Delivery of an enzyme-IGFII fusion protein to the mouse brain is therapeutic for mucopolysaccharidosis type IIIB Shih-hsin Kana, Mika Aoyagi-Scharberb,1, Steven Q. Lea, Jon Vinceletteb, Kazuhiro Ohmic, Sherry Bullensb, Daniel J. Wendtd, Terri M. Christiansonb, Pascale M. N. Tigerb, Jillian R. Brownb, Roger Lawrenceb, Bryan K. Yipb, John Holtzingerb, Anil Bagrib, Danielle Crippen-Harmonb, Kristen N. Vondraka, Zhi Chend, Chuck M. Hagued, Josh C. Woloszynekb, Diana S. Cheungb, Katherine A. Websterb, Evan G. Adintorib, Melanie J. Lob, Wesley Wongb, Paul A. Fitzpatrickb, Jonathan H. LeBowitzb, Brett E. Crawfordb, Stuart Buntingb, Patricia I. Dicksona,1, and Elizabeth F. Neufeldc,1 aDepartment of Pediatrics, Los Angeles Biomedical Research Institute at Harbor–UCLA Medical Center, Torrance, CA 90502; bResearch and Drug Discovery and dAnalytical Chemistry, BioMarin Pharmaceutical, Inc., Novato, CA 94949; and cDepartment of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095 Contributed by Elizabeth F. Neufeld, August 29, 2014 (sent for review August 3, 2014; reviewed by Roscoe O. Brady and William S. Sly) Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome (NAGLU), there is an additional difficulty in that, in contrast to type B) is a lysosomal storage disease characterized by profound in- most other soluble lysosomal enzymes, recombinant NAGLU tellectual disability, dementia, and a lifespan of about two decades. produced in Chinese hamster ovary (CHO) cells contains little or Thecauseismutationinthegeneencodingα–N-acetylglucosaminidase no mannose 6-phosphate (Man6-P) (9–11), the signal for receptor- (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. mediated endocytosis and targeting to lysosomes (12, 13). The Impediments to enzyme replacement therapy are the absence of man- reason for the lack of the Man6-P modification is not understood – nose 6-phosphate on recombinant human NAGLU and the blood and appears to apply only to the recombinant enzyme, as human brain barrier. To overcome the first impediment, a fusion protein of urinary NAGLU (14) and endogenous NAGLU made by CHO recombinant NAGLU and a fragment of insulin-like growth factor II cells (9) contain Man6-P. (IGFII) was prepared for endocytosis by the mannose 6-phosphate/ The lack of Man6-P on the enzyme can be overcome by taking IGFII receptor. To bypass the blood–brain barrier, the fusion protein (“enzyme”) in artificial cerebrospinal fluid (“vehicle”) was adminis- advantage of the ability of the cation-independent Man6-P re- tered intracerebroventricularly to the brain of adult MPS IIIB mice, ceptor to bind insulin-like growth factor II (IGFII) at a site – four times over 2 wk. The brains were analyzed 1–28 d later and distinct from the Man6-P binding sites (15 19). Fusion proteins compared with brains of MPS IIIB mice that received vehicle alone consisting of a fragment of IGFII linked to a lysosomal enzyme or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the Significance brain, where it persisted with a half-life of approximately 10 d. Hep- aran sulfate, and especially disease-specific heparan sulfate, was Mucopolysaccharidosis type IIIB (MPS IIIB) is a devastating and reduced to control level. A number of secondary accumulations currently untreatable disease affecting mainly the brain. The in neurons [β-hexosaminidase, LAMP1(lysosome-associated mem- cause is lack of the lysosomal enzyme, α–N-acetylglucosami- brane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), nidase (NAGLU), and storage of heparan sulfate. Using a mouse glypican 5, β-amyloid, P-tau] were reduced almost to control level. model of MPS IIIB, we administered a modified NAGLU by in- CD68, a microglial protein, was reduced halfway. A large amount of jection into the left ventricle of the brain, bypassing the blood– enzyme also appeared in liver cells, where it reduced heparan sulfate brain barrier. The modification consisted of a fragment of IGFII, and β-hexosaminidase accumulation to control levels. These results which allows receptor-mediated uptake and delivery to lyso- suggest the feasibility of enzyme replacement therapy for MPS IIIB. somes. The modified enzyme was taken up avidly by cells in both brain and liver, where it reduced pathological accumula- ucopolysaccharidosis type III (MPS III, Sanfilippo syn- tion of heparan sulfate and other metabolites to normal or Mdrome) is a heritable lysosomal disorder of heparan sulfate near-normal levels. The results suggest the possibility of treat- degradation, divided into four types (A–D), depending on the ment for MPS IIIB. enzyme deficiency (1, 2). All four MPS III types are characterized Author contributions: M.A.-S., J.V., S. Bullens, D.J.W., T.M.C., P.A.F., J.H.L., B.E.C., S. Bunting, by severe neurologic problems and relatively mild somatic ones. P.I.D., and E.F.N. designed research; S.-h.K., S.Q.L., J.V., K.O., D.J.W., T.M.C., P.M.N.T., J.R.B., Profound intellectual disability that progresses to dementia, be- R.L., B.K.Y., J.H., A.B., D.C.-H., K.N.V., Z.C., C.M.H., J.C.W., D.S.C., K.A.W., E.G.A., M.J.L., and havioral disturbances, and death in the second or third decade W.W. performed research; S.-h.K., M.A.-S., S.Q.L., K.O., S. Bullens, D.J.W., T.M.C., J.R.B., R.L., bring untold suffering to the MPS III patients and their families. B.K.Y., J.H., A.B., D.C.-H., Z.C., C.M.H., J.C.W., K.A.W., E.G.A., B.E.C., P.I.D., and E.F.N. ana- lyzed data; and S.-h.K., M.A.-S., and E.F.N. wrote the paper. Despite the dire need, treatment for the MPS III disorders has Reviewers: R.O.B., National Institute of Neurological Disorders and Stroke; and W.S.S., lagged behind other MPS diseases. Hematopoietic stem cell trans- Saint Louis University School of Medicine. plantation, an effective procedure for MPS I patients with CNS in- Conflict of interest statement: M.A.-S., J.V., S. Bullens., D.J.W., T.M.C., P.M.N.T., J.R.B., R.L., volvement (3), is not effective for MPS III (4). Enzyme replacement B.K.Y., J.H., A.B., D.C.-H., Z.C., C.M.H., J.C.W., D.S.C., K.A.W., E.G.A., M.J.L., W.W., P.A.F., therapy has been available for some years for several MPS with J.H.L., B.E.C., and S. Bunting are employees of BioMarin Pharmaceutical, Inc., which is developing a commercial treatment for mucopolysaccharidosis type IIIB. E.F.N. is principal extensive somatic involvement [MPS I (5, 6), II (7), and VI (8)], or is investigator of a sponsored research agreement with BioMarin Pharmaceutical, Inc. newly approved (MPS IVA), or in clinical trial (MPS VII). However, Freely available online through the PNAS open access option. development of enzyme replacement for MPS III did not seem 1To whom correspondence may be addressed. Email: [email protected], promising because access to therapeutic enzyme to brain paren- [email protected], or [email protected]. – chyma would be limited by the blood brain barrier. With respect to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. MPS IIIB, a deficiency of α–N-acetylglucosaminidase, EC 3.2.1.50) 1073/pnas.1416660111/-/DCSupplemental. 14870–14875 | PNAS | October 14, 2014 | vol. 111 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1416660111 Downloaded by guest on October 1, 2021 with no Man6-P have been made for β-glucuronidase (20), α-glu- cosidase (21), and NAGLU (11), and shown to have enzymatic activity similar to that of the original lysosomal enzyme and to be taken up by cultured cells in a manner that is dependent on IGFII and independent of Man6-P. In addition, the β-glucuronidase– and α-glucosidase–IGFII fusion proteins, administered i.v. to de- ficient mice, were found to be taken up by major somatic organs and muscles, respectively, in which they functioned to reduce storage and pathology (20, 21). On the basis of these promising earlier studies, we treated the brain of the MPS IIIB mouse by administering a NAGLU–IGFII fusion protein directly into the left cerebral ventricle, bypassing the blood–brain barrier. The modified enzyme was endocytosed mainly into neurons, where it Fig. 2. Uptake of NAGLU–IGFII by MPS IIIB mouse brain after intra- functioned to reduce the level of stored heparan sulfate and of cerebroventricular administration of NAGLU–IGFII. (A) NAGLU taken up, other accumulated substances to a normal or near-normal level. It measured as NAGLU catalytic activity in homogenates of slice 1. Measure- also spread to the liver, where it was endocytosed into vascular cells ments are in units per milligram of protein, expressed as mean ± SD. The and hepatocytes and eliminated storage of heparan sulfate. The brains were taken at the times indicated (in days from the last injection) from – results suggest that the combined use of the IGFII signal for en- mice treated with NAGLU IGFII (ENZ) or vehicle (VEH). The number of mouse docytosis with administration directly into the brain may overcome brains analyzed at each time point, going from left to right, was 7, 4, 8, 4, 4, 4, and 8. (B) Immunohistochemical staining for NAGLU in cortex (slice 2) and in the major obstacles to enzyme replacement therapy for MPS IIIB. CA3 layer of the hippocampus (slice 4), in the mutant mice treated with ve- A preliminary account of this work has been presented in hicle or with enzyme, as indicated.