Macrophage enzyme and reduced inflammation drive brain correction of mucopolysaccharidosis IIIB by stem cell gene therapy

doi:10.1093/brain/awx311 BRAIN 2018: 141; 99–116 | 99 Macrophage enzyme and reduced inflammation drive brain correction of mucopolysaccharidosis IIIB by stem cell gene therapy Rebecca J. Holley,1 Stuart M. Ellison,1 Daniel Fil,1 Claire O’Leary,1 John McDermott,1 Nishanthi Senthivel,1 Alexander W. W. Langford-Smith,1,2 Fiona L. Wilkinson,1,2 Zelpha D’Souza,1 Helen Parker,1 Aiyin Liao,1 Samuel Rowlston,1 Hélène F. E. Gleitz,1 Shih-Hsin Kan,3 Patricia I. Dickson3 and Brian W. Bigger1 Mucopolysaccharidosis IIIB is a paediatric lysosomal storage disease caused by deficiency of the enzyme a-N-acetylglucosaminidase (NAGLU), involved in the degradation of the glycosaminoglycan heparan sulphate. Absence of NAGLU leads to accumulation of partially degraded heparan sulphate within lysosomes and the extracellular matrix, giving rise to severe CNS degeneration with progressive cognitive impairment and behavioural problems. There are no therapies. Haematopoietic stem cell transplant shows great efficacy in the related disease mucopolysaccharidosis I, where donor-derived monocytes can transmigrate into the brain following bone marrow engraftment, secrete the missing enzyme and cross-correct neighbouring cells. However, little neurological correction is achieved in patients with mucopolysaccharidosis IIIB. We have therefore developed an ex vivo haematopoietic stem cell gene therapy approach in a mouse model of mucopolysaccharidosis IIIB, using a high-titre lentiviral vector and the myeloidspecific CD11b promoter, driving the expression of NAGLU (LV.NAGLU). To understand the mechanism of correction we also compared this with a poorly secreted version of NAGLU containing a C-terminal fusion to IGFII (LV.NAGLU-IGFII). Mucopolysaccharidosis IIIB haematopoietic stem cells were transduced with vector, transplanted into myeloablated mucopolysaccharidosis IIIB mice and compared at 8 months of age with mice receiving a wild-type transplant. As the disease is characterized by increased inflammation, we also tested the anti-inflammatory steroidal agent prednisolone alone, or in combination with LV.NAGLU, to understand the importance of inflammation on behaviour. NAGLU enzyme was substantially increased in the brain of LV.NAGLU and LV.NAGLU-IGFII-treated mice, with little expression in wild-type bone marrow transplanted mice. LV.NAGLU treatment led to behavioural correction, normalization of heparan sulphate and sulphation patterning, reduced inflammatory cytokine expression and correction of astrocytosis, microgliosis and lysosomal compartment size throughout the brain. The addition of prednisolone improved inflammatory aspects further. Substantial correction of lysosomal storage in neurons and astrocytes was also achieved in LV.NAGLU-IGFII-treated mice, despite limited enzyme secretion from engrafted macrophages in the brain. Interestingly both wild-type bone marrow transplant and prednisolone treatment alone corrected behaviour, despite having little effect on brain neuropathology. This was attributed to a decrease in peripheral inflammatory cytokines. Here we show significant neurological disease correction is achieved using haematopoietic stem cell gene therapy, suggesting this therapy alone or in combination with anti-inflammatories may improve neurological function in patients. 1 Stem Cell and Neurotherapies, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK 2 Vascular Pathology Group, Centre for Biomedicine, School of Healthcare Science, Faculty of Science and Engineering, Manchester Metropolitan University, John Dalton Building, Chester Street, Manchester, M1 5GD, UK 3 Department of Paediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, CA, 90502, USA Correspondence to: Professor Brian Bigger, Stem Cell and Neurotherapies, Division of Cell Matrix Biology and Regenerative Medicine, Received June 26, 2017. Revised September 27, 2017. Accepted September 29, 2017. Advance Access publication November 27, 2017 ß The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: 100 | BRAIN 2018: 141; 99–116 R. J. Holley et al. Faculty of Biology, Medicine and Health, 3.721 Stopford Building, Oxford Road, University of Manchester, Manchester, M13 9PT, UK E-mail: Keywords: lysosomal storage disease; microglia; mucopolysaccharidosis; neurodegeneration; stem cells Abbreviations: AAV = adeno-associated virus; CS = chondroitin sulphate; DS = dermatan sulphate; HSCGT = haematopoietic stem cell gene therapy; HSCT = haematopoietic stem cell transplant; IB4 = isolectin B4; IGFII = insulin-like growth factor 2; LV = lentiviral vector; MPSIIIB = mucopolysaccharidosis IIIB; UA = uronic acid Introduction Mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo B syndrome) is a neurodegenerative lysosomal storage disease caused by a deficiency in the glycosaminoglycan degrading enzyme, a-N-acetylglucosaminidase (NAGLU). NAGLU deficiency results in the global accumulation of partially degraded heparan sulphate in cells, leading to cellular dysfunction, which is particularly apparent in the brain. Symptom onset begins within the first 2 to 4 years of life, characterized by a plateau in development, speech delay, and worsening behavioural problems, together with minor somatic indicators such as organomegaly and facial dysmorphia. Progressive motor impairment follows leading to greatly shortened life expectancy. There are currently no effective treatments. Enzyme replacement therapy has successfully treated somatic disease in other mucopolysaccharidosis disorders. Crosscorrection occurs via mannose-6-phosphate mediated enzyme uptake by deficient cells. However, the inability of delivered enzyme to cross the blood–brain barrier limits its worth in MPSIIIB, where the main pathology is neurological. Approaches to deliver enzyme via an intracerebroventricular route are in progress, but these methods are invasive. A C-terminal fusion of NAGLU to insulin-like growth factor 2 (rhNAGLU-IGFII), which also binds the mannose-6-phosphate receptor leads to more efficient uptake, lysosomal targeting and activity following intracerebroventricular injection in MPSIIIB mice (Kan et al., 2014a, b). Another option for patients with MPSIIIB is delivery of adeno-associated virus (AAV)-based gene therapy vectors via a variety of routes to treat the brain (Fu et al., 2002, 2007, 2010; Cressant et al., 2004). One hurdle in developing these therapies is achieving effective vector distribution from the sites of injection, something that can be effectively achieved in rodents but not in patients to date (Tardieu et al., 2014, 2017). Haematopoietic stem cell transplant (HSCT), enables donor haematopoietic stem cells to repopulate bone marrow of myeloablated recipients, with donor-engrafted leucocytes secreting enzyme to cross-correct somatic cells. Bone marrow-derived monocytes are also able to cross the blood (...truncated)