Galactocerebrosidase Research Articles

Krabbe disease: One Hundred years from the bedside to the bench to the bedside.

This Review summarizes the progress in understanding the pathogenesis and treatment of Krabbe disease from the description of five patients in by Knud Krabbe until 2016. To determine the cause of this genetic disease, pathological and chemical analyses of tissues from the nervous systems of patients were performed. It was determined that these patients had a pathological feature known as globoid cell in the brain and that this consisted partially of galactosylceramide, a major sphingolipid component of myelin. The finding that these patients had a deficiency of galactocerebrosidase (GALC) activity opened the way to relatively simple diagnostic testing with easily obtainable tissue samples, studies leading to the purification of GALC, and cloning of the GALC cDNA and gene. The availability of the gene sequence led to the identification of mutations in patients and to the current studies involving the use of viral vectors containing the GALC cDNA to treat experimentally naturally occurring animal models, such as twitcher mice. Currently, treatment of presymptomatic human patients is limited to hematopoietic stem cell transplantation (HSCT). With recent studies showing successful treatment of animal models with a combination of HSCT and viral gene therapy, it is hoped that more effective treatments will soon be available for human patients. For this Review, it is not possible to reference all of the articles contributing to our current state of knowledge about this disease; however, we have chosen those that have influenced our studies by suggesting research paths to pursue. © 2016 Wiley Periodicals, Inc.

Chaperones as potential therapeutics for Krabbe disease.

Krabbe's disease (KD) is an autosomal recessive, neurodegenerative disorder. It is classified among the lysosomal storage diseases (LSDs). It was first described in , but the genetic defect for the galactocerebrosidase (GALC) gene was not discovered until the beginning of the 1970s, 20 years before the GALC cloning. Recently, in 2011, the crystal structures of the GALC enzyme and the GALC-product complex were obtained. For this, compared with other LSDs, the research on possible therapeutic interventions is much more recent. Thus, it is not surprising that some treatment options are still under preclinical investigation, whereas their relevance for other pathologies of the same group has already been tested in clinical studies. This is specifically the case for pharmacological chaperone therapy (PCT), a promising strategy for selectively correcting defective protein folding and trafficking and for enhancing enzyme activity by small molecules. These compounds bind directly to a partially folded biosynthetic intermediate, stabilize the protein, and allow completion of the folding process to yield a functional protein. Here, we review the chaperones that have demonstrated potential therapeutics during preclinical studies for KD, underscoring the requirement to invigorate research for KD-addressed PCT that will benefit from recent insights into the molecular understanding of GALC structure, drug design, and development in cellular models. © 2016 Wiley Periodicals, Inc.

Metabolic profiling reveals biochemical pathways and potential biomarkers associated with the pathogenesis of Krabbe disease.

Krabbe disease (KD) is caused by mutations in the galactosylceramidase (GALC) gene, which encodes a lysosomal enzyme that degrades galactolipids, including galactosylceramide and galactosylsphingosine (psychosine). GALC deficiency results in progressive intracellular accumulation of psychosine, which is believed to be the main cause for the demyelinating neurodegeneration in KD pathology. Umbilical cord blood transplantation slows disease progression when performed presymptomatically but carries a significant risk of morbidity and mortality. Accurate presymptomatic diagnosis is therefore critical to facilitate the efficacy of existing transplant approaches and to avoid unnecessary treatment of children who will not develop KD. Unfortunately, current diagnostic criteria, including GALC activity, genetic analysis, and psychosine measurement, are insufficient for secure presymptomatic diagnosis. This study performs a global metabolomic analysis to identify pathogenetic metabolic pathways and novel biomarkers implicated in the authentic mouse model of KD known as twitcher. At a time point before onset of signs of disease, twitcher hindbrains had metabolic profiles similar to WT, with the exception of a decrease in metabolites related to glucose energy metabolism. Many metabolic pathways were altered after early signs of disease in the twitcher, including decreased phospholipid turnover, restricted mitochondrial metabolism of branched-chain amino acids, increased inflammation, and changes in neurotransmitter metabolism and osmolytes. Hypoxanthine, a purine derivative, is increased before signs of disease appear, suggesting its potential as a biomarker for early diagnosis of KD. Additionally, given the early changes in glucose metabolism in the pathogenesis of KD, diagnostic modalities that report metabolic function, such as positron emission tomography, may be useful in KD. © 2016 Wiley Periodicals, Inc.

Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an autosomal recessive neurodegenerative disease caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). Hematopoietic stem cell transplantation (HSCT) provides modest benefit in presymptomatic patients but is well short of a cure. Gene transfer experiments using viral vectors have shown some success in extending the survival in the mouse model of GLD, twitcher mice. The present study compares three single-stranded (ss) AAV serotypes, two natural and one engineered (with oligodendrocyte tropism), and a self-complementary (sc) AAV vector, all packaged with a codon-optimized murine GALC gene. The vectors were delivered via a lumbar intrathecal route for global CNS distribution on PND10-11 at a dose of 2 × 10(11) vector genomes (vg) per mouse. The results showed a similar significant extension of life span of the twitcher mice for all three serotypes (AAV9, AAVrh10, and AAV-Olig001) as well as the scAAV9 vector, compared to control cohorts. The rAAV gene transfer facilitated GALC biodistribution and detectable enzymatic activity throughout the CNS as well as in sciatic nerve and liver. When combined with BMT from syngeneic wild-type mice, there was significant improvement in survival for ssAAV9. Histopathological analysis of brain, spinal cord, and sciatic nerve showed significant improvement in preservation of myelin, with ssAAV9 providing the greatest benefit. In summary, we demonstrate that lumbar intrathecal delivery of rAAV/mGALCopt can significantly enhance the life span of twitcher mice treated at PND10-11 and that BMT synergizes with this treatment to improve the survival further. © 2016 Wiley Periodicals, Inc.

Cellular transplant therapies for globoid cell leukodystrophy: Preclinical and clinical observations.

Globoid cell leukodystrophy (GLD) is a progressive neurodegenerative disorder caused by the deficiency of galactocerebrosidase (GALC), resulting in accumulation of toxic metabolites in neural tissues. Clinically variable based on age of onset, infantile GLD is generally a rapidly fatal syndrome of progressive neurologic and cognitive decline, whereas later-onset GLD has a more indolent, protracted clinical course. Animal models, particularly the twitcher mouse, have allowed investigation of both the pathophysiology of and the potential treatment modalities for GLD. Cellular therapy for GLD, notably hematopoietic cell transplantation (HCT; transplantation of bone marrow, peripheral blood stem cells, or umbilical cord blood cells) from a normal related or unrelated allogeneic donor provides a self-renewing source of GALC in donor-derived cells. The only currently available treatment option in human GLD, allogeneic HCT, can slow the progression of the disease and improve survival, especially when performed in presymptomatic infants. Because persistent neurologic dysfunction still occurs after HCT in GLD, preclinical studies are evaluating combinations of HCT with other treatment modalities. © 2016 Wiley Periodicals, Inc.

Lithium improves cell viability in psychosine-treated MO3.13 human oligodendrocyte cell line via autophagy activation.

Globoid cell leukodystrophy (GLD) is a rare, rapidly progressing childhood leukodystrophy triggered by deficit of the lysosomal enzyme galactosylceramidase (GALC) and characterized by the accumulation of galactosylsphingosine (psychosine; PSY) in the nervous system. PSY is a cytotoxic sphingolipid, which leads to widespread degeneration of oligodendrocytes and Schwann cells, causing demyelination. Here we report on autophagy in the human oligodendrocyte cell line MO3.13 treated with PSY and exploitation of Li as an autophagy modulator to rescue cell viability. We demonstrate that PSY causes upregulation of the autophagic flux at the level of autophagosome and autolysosome formation and LC3-II expression. We show that pretreatment with Li, a drug clinically used to treat bipolar disorders, can further stimulate autophagy, improving cell tolerance to PSY. This Li protective effect is found not to be linked to reduction of PSY-induced oxidative stress and might not stem from a reduction of PSY accumulation. These data provide novel information on the intracellular pathways activated during PSY-induced toxicity and suggest the autophagy pathway as a promising novel therapeutic target for ameliorating the GLD phenotype. © 2016 Wiley Periodicals, Inc.

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

Globoid cell leukodystrophy (GLD), or Krabbe's 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. © 2016 Wiley Periodicals, Inc.

Myelin repair by transplantation of myelin-forming cells in globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is a devastating demyelinating disease that affects both the central and peripheral nervous systems. It is caused by genetic deficiency in the activity of a lysosomal enzyme, galactocerebrosidase (GALC), which is necessary for the maintenance of myelin. Hematopoietic stem cell transplantation (HSCT) including umbilical cord stem cell transplantation is the only effective therapy available to date. HSCT significantly prolongs the life span of patients with GLD when performed before disease onset, although it is not curative. In HSCT, infiltrating donor-derived macrophages are thought to indirectly supply the enzyme (called "cross-correction") to the host's myelinating cells. Given the limitation in treating GLD, it is hypothesized that remyelinating demyelinated axons with GALC-competent myelinating cells by transplantation will result in more stable myelination than endogenous myelin repair supported by GALC cross-correction. Transplantation of myelin-forming cells in a variety of animal models of dysmyelinating and demyelinating disorders suggests that this approach is promising in restoring saltatory conduction and protecting neurons by providing new healthy myelin. However, GLD is one of the most challenging diseases in terms of the aggressiveness of the disease and widespread pathology. Experimental transplantation of myelin-forming cells in the brain of a mouse model of GLD has been only modestly effective to date. Thus, a practical strategy for myelin repair in GLD would be to combine the rapid and widespread cross-correction of GALC by HSCT with the robust, stable myelination provided by transplanted GALC-producing myelin-forming cells. This short review will discuss such possibilities. © 2016 Wiley Periodicals, Inc.

Neuroradiological, neurophysiological and molecular findings in infantile Krabbe disease: two case reports.

Krabbe disease is an autosomal recessive neurodegenerative disorder due to a defect of the lysosomal enzyme β-galactocerebrosidase (β-GALC). Depending on the age of onset, the disease is classified into infantile and later-onset forms. We report neuroradiological, neurophysiological and molecular findings in two Greek patients with the infantile form of Krabbe disease. The index patients presented at the age of 3.5 and 6 months, respectively, due to developmental delay. Magnetic resonance imaging (MRI) of the first patient’s brain demonstrated signs of leukodystrophy, while nerve conduction velocities (NCVs) were significantly decreased. The second patient’s MRI at the age of 4 months was initially normal, but at 18 months demonstrated leukodystrophic alterations as well, whereas NCVs were also significantly delayed. In both patients, a severe decrease in β-GALC, activity supported the diagnosis of Krabbe disease, while the final diagnosis was confirmed by molecular genetic testing. Two homozygous mutations of the GALC gene, the c.411_413delTAA [p.K139del] mutation in the first patient, and the c.749T>C [p.I250T] mutation in the second patient, were identified. At their last follow-up visit at the age of 4 and 6 years, respectively, both patients were bedridden and quadri-plegic, suffering from frequent respiratory tract infections and fed through a gastrostomy. Both mutations found in homozygosity in these two unrelated patients of Greek ancestry, could pinpoint a common origin. Genotyping of patients with Krabbe disease is important, in order to contribute to the creation of a European mutation database and to further study possible genotype-phenotype correlations of the disease.

Molecular Mechanisms of Disease Pathogenesis Differ in Krabbe Disease Variants.

Krabbe disease is a severe, fatal neurodegenerative disorder caused by defects in the lysosomal enzyme galactocerebrosidase (GALC). The correct targeting of GALC to the lysosome is essential for the degradation of glycosphingolipids including the primary lipid component of myelin. Over 100 different mutations have been identified in GALC that cause Krabbe disease but the mechanisms by which they cause disease remain unclear. We have generated monoclonal antibodies against full‐length human GALC and used these to monitor the trafficking and processing of GALC variants in cell‐based assays and by immunofluorescence microscopy. Striking differences in the secretion, processing and endosomal targeting of GALC variants allows the classification of these into distinct categories. A subset of GALC variants are not secreted by cells, not proteolytically processed, and remain trapped in the ER; these are likely to cause disease due to protein misfolding and should be targeted for pharmacological chaperone therapies. Other GALC variants can be correctly secreted by cells and cause disease due to catalytic defects in the enzyme active site, inappropriate post‐translational modification or a potential inability to bind essential cofactors. The classification of disease pathogenesis presented here provides a molecular framework for appropriate targeting of future Krabbe disease therapies.

Pilot study of newborn screening for six lysosomal storage diseases using Tandem Mass Spectrometry.

BackgroundThere is current expansion of newborn screening (NBS) programs to include lysosomal storage disorders because of the availability of treatments that produce an optimal clinical outcome when started early in life.ObjectiveTo evaluate the performance of a multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assay of 6 lysosomal enzymes in a NBS laboratory for the identification of newborns at risk for developing Pompe, Mucopolysaccharidosis-I (MPS-I), Fabry, Gaucher, Niemann Pick-A/B, and Krabbe diseases.Methods and ResultsEnzyme activities (acid α-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), α-galactosidase A (GLA), α-iduronidase (IDUA) and sphingomyeline phosphodiesterase-1 (SMPD-1)) were measured on ~43,000 de-identified dried blood spot (DBS) punches, and screen positive samples were submitted for DNA sequencing to obtain genotype confirmation of disease risk. The 6-plex assay was efficiently performed in the Washington state NBS laboratory by a single laboratory technician at the bench using a single MS/MS instrument. The number of screen positive samples per 100,000 newborns were as follows: GAA (4.5), IDUA (13.6), GLA (18.2), SMPD1 (11.4), GBA (6.8), and GALC (25.0).DiscussionA 6-plex MS/MS assay for 6 lysosomal enzymes can be successfully performed in a NBS laboratory. The analytical ranges (enzyme-dependent assay response for the quality control HIGH sample divided by that for all enzyme-independent processes) for the 6-enzymes with the MS/MS is 5- to 15-fold higher than comparable fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection is consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform.

57. Intrathecal Administration of AAV/GALC Vectors in Juvenile Twitcher Mice Improves Survival and Is Enhanced by Bone Marrow Transplant

Globoid cell Leukodystrophy (GLD) or Krabbe disease is a rapidly progressing, neurodegenerative disease caused by the deficiency of the lysosomal enzyme Galactocerebrosidase (GALC). The pathological characteristics include presence of globoid cells and decreased myelin. In its severe infantile form, the symptoms appear within the first 6 months of life and complete loss of GALC function is fatal by 2-3 years old. The murine model of infantile GLD, the twitcher mouse, has been used to evaluate potential therapeutic approaches for GLD. Hematopoietic stem cell transplantation (HSCT) provides modest benefit in presymptomatic patients and mice indicating a slowdown in the progression of the disease, but no complete cure. Neonatal gene transfer experiments using viral vectors also has shown some limited success in extending the survival of the twitcher mice. The translatability of neonatal therapy in mice to human has met with difficulties as the stage of the disease in human and mice differ due to the difference in their gestation periods. So there is a need for testing out later stage interventions for GLD treatment. In the present study, we compare multiple vector designs along with a combination treatment of AAV plus bone marrow transplant (BMT) in juvenile twitcher mice. Initially, three single stranded (ss) AAV serotypes, two natural and one engineered (with oligodendrocyte tropism), were packaged with a codon-optimized murine GALC gene driven by the beta actin promoter. The vectors were delivered via a lumbar intrathecal route for global CNS distribution on post-natal day (PND) 10-11, at a dose of 2×1011 vg per mouse. The results show a significant extension of life span of the twitcher mice for all three serotypes (AAV9, AAVrh10, and AAV-Olig001) when compared to control cohorts. The treatment produced similar survival benefit regardless of which capsid was used. The rAAV gene transfer facilitates GALC biodistribution and detectable enzymatic activity throughout the CNS as well as in sciatic nerve and liver. When combined with BMT from syngeneic wild type mice, there was significant improvement in survival and enzymatic activity over either treatment alone. Immunohistochemical analysis of the brain and spinal cord showed reduced inflammation and pathology. Additionally, we have also tested a novel self-complementary (sc) AAV vector with a minimal synthetic promoter, which would mediate a weaker overall level of GALC expression but express in more cells. Preliminary results indicate that this vector design provides a survival advantage over the ssAAV vector designs. In summary, we demonstrate that lumbar intrathecal delivery of rAAV/mGALCopt can significantly enhance the life span of twitcher mice treated at juvenile stage (PND10-11) and BMT synergizes with this treatment to further improve the survival. This effect is mediated by increased GALC activity in various parts of the nervous system as well as by the reduction in neuro-inflammation. Together, these studies detail a therapeutic approach for GLD in mice which is feasible and relevant for human translation.

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.

No Evidence of Association of Heterozygous Galactosylceramidase Deletion With Normal-Tension Glaucoma in a Korean Population.

A significant association between primary open-angle glaucoma risk and copy-number variation in the galactosylceramidase (GALC) gene was reported recently. This study investigated whether a heterozygous deletion of the GALC gene plays a significant role in normal-tension glaucoma (NTG) in Koreans. A 3-primer polymerase chain reaction assay was used to examine the heterozygous deletion of GALC in all Korean NTG cases (n=276) and controls (n=135). We did not identify any deletion variant of GALC gene in the NTG patients. This is the first copy-number variation study of the GALC gene in the Korean population with NTG. We demonstrated that a heterozygous GALC deletion does not play a significant role in the pathogenesis of NTG in a representative clinic-based population of South Koreans, unlike whites.

Atypical presentation of infantile Alexander disease without macrocephaly

Abstract Background Alexander disease is a rare form of leukodystrophy that involves mainly astrocytes; it is inherited in an autosomal recessive manner and occurs by mutations in the GFAP gene, located on chromosome 17q21. It can occur at any age, and its infantile form is characterized by macrocephaly, seizures, severe motor and cognitive delay, and progressive spasticity or ataxia. Case report An 8-month-old female was evaluated with a history of neurodevelopmental delay and unprovoked focal motor seizures. Physical examination showed normal head circumference, increased motor responses to tactile and noise stimuli, pyramidal signs and no visceromegaly. The widespread hypodense white matter was found on magnetic resonance, and lumbar puncture showed hyperproteinorrachia. Krabbe disease was ruled out by enzymatic assay and sequencing of GALC gene. In the reassessment of the case, abnormalities in neuroimaging lead to suspicion of Alexander disease, and GFAP gene sequencing reported a pathogenic mutation in exon 4 c.716G > A, which caused a change of arginine to histidine at position 239 of the protein (p.Arg239His). Conclusions The neuroradiology signs observed in the resonance were decisive for the diagnosis later confirmed by molecular techniques. It is important to consider that certain mutations are not associated with macrocephaly, which may cause a delay in the diagnosis.

Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy.

Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non‐human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe‐affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD.

RP-CARS reveals molecular spatial order anomalies in myelin of an animal model of Krabbe disease.

Krabbe disease (KD) is a rare demyelinating sphingolipidosis, often fatal in the first years of life. It is caused by the inactivation of the galactocerebrosidase (GALC) enzyme that causes an increase in the cellular levels of psychosine considered to be at the origin of the tissue-level effects. GALC is inactivated also in the Twitcher (TWI) mouse: a genetic model of KD that is providing important insights into the understating of the pathogenetic process and the development of possible treatments. In this article an innovative optical technique, RP-CARS, is proposed as a tool to study the degree of order of the CH2 bonds inside the myelin sheaths of TWI-mice sciatic-nerve fibres. RP-CARS, a recently developed variation of CARS microscopy, is able to combine the intrinsic chemical selectivity of CARS microscopy with molecular-bond-spatial-orientation sensibility. This is the first time RP-CARS is applied to the study of a genetic model of a pathology, leading to the demonstration of a post-onset progressive spatial disorganization of the myelin CH2 bonds. The presented result could be of great interest for a deeper understanding of the pathogenic mechanisms underlying the human KD and, moreover, it is an additional proof of the experimental validity of this microscopy technique. RP-CARS image (2850cm-1 , CH2 bonds) of a sciatic-nerve optical longitudinal section from a Twitcher P23 (symptomatic) mouse. Scale bar: 10microns. The image was constructed by colour-mapping the degree of molecular order of the CH2 bonds inside the myelin walls, as displayed in the colour bar on the right.

Altered Trafficking and Processing of GALC Mutants Correlates with Globoid Cell Leukodystrophy Severity.

Globoid cell leukodystrophy (GLD, Krabbe disease) is diagnosed by measuring galactosylceramidase (GALC) activity and DNA analysis. However, genotype and phenotype often do not correlate due to considerable clinical variability, even for the same mutation, for unknown reasons. We find that altered trafficking to the lysosome and processing of GALC correlates with GLD severity and is modulated by cis-polymorphisms. Current diagnosis of GLD is based on GALC activity of total cell lysates from blood, which does not discriminate whether the activity comes from the lysosome or other subcellular organelles. Measurement of GALC activity in lysosomes may predict which infants are at high risk for the infantile phenotype while distinguishing other children who will develop later-onset phenotypes without onset of symptoms for years.

Krabbe and Niemann-Pick disorders: Development of novel fluorimetric assays using dried blood spots

Deficiencies in the lysosomal storage enzymes galactocerebrosidase (GALC) and acid sphingomyelinase (ASM) are causative for the devastating metabolic disorders Krabbe and Niemann-Pick, respectively. Neurological symptoms of both disorders often present in infancy and progress quickly. Timely disease detection is therefore a critical component of patient care. Dried blood spot (DBS) cards are the current standard specimen for newborn screening assays in state public health laboratories. Here, we present fluorimetric assays to measure GALC and ASM enzyme activity in dried blood spot specimens. Introduction

Newborn screening for Krabbe disease in New York State: the first eight years' experience.

Krabbe disease (KD) results from galactocerebrosidase (GALC) deficiency. Infantile KD symptoms include irritability, progressive stiffness, developmental delay, and death. The only potential treatment is hematopoietic stem cell transplantation. New York State (NYS) implemented newborn screening for KD in 2006. Dried blood spots from newborns were assayed for GALC enzyme activity using mass spectrometry, followed by molecular analysis for those with low activity (≤12% of the daily mean). Infants with low enzyme activity and one or more mutations were referred for follow-up diagnostic testing and neurological examination. Of >1.9 million screened, 620 infants were subjected to molecular analysis and 348 were referred for diagnostic testing. Five had enzyme activities and mutations consistent with infantile KD and manifested clinical/neurodiagnostic abnormalities. Four underwent transplantation, two are surviving with moderate to severe handicaps, and two died from transplant-related complications. The significance of many sequence variants identified is unknown. Forty-six asymptomatic infants were found to be at moderate to high risk for disease. The positive predictive value of KD screening in NYS is 1.4% (5/346) considering confirmed infantile cases. The incidence of infantile KD in NYS is approximately 1 in 394,000, but it may be higher for later-onset forms.