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High and Prolonged Sulfamidase Secretion by the Liver of MPS-IIIA Mice Following Hydrodynamic Tail Vein Delivery of Antibiotic-Free Pfar4 Plasmid Vector

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High and Prolonged Sulfamidase Secretion by the Liver of MPS-IIIA Mice Following Hydrodynamic Tail Vein Delivery of Antibiotic-Free Pfar4 Plasmid Vector Gene Therapy (2014) 21, 1001–1007 © 2014 Macmillan Publishers Limited All rights reserved 0969-7128/14 www.nature.com/gt ORIGINAL ARTICLE High and prolonged sulfamidase secretion by the liver of MPS-IIIA mice following hydrodynamic tail vein delivery of antibiotic-free pFAR4 plasmid vector M Quiviger1,2,3,4,AArfi1,2,3,4, D Mansard1,2,3,4, L Delacotte1,2,3,4, M Pastor1,2,3,4, D Scherman1,2,3,4 and C Marie1,2,3,4 Mucopolysaccharidosis type IIIA (MPS-IIIA) or Sanfilippo A syndrome is a lysosomal storage genetic disease that results from the deficiency of the N-sulfoglucosamine sulfohydrolase (SGSH) protein, a sulfamidase required for the degradation of heparan sulfate glycosaminoglycans (GAGs). The accumulation of these macromolecules leads to somatic organ pathologies, severe neurodegeneration and death. To assess a novel gene therapy approach based on prolonged secretion of the missing enzyme by the liver, mediated by hydrodynamic gene delivery, we first compared a kanamycin and an antibiotic-free expression plasmid vector, called pFAR4. Thanks to the reduced vector size, pFAR4 derivatives containing either a ubiquitous or a liver-specific promoter mediated a higher reporter gene expression level than the control plasmid. Hydrodynamic delivery of SGSH-encoding pFAR4 into MPS-IIIA diseased mice led to high serum levels of sulfamidase protein that was efficiently taken up by neighboring organs, as shown by the correction of GAG accumulation. A similar reduction in GAG content was also observed in the brain, at early stages of the disease. Thus, this study contributes to the effort towards the development of novel biosafe non-viral gene vectors for therapeutic protein expression in the liver, and represents a first step towards an alternative gene therapy approach for the MPS-IIIA disease. Gene Therapy (2014) 21, 1001–1007; doi:10.1038/gt.2014.75; published online 21 August 2014 INTRODUCTION decreased content in prokaryotic sequences, as the size of the Gene therapy involves the use of DNA, RNA, viral or non-viral- t-RNA gene is smaller than that of the kanamycin-resistance gene. based vectors. For non-viral approaches, naked plasmid DNA In the present study, our first objective was to identify a potent fi represent the most popular gene vectors as this subclass now expression vector for ef cient transfection of hepatocytes follow- accounts for more than 18% of all gene therapy clinical trials, just ing hydrodynamic tail vein injection, with the aim of proposing an behind those involving adenoviruses and retroviruses.1 This alternative gene therapy approach for the treatment of the fi relatively high percentage most probably results from the San lippo A or mucopolysaccharidosis type IIIA (MPS-IIIA) disease. continuous improvement of both gene carriers and physical MPS-IIIA belongs to the lysosomal storage disease (LSD) family that regroups more than 60 inherited metabolic monogenic delivery techniques such as electroporation, sonoporation or – diseases specified by the types of accumulating macromolecules hydrodynamics-based techniques.2 7 in lysosomal organelles. Patients suffering from MPS-IIIA are Recently, we developed a novel antibiotic-free expression deficient in the sulfamidase protein (SGSH, EC3.10.1.1) that cleaves vector, called pFAR4.8 With this system, selection of plasmid- the glucosamine-N-sulfate bonds at the non-reducing end of containing bacteria relies on the suppression, by a plasmid-borne glycosaminoglycan heparan sulfate (GAG-HS). The resulting function, of an amber nonsense mutation introduced into an undigested GAG-HS accumulates in all body cells generating essential Escherichia coli gene. The 1.1-kb pFAR4 vector was de severe and progressive neurodegeneration, hepatosplenomegaly novo entirely synthesized, thus allowing the removal of redundant as well as skeletal and joint malformations.11,12 First clinical signs sequences to mostly conserve a replication origin and a are usually noted at a median age of 2.5 years and consist of suppressor t-RNA selection marker regulated by prokaryotic 8 developmental delay (for example, speech) and/or behavioral regulatory elements. The potency of pFAR4 plasmids as problems (such as sleep disturbances, hyperactivity, aggressive- eukaryotic expression vectors was demonstrated in muscle, tumor ness and anxiety). Other symptoms include recurrent diarrhea, and skin cells, in which transgene expression was found to be frequent upper airway infections, hearing and visual impairment, higher, prolonged or both, as compared with a kanamycin- and epilepsy. Full stagnation of development occurs at around the resistance vector containing the same eukaryotic expression age of 3–4 years, and patients with severe or less attenuated form cassette.8 Thus, not only are pFAR4 derivatives safer, but they of the disease usually die by the mid to late teenage years.11,13 also display a higher efficiency for gene delivery and expression, Several therapeutic approaches have been evaluated. which most probably results from their reduced size8–10 and a They include gene therapies (using either adeno-associated14–16 1Unité de Technologies Chimiques et Biologiques pour la Santé, Faculté de Pharmacie, Université Paris Descartes, Paris, France; 2Ecole Nationale Supérieure de Chimie de Paris, Chimie Paristech, Paris, France; 3CNRS, UMR8258, Paris, France and 4Inserm, U1022, Paris, France. Correspondence: Dr C Marie, INSERM U1022 – CNRS UMR8258 - Unité de Technologies Chimiques et Biologiques pour la Santé, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, 4 avenue de l’Observatoire, Paris F-75270, France. E-mail: [email protected] Received 16 August 2013; revised 17 May 2014; accepted 15 July 2014; published online 21 August 2014 Delivery of pFAR4 plasmids to MPS-IIIA mouse liver M Quiviger et al 1002 or lentivirus vectors17), SGSH-transduced hematopoietic stem liver-specific promoter in combination with pFAR4 was selected as cells,18 enzyme replacement therapy (ERT)19,20 or substrate reduc- this combination mediated a prolonged transgene expression. tion therapy (SRT).21–23 From these studies, two clinical trials have In order to determine whether similar results could be obtained been launched. The first one is based on the administration of the with a therapeutic gene product, the murine SGSH cDNA recombinant protein into the cerebrospinal fluid (CSF) via an expressed from the hAAT promoter was introduced into either implanted intrathecal drug delivery device (#NCT01155778 and the pFAR4 or the pKAR4 vector. Hydrodynamic injection of the #NCT01299727, clinical trials.gov), whereas the second one is based pFAR4 derivative led to a higher level of serum sulfamidase on an intracerebral injection of sulfamidase-expressing adeno- activity, which was remarkably prolonged over time, in contrast to associated vector to four patients in a phase I/II clinical trial the rapid decline obtained after delivery of the pKAR4 derivative (#NCT01474343). After a year of follow-up, treatment was estimated (Figure 1b). This tendency lasted for more than 4 months and to be safe and well tolerated but requiring some adjustments, as could not be attributed to a loss in plasmid copy number neuropsychological evaluations revealed a possible albeit moderate (Supplementary Information 1). When compared with the pKAR4 improvement.24 As there is no widely available safe and effective derivative expressing SGSH, analyses of serum cytokine (inter- treatment for this disease, the objectives of our work were therefore leukin-12 and tumor necrosis factor-α) and aspartate transaminase dual: (a) to assess the potency of pFAR4 derivatives as expression levels showed that pFAR4-hAAT-SGSH plasmid did not exert any vectors in hepatocytes after plasmid hydrodynamic delivery and (b) adverse effect in liver (Supplementary Information 2A–C). to use the pFAR4 vector in a therapeutic context, namely in an MPS- Furthermore, in contrast to what was observed with the mSEAP- IIIA mouse model, which contains a spontaneous mutation in the encoding plasmid, an ~ 50% initial reduction of serum sulfamidase SGSH gene and exhibits many of the clinical manifestations of the activity was not observed at early time points. This could neither human disease.25 In this approach, liver is used as a manufacturing be attributed to the plasmid amount used (as a similar result was organ to secrete the sulfamidase protein, taking advantage of the obtained after the injection of 100 μg of pFAR4-hAAT-mSEAP, property of lysosomal proteins, which are glycosylated and Supplementary Information 3A) nor to a shorter protein half-life, endocytosed by mannose 6-phosphate receptor-expressing cells estimated to be o13 h for mSEAP and o7 h for mSGSH and thereafter trafficked to the lysosomes to perform its enzymatic (Supplementary Information 3B and C). Instead, it could be function, thus allowing cross correction in distant cells.26 attributed to a decrease in plasmid copy number in transfected hepatocytes (Supplementary Information 3A), which do not naturally secrete the embryonic SEAP protein. RESULTS Data in the literature report that plasmid hydrodynamic delivery 27,28 fi fi predominantly leads to the transfection of liver cells. In order Identi cation of the optimal non-viral vector for ef cient gene fi expression in the liver to challenge the speci city of the hAAT promoter carried by the newly designed pFAR4 vector, the luciferase gene encoding a In order to assess the potency of pFAR4 to deliver and express non-secreted protein was introduced downstream from the liver- eukaryotic genes in the liver, the murine secreted alkaline specific promoter. The pFAR4-hAAT-Luc plasmid was tail vein fi phosphatase (mSEAP) reporter protein was rst used to long- hydrodynamically injected into both healthy and diseased mice, in itudinally monitor transgene expression. The mSEAP gene was order to assess whether the pathology of MPS-IIIA mice (which cloned downstream from either the ubiquitous CAG (CMV early includes hepatosplenomegaly) could affect plasmid biodistribu- enhancer/chicken β actin promoter) or the 415-bp liver-specific tion. Monitoring of luciferase expression 2 or 8 days after plasmid hAAT (human alpha-1 antitrypsin) promoter.
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