Maria Prociuk

Miglustat Improves Purkinje Cell Survival and Alters Microglial Phenotype in Feline Niemann-Pick Disease Type C

Abstract Niemann-Pick disease type C (NPC disease) is an incurable cellular lipid-trafficking disorder characterized by neurodegeneration and intralysosomal accumulation of cholesterol and glycosphingolipids. Treatment with miglustat, a small imino sugar that reversibly inhibits glucosylceramide synthase, which is necessary for glycosphingolipid synthesis, has been shown to benefit patients with NPC disease. The mechanism(s) and extent of brain cellular changes underlying this benefit are not understood. To investigate the basis of the efficacy of miglustat, cats with disease homologous to the juvenile-onset form ofhuman NPC disease received daily miglustat orally beginning at 3weeks of age. The plasma half-life of miglustat was 6.6 ± 1.1 hours, with a tmax, Cmax, and area under the plasma concentration-time curve of 1.7 ± 0.6 hours, 20.3 ± 4.6 &mgr;g/mL, and 104.1 ± 16.6 &mgr;g hours/mL, respectively. Miglustat delayed the onset of neurological signs and increased the lifespan of treated cats and was associated with decreased GM2 ganglioside accumulation in the cerebellum and improved Purkinje cell survival. Ex vivo examination of microglia from the brains oftreated cats revealed normalization of CD1c and class II major histocompatibility complex expression, as well as generation of reactive oxygen species. Together, these results suggest that prolonged Purkinje cell survival, reduced glycosphingolipid accumulation, and/or the modulation of microglial immunophenotype and function contribute to miglustat-induced neurological improvement in treated cats.

Adeno-associated virus serotypes 9 and rh10 mediate strong neuronal transduction of the dog brain

Canine models have many advantages for evaluating therapy of human central nervous system (CNS) diseases. In contrast to nonhuman primate models, naturally occurring canine CNS diseases are common. In contrast to murine models, the dog’s lifespan is long, its brain is large and the diseases affecting it commonly have the same molecular, pathological and clinical phenotype as the human diseases. We compared the ability of four intracerebrally injected adeno-associated virus vector (AAV) serotypes to transduce the dog brain with green fluorescent protein as the first step in using these vectors to evaluate both delivery and efficacy in naturally occurring canine homologs of human diseases. Quantitative measures of transduction, maximum diameter and area, identified both AAV2/9 and AAV2/rh10 as significantly more efficient than either AAV2/1 or AAV2/5 at transducing cerebral cortex, caudate nucleus, thalamus and internal capsule. Fluorescence co-labeling with cell-type-specific antibodies demonstrated that AAV2/9 and AAV2/rh10 were capable of primarily transducing neurons, although glial transduction was also identified and found to be more efficient with the AAV2/9 vector. These data are a prerequisite to evaluating the efficacy of recombinant AAV vectors carrying disease-modifying transgenes to treat naturally occurring canine models in preclinical studies of human CNS disease therapy.

Cerebrospinal Fluid Calbindin D Concentration as a Biomarker of Cerebellar Disease Progression in Niemann-Pick Type C1 Disease.

Niemann-Pick type C (NPC) 1 disease is a rare, inherited, neurodegenerative disease. Clear evidence of the therapeutic efficacy of 2-hydroxypropyl-β-cyclodextrin (HPβCD) in animal models resulted in the initiation of a phase I/IIa clinical trial in 2013 and a phase IIb/III trial in 2015. With clinical trials ongoing, validation of a biomarker to track disease progression and serve as a supporting outcome measure of therapeutic efficacy has become compulsory. In this study, we evaluated calcium-binding protein calbindin D-28K (calbindin) concentrations in the cerebrospinal fluid (CSF) as a biomarker of NPC1 disease. In the naturally occurring feline model, CSF calbindin was significantly elevated at 3 weeks of age, prior to the onset of cerebellar dysfunction, and steadily increased to >10-fold over normal at end-stage disease. Biweekly intrathecal administration of HPβCD initiated prior to the onset of neurologic dysfunction completely normalized CSF calbindin in NPC1 cats at all time points analyzed when followed up to 78 weeks of age. Initiation of HPβCD after the onset of clinical signs (16 weeks of age) resulted in a delayed reduction of calbindin levels in the CSF. Evaluation of CSF from patients with NPC1 revealed that calbindin concentrations were significantly elevated compared with CSF samples collected from unaffected patients. Off-label treatment of patients with NPC1 with miglustat, an inhibitor of glycosphingolipid biosynthesis, significantly decreased CSF calbindin compared with pretreatment concentrations. These data suggest that the CSF calbindin concentration is a sensitive biomarker of NPC1 disease that could be instrumental as an outcome measure of therapeutic efficacy in ongoing clinical trials.

Electrodiagnostic testing and histopathologic changes confirm peripheral nervous system myelin abnormalities in the feline model of niemann-pick disease type C.

Niemann-Pick disease type C (NPC disease) is an incurable, neurodegenerative, autosomal recessive disease caused by mutations in either the NPC1 or the NPC2 gene. These mutations affect the intracellular trafficking of lipids and cholesterol, resulting in the intralysosomal accumulation of unesterified cholesterol and glycosphingolipids. These abnormalities are associated with clinical ataxia and impaired motor and intellectual development, and death frequently occurs in adolescence. The incidence of peripheral neuropathy in NPC patients is not known. We investigated peripheral nerves in the naturally occurring feline model of NPC disease, which has proven to be critical for understanding both disease pathogenesis and for evaluating experimental therapies. Electrodiagnostic studies revealed significantly slowed motor and sensory nerve conduction velocities in affected cats in the absence of altered M-wave amplitude. Histologic and ultrastructural analyses showed thin myelin sheaths, membranous debris, myelin figures, lipid vacuolization of Schwann cell cytoplasm, and expanded paranodal areas. Axonal degeneration was not identified. There was a shift to small myelinated fibers in affected cats, and there were significant decreases in fiber diameter, axon diameter, and myelin thickness. These changes were similar to those described in the murine NPC disease model and in rare patients in whom nerve biopsy has been performed. Characterization of the demyelinating neuropathy is necessary for evaluating clinical trials that target only the CNS aspects of NPC.

Clinical, electrophysiological, and biochemical markers of peripheral and central nervous system disease in canine globoid cell leukodystrophy (Krabbe's disease)

Globoid cell leukodystrophy (GLD), or Krabbes disease, is a debilitating and always fatal pediatric neurodegenerative disease caused by a mutation in the gene encoding the hydrolytic enzyme galactosylceramidase (GALC). In the absence of GALC, progressive loss of myelin and accumulation of a neurotoxic substrate lead to incapacitating loss of motor and cognitive function and death, typically by 2 years of age. Currently, there is no cure. Recent convincing evidence of the therapeutic potential of combining gene and cell therapies in the murine model of GLD has accelerated the requirement for validated markers of disease to evaluate therapeutic efficacy. Here we demonstrate clinically relevant and quantifiable measures of central (CNS) and peripheral (PNS) nervous system disease progression in the naturally occurring canine model of GLD. As measured by brainstem auditory‐evoked response testing, GLD dogs demonstrated a significant increase in I‐V interpeak latency and hearing threshold at all time points. Motor nerve conduction velocities (NCVs) in GLD dogs were significantly lower than normal by 12–16 weeks of age, and sensory NCV was significantly lower than normal by 8–12 weeks of age, serving as a sensitive indicator of peripheral nerve dysfunction. Post‐mortem histological evaluations confirmed neuroimaging and electrodiagnostic assessments and detailed loss of myelin and accumulation of storage product in the CNS and the PNS. Additionally, cerebrospinal fluid psychosine concentrations were significantly elevated in GLD dogs, demonstrating potential as a biochemical marker of disease. These data demonstrate that CNS and PNS disease progression can be quantified over time in the canine model of GLD with tools identical to those used to assess human patients.

AAVrh10 Gene Therapy Ameliorates Central and Peripheral Nervous System Disease in Canine Globoid Cell Leukodystrophy (Krabbe Disease)

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an inherited, neurologic disorder that results from deficiency of a lysosomal enzyme, galactosylceramidase. Most commonly, deficits of galactosylceramidase result in widespread central and peripheral nervous system demyelination and death in affected infants typically by 2 years of age. Hematopoietic stem-cell transplantation is the current standard of care in children diagnosed prior to symptom onset. However, disease correction is incomplete. Herein, the first adeno-associated virus (AAV) gene therapy experiments are presented in a naturally occurring canine model of GLD that closely recapitulates the clinical disease progression, neuropathological alterations, and biochemical abnormalities observed in human patients. Adapted from studies in twitcher mice, GLD dogs were treated by combination intravenous and intracerebroventricular injections of AAVrh10 to target both the peripheral and central nervous systems. Combination of intravenous and intracerebroventricular AAV gene therapy had a clear dose response and resulted in delayed onset of clinical signs, extended life-span, correction of biochemical defects, and attenuation of neuropathology. For the first time, therapeutic effect has been established in the canine model of GLD by targeting both peripheral and central nervous system impairments with potential clinical implications for GLD patients.

611. Promoter Evaluation of AAV Gene Therapy in the Central Nervous System for Feline Niemann-Pick Type C Disease

Niemann-Pick type C1 (NPC1) disease is characterized by progressive cerebellar ataxia, dementia, and death in adolescence. It is caused by mutations that result in deficient function of lysosomal, membrane-bound NPC1, resulting in the intralysosomal accumulation of cholesterol and sphingolipids. There are no FDA-approved therapies for NPC1 disease, however, we have previously shown that administration of 2-hydroxypropyl-beta-cyclodextrin (HPsCD) via the cerebromedullary cistern (CBMC) in presymptomatic cats with NPC1 disease prevented the onset of cerebellar tremor and resulted in Purkinje cell (PC) survival and near normal concentrations of cerebral cortex and cerebellar cholesterol and sphingolipids. Remarkably, these cats remained alive for greater than three years of age, in contrast to untreated NPC1 cats that died before six months of age. This therapy has advanced to clinical trials where patients receive biweekly HPsCD. In an attempt to both reduce the number of HPsCD injections and to treat uncorrected brain regions, we studied the feasibility of viral gene therapy, providing a one-time injection, in NPC1 cats. Preliminary studies utilized administration of an adeno-associated virus serotype 9 carrying the feline NPC1 transgene (fNPC1) via the CBMC of pre-symptomatic NPC1 cats. These treated cats showed reduced ataxia compared to untreated cats. Histologic evaluation of the brains revealed that NPC1 expression had been restored to some PCs and that this was associated with increased PC survival. This initial success prompted us to further explore the potential of various promoters to express functional NPC1 protein in the brain. In this study we evaluated the ability of four different promoters, three ubiquitous (CB7, GUSB, and JeTi) and one neuron-specific (CamKII), to drive GFP expression in the central nervous system of normal cats. Our preliminary therapeutic studies used the GUSB promoter (pGUSB) to mediate fNPC1 expression. Expression of both fNPC1 and a GFP reporter under pGUSB were low in PCs, and in cerebrocortical neurons and astrocytes. Overall, the CB7 promoter drove the highest level of expression in similar regions; however, this large promoter (~970bp) limits transgene size (where NPC is ~4.3Kbp) and led us to examine a smaller exogenous promoter based on CB7, termed JeTi. This promoter was similar in expression levels to the pGUSB, leading to low-level expression at our limit of detection using DAB-based immunohistochemistry. Interestingly, a six-month therapeutic study using the JeTi promoter had a similar outcome as pGUSB where treated cats had reduced clinical signs at the end of study. Expression driven by the neuron-specific promoter CamKII produced levels similar to the CB7 promoter, although PCs of the cerebellum had low to undetectable expression levels. These data and preliminary therapeutic data in AAV-treated NPC1 cats will be further explained in this presentation.

56. Intracerebroventricular and Intravenous AAV Gene Therapy in Canine Globoid Cell Leukodystrophy

Globoid cell leukodystrophy (GLD), or Krabbe disease, results from a deficiency in the hydrolytic enzyme galactosylceramidase (GALC), which is responsible for degrading the central and peripheral nervous system (CNS, PNS) myelin lipids galactosylceramide and galactosylsphingosine (psychosine). Symptoms in infants include irritability and stiffness with disease progressing rapidly to a vegetative state and death typically resulting by 2 years of age. GLD is naturally occurring in dogs and signs include ataxia, tremors, pelvic limb paralysis, loss of hearing, and blindness, with disease progression warranting euthanasia at 15.9 ± 4.6 weeks. In GLD dogs, GALC concentrations are decreased in leukocytes, CSF, and tissues and psychosine concentrations are elevated in serum and CSF. Conduction velocities in peripheral nerves of GLD dogs are < 50% of normal and G-ratios of the sciatic nerve are significantly higher than normal. Brain stem auditory evoked response testing exhibits a loss of waveform integrity and increased central conduction time consistent with auditory system demyelination. MRI of the brain shows T2-weighted hyperintensity of the white matter, widened sulci, and enlarged ventricles and MRS identified decreases in N-acetylaspartate and increases in choline. Diffusion tensor imaging established decreased fractional anisotropy and increased radial diffusivity in multiple white matter tracts including the corpus callosum and posterior internal capsule. Histologically, GLD dogs revealed severe loss of myelin, a decrease in oligodendrocytes, astrogliosis, and microgliosis. Combination gene therapy has been evaluated in GLD affected dogs using AAVrh10 encoding canine GALC (AAVrh10-cGALC). Two GLD dogs received a low dose of AAVrh10-cGALC, 1.2E12, by combination intravenous (IV) and intracerebroventricular (ICV) injection routes and displayed a modest increase in survival to 17.9 and 22.1 weeks of age. Two additional GLD dogs were treated with an increased dose of AAVrh10-cGALC, 1.9E13, and survival was further increased to 30.3 and 43.1 weeks of age. Both low and high dose combination IV and ICV therapy delayed the onset of neurological signs and prevented the onset of tremors, one of the debilitating neurologic signs in untreated GLD dogs. High dose AAVrh10-cGALC, but not low dose, resulted in complete normalization of pelvic and thoracic limb NCV and near normal sensory NCV. Combination therapy of either dose had negligible effect on the auditory system, as treated animals showed little to no improvement in distance between waveform peaks or hearing threshold over untreated animals. After high dose treatment with AAVrh10-cGALC, GALC activity reached near normal levels in the cerebellum and sciatic nerve, with levels decreasing in more distal brain regions. GALC levels in the liver and heart were intermediate between affected untreated GLD and normal control dogs. Interestingly, the highest GALC activity was found in the quadriceps muscle. GLD dogs treated with high dose AAV had CSF psychosine concentrations lower than untreated and low dose AAV-treated GLD dogs, despite being substantially older. Efficacy of hematopoietic stem cell transplant (HSCT) alone and in combination with IV AAVrh10-cGALC is currently being evaluated in GLD dogs. Ongoing studies suggest that addition of IV infusion of AAVrh10-cGALC substantially increases survival as compared to HSCT alone.