Mannitol-facilitated CNS entry of rAAV2 vector significantly delayed the neurological disease progression in MPS IIIB mice

Gene Therapy (2009) 16, 1340–1352 & 2009 Macmillan Publishers Limited All rights reserved 0969-7128/09 $32.00 www.nature.com/gt ORIGINAL ARTICLE Mannitol-facilitated CNS entry of rAAV2 vector significantly delayed the neurological disease progression in MPS IIIB mice DM McCarty1,2, J DiRosario1, K Gulaid1, J Muenzer3 and H Fu1,2 1 The Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, OH, USA, 2Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, OH, USA and 3Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA The presence of the blood–brain barrier (BBB) presents the most critical challenge in therapeutic development for mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease with severe neurological manifestation, because of a-N-acetylglucosaminidase (NaGlu) deficiency. Earlier, we showed a global central nervous system (CNS) transduction in mice by mannitol-facilitated entry of intravenous (IV)delivered recombinant adeno-associated viral serotype 2 (rAAV2) vector. In this study, we optimized the approach and showed that the maximal transduction in the CNS occurred when the rAAV2 vector was IV injected at 8 min after mannitol administration, and was approximately 10-fold more efficient than IV delivery of the vector at 5 or 10 min after mannitol infusion. Using this optimal (8 min) regimen, a single IV infusion of rAAV2-CMV-hNaGlu vector is therapeutically beneficial for treating the CNS disease of MPS IIIB in adult mice, with significantly extended survival, improved behavioral performance, and reduction of brain lysosomal storage pathology. The therapeutic benefit correlated with maximal delivery to the CNS, but not peripheral tissues. This milestone data shows the first effective gene delivery across the BBB to treat CNS disease. The critical timing of vector delivery and mannitol infusion highlights the important contribution of this pretreatment to successful intervention, and the long history of safe use of mannitol in patients bodes well for its application in CNS gene therapy. Gene Therapy (2009) 16, 1340–1352; doi:10.1038/gt.2009.85; published online 9 July 2009 Keywords: lysosomal storage disease; neuropathology; BBB; mannitol; rAAV; CNS gene delivery Introduction Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive lysosomal storage disease (LSD) caused by defects of a-N-acetylglucosaminidase (NaGlu), an enzyme, involved in degrading one of a group of biologically important glycosaminoglycans (GAG) in lysosomes.1 The primary pathology of MPS IIIB is lysosomal accumulation of heparan sulfate in somatic cells and the central nervous system (CNS), especially cells throughout the CNS, with complex secondary pathology manifestations in MPS IIIB mouse brain.2–8 MPS IIIB infants appear normal at birth, but develop progressive severe neurological manifestations, causing high mortality and premature death. The somatic manifestation in MPS IIIB is mild relative to other MPS, such as MPS I, II, and VII. No treatment is currently available for MPS IIIB. The disease is not amenable to either recombinant enzyme replacement therapy or hematopoietic stem cell transplantation, which have Correspondence: Dr H Fu, The Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, 700 Children’s Drive, Columbus, OH 43205, USA. E-mail: Received 6 January 2009; revised 2 June 2009; accepted 6 June 2009; published online 9 July 2009 been used to treat mostly somatic disorders in patients with MPS I, II, and IV.1,9,10 This is because the neurological pathology in MPS IIIB is global and the blood–brain barrier (BBB) precludes effective CNS access. Effective treatments for the majority of CNS diseases are rare, as the CNS is a unique system, located in a wellprotected environment, and isolated by a highly defined anatomical/functional barrier. The BBB is a cellular interface between the blood circulation and the CNS, formed mainly by capillary endothelial cells with tight junctions, and enhanced by surrounding cells.11,12 The BBB is completely formed at birth in human beings. In general, the BBB protects the CNS by selectively regulating the transport of molecules/agents from the blood circulation into the CNS or vice versa. Likewise, it also prevents potential therapeutic materials from entering the CNS. The presence of the BBB is the most critical challenge to developing therapies for CNS diseases, especially global CNS disorders, as targeting the entire CNS can be most effectively achieved only by systemic delivery through vasculature.13 Over the years, many strategies have been developed to deliver therapeutic agents into the CNS, though detailed mechanisms of BBB function remain to be largely unknown.11,14 These strategies include osmotic disruption of the BBB, receptor-mediated delivery by Mannitol-facilitated CNS entry of rAAV2 vector DM McCarty et al drug manipulation, and direct delivery bypassing the BBB.13,15–17 Mannitol, a well-characterized osmotic agent, has long been administered by intravascular infusion in routine medical practice for various purposes, the most important of which has been the temporary opening of the BBB.13 Earlier animal experiments have shown that disrupting the BBB with mannitol by an intra (carotid)arterial (IA) injection made the BBB permeable to a wide range of substances, including antibodies, enzymes, and viral vectors.13,18–23 Clinical studies in patients with brain tumors showed improved survival through mannitolfacilitated CNS delivery of chemotherapeutic drugs by IA injection through the carotid artery.15 Intravenous (IV) delivery offers potential benefits for treating global CNS diseases, as it would result in non-differential distribution throughout the CNS. Earlier studies showed that the peak opening of BBB in rats was 5 min after an IA infusion of mannitol and the opening lasted 20–30 min.24 However, the optimal conditions for IV injection of mannitol to disrupt the BBB, to enhance CNS entry of IVdelivered substances, were unclear. Gene therapy has great potential for treating LSDs, because of possible long-term transduction of affected cells, and the by-stander effect of secreted lysosomal enzymes, including NaGlu.25–27 The adeno-associated viral (AAV) vector system has been widely studied as a gene delivery tool for treating various diseases, with presented therapeutic effect. The recombinant AAV (rAAV) vectors have a broad spectrum of tissue tropism, which can be varied through the use of different serotypes. To date, no known pathogenesis has been linked to AAV in human beings.28 Earlier studies showed that rAAV vectors target both neuronal and nonneuronal (...truncated)