissues (light microscopy) At the end point (30-day post injection) the mice were terminated by cervical dislocation and liver, spleen, brain, femur and knee joint was collected in all mice. The tissue samples were immersed in 4% formaldehyde and embedded in paraffin. Paraffin blocks were sectioned (5 mm-thick sections), sections were mounted on microscope slides, deparaffinized and stained with eosin–hematoxylin–saffron. Microscopic analysis and histopathological changes were evaluated by light microscopy using an Olympus BH2 microscope.

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We thank Dr B Soper for generously providing the gus / gusmps mice, as well as Dr J Grubb for kindly providing the murine b-glucuronidase protein. We thank also Dr J-M Caillaud (Biodoxis, Romainville, France) for his support in performing bright field microscopy histological analyses of tissues. This work was supported by European ‘MOLEDA’ STREP and European ‘CLINIGENE’ Network of Excellence. MR and AA received sponsorship from MOLEDA and CLINIGENE, respectively.

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