36. Intrathecal enzyme replacement therapy treats meningeal storage and spinal cord compression in MPS I dogs

36. Intrathecal enzyme replacement therapy treats meningeal storage and spinal cord compression in MPS I dogs

Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41 that CLN3 and CLN6 proteins function at distinct steps in a common pathway. Cell biologic...

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Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41 that CLN3 and CLN6 proteins function at distinct steps in a common pathway. Cell biological screening of the mutant neuronal precursor cells (CbCln6 and CbCln3 cells) revealed altered ER marker stain in homozygous CbCln6 mutant cells, and both genetic models exhibited reduced endocytosis and altered Lysotracker stain but with distinct subcellular rearrangements of these organelles. Pathways-based global gene expression analyses further identified overlapping yet distinct alterations, including commonly altered pathways related to energy metabolism, ion transport, proteolysis/degradation, and trafficking, and unique ER-related changes in CbCln6 mutant cells. Investigation of abnormal ATP synthase subunit c accumulation revealed that autophagy is disrupted by CLN3 and CLN6 NCL disease mutations, though likely via distinct mechanisms. Therefore, these data support the hypothesis that the common disease features of variant late-infantile NCL and juvenile NCL result from defects at different steps in common or overlapping pathways that lead to abnormal accumulation of ATP synthase subunit c protein and neuronal cell dysfunction. doi:10.1016/j.ymgme.2009.10.049

33. Assessment of neurological deterioration in subjects with LINCL Ronald Crystal, Joan & Stanford Weill Medical College of Cornell University, New York, NY, USA Late infantile ceroid lipofuscinosis (LINCL) is a rare, rapidly progressing lysosomal storage disease. The rareness of the patients as well as the possibility of non-uniform progression depending on genotype mean that limited data is available that delineates the natural history of disease progression. The primary focus of this study is to use clinical rating scales and magnetic resonance imaging methods to define the natural history of LINCL and to provide objective and sensitive surrogates for neurological status and for the assessment of the impact of experimental treatments in children with LINCL. To achieve this goal we have three aims: (1) recruit children with LINCL and perform serial neurological assessments and MRI studies; (2) using this data, expand the spectrum of existing quantitative MRI parameters and derive normal ranges and correlate with neurological status and specific mutations; and (3) extract additional parameters from MRI data including volumes of brain substructures, local metabolite levels by magnetic resonance spectroscopy and local diffusion weighted imaging. Together, these parameters will be applicable to future clinical studies of novel therapies for LINCL, and should be transferable to other neurological lysosomal storage diseases. doi:10.1016/j.ymgme.2009.10.050

34. Macroautophagy is defective in mucolipin 1-deficient mouse neurons Cyntia Curcio-Morellia, Florie A. Charlesa, Matthew C. Micsenyib, Yi Caoa, Bhuvarahamurthy Venugopala, Marsha F. Browninga, Kostantin Dobrenisb, Susan L. Cotmana, Steven U. Walkleyb, Susan A. Slaugenhaupta, aCenter for Human Genetic Research, Massachusetts General Hospital/Harvard Medical School, Boston, USA, b Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, USA Mucolipidosis Type IV is a neurodegenerative lysosomal disease clinically characterized by psychomotor retardation, visual impairment and achlorhydria. Prior to this study, functional characterization in MLIV cells has been limited to fibroblast cultures gleaned from patients. Here we report the generation and characterization of neuronal cultures from the cerebrum of Mcoln1 / embryos. The Mcoln1 / neuronal cultures show an increase in the number and size of LysoTracker positive-vesicles. Electron microscopy studies reveal significant membranous intracytoplasmic storage bodies, which correlate with the storage morphology observed in cerebral cortex of Mcoln1 / pups. Using this neuronal model system, we show that macroautophagy is defective in mucolipin-1 deficient neurons and that LC3-II levels are significantly elevated. This increase appeared to be mediated by Beclin-1 and not by the mammalian target of rapamycin. P62/SQSTM1 levels were increased in Mcoln1 / neuronal cultures. Treatment with rapamycin plus protease inhibitors failed to increase levels of LC3-II in Mcoln1 / neuronal cultures, suggesting that the lack of mucolipin-1 affects macroautophagy flux. This study describes, for the first time, a defect in autophagy in mucolipin-1 deficient neurons, which provides new insight into the neuronal pathogenesis of this disease. doi:10.1016/j.ymgme.2009.10.051

35. Disease brain endothelia provide unique molecular signatures for CNSdirected enzyme therapy Beverly Davidson, Yong-Hong Chen, Michael Chang, University of Iowa, Iowa City, IA, USA


Vascular endothelial cells vary among organs and as a consequence of disease status. Moreover, transduced endothelial cells demonstrate basolateral secretion of gene product, illustrating the potential for correction of the vascularized tissue. Therefore, the brain vascular endothelium represents a promising target for gene therapies of neurodegenerative disorders caused by lysosomal storage disease (LSD), in which the recombinant enzyme expressed in, and secreted from, the vascular endothelia will be endocytosed by underlying neurons and glia, decreasing neuropathology. Here we tested the hypothesis that endothelia lining these vessels can be harnessed to create a cellular reservoir of enzyme replacement therapy to diseased brain. In our approach to develop brain vascular endothelium targeted AAV, we used animals with central nervous system (CNS) deficits due to LSD. We screened a phage library invivo to identify peptides that mediate selective and efficient binding to brain endothelial cells in diseased and wildtype mice. Surprisingly, epitopes binding diseased brain were distinct from those panned from normal brain. Moreover, different epitopes were panned out of different disease models, implying a unique vascular signature imparted by the disease state. Importantly, presentation of these epitopes on the capsid of adeno-associated virus (AAV) expanded the biodistribution of IV-injected AAV from predominantly liver to include the CNS. Peripheral injection of the epitope-modified AAVs expressing the enzymes lacking in LSD mice reconstituted enzyme activity throughout the brain and improved disease phenotypes in two distinct models. doi:10.1016/j.ymgme.2009.10.052

36. Intrathecal enzyme replacement therapy treats meningeal storage and spinal cord compression in MPS I dogs Patricia Dicksona, N.M. Ellinwoodb, A. Dierenfeldb, K. Klineb, J. Parkesb, S. Hansonc, C. Vited, A. Mlikotica, A. Chena, W. Grossb, M. Haskinsd, K. Pondere, S. Lea, aLA Biomed at Harbor-UCLA, Torrance, USA, bAnimal Science, Iowa State University, Ames, USA, c Veterinary Neurology Center, Tustin, USA, dSchool of Veterinary Medicine, University of Pennsylvania, Philadelphia, USA, eWashington University School of Medicine, St. Louis, USA Intrathecal enzyme replacement therapy (IT ERT) was studied long-term and from birth in MPS I dogs for treatment of spinal cord compression. Four MPS I dogs age 15–16 mo. received weekly IV ERT + IT ERT (duration 1 year, group Adult IV + IT). Four MPS I dogs received only weekly IV ERT (group Adult IV). Four MPS I dogs 7– 23 days old received weekly IV ERT + IT ERT for 65–81 weeks (group Early IV + IT). Eight MPS I dogs 3–9 days old received IV ERT weekly at 0.58 mg/kg/wk (n = 4, Early Low IV) or 1.57 mg/kg/wk (n = 4, Early High IV). At study start, all adult dogs had compression of their spinal cord, three of four had a large third ventricle and all had minor neurologic signs. Two Adult IV + IT dogs showed improvement in neurologic examination; two showed no change. All four Adult IV dogs had worsened neurologic examination. No Early treated dog developed myelopathy, though four in Early Low IV or IV + IT group developed tenderness over areas of intervertebral narrowing. Meninges GAG was lower than age-matched untreated MPS I dogs in Early Low IV and Early High IV groups, but was lowest in IV + IT groups, reaching normal levels in two Early IV + IT dogs (mean of cervical, thoracic, and lumbar areas). Spinal MRI are being evaluated. IT ERT may stabilize or reverse neurologic signs of spinal cord compression and prevent meningeal GAG storage in MPS I dogs. doi:10.1016/j.ymgme.2009.10.053

37. Cervical cord compression in mucopolysaccharidosis diseases Brenda Diethelm-Okita, Chester B. Whitley, University of Minnesota, Minneapolis, USA Background: Because of its debilitative effect, cervical cord compression is an important complication of mucopolysaccharidosis (MPS) conditions. Via pachymeningitis cervicalis, cervical cord compression leads to loss of sensation and function in the extremities causing, impaired gait, loss of manual dexterity, and decreased bowel and bladder control. The standard treatment has been decompressive laminectomy, a surgical procedure with uncertain outcomes, which may be syndrome dependant. Of the new treatment modalities, hematopoietic stem cell transplantation (HSCT) reduces the GAG level in the CNS but, because of the blood–brain barrier, enzyme replacement therapy (ERT) does not. Hypothesis: We assess the outcomes associated with surgical de-compression for a cohort of patients with MPS at the University of Minnesota, and from the literature review. Outcomes were assessed through a ratings scale ranging from 0 to 4, where 0 equals death, 1 equals significant loss of function, 2 equals minor loss of function, 3 equals no change, and 4 is equivalent to improvement over pre-operative condition. Scores were assigned by physician assessment. Results: Literature review suggests that surgical correction for cord compression is successful over 90% of the time (19 of 22 patients, average score = 3.7). In contrast 75% of the cohort of patients at the University of Minnesota had less than favorable outcomes (6 of 8, x = 1.3). Our results indicate that surgical correction of cervical cord compression in MPS disorders may have poorer outcomes than previously reported,