Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC) and the progressive accumulation of the toxic metabolite psychosine. We showed previously that central nervous system (CNS)-directed, adeno-associated virus (AAV)2/5-mediated gene therapy synergized with bone marrow transplantation and substrate reduction therapy (SRT) to greatly increase therapeutic efficacy in the murine model of Krabbe disease (Twitcher). However, motor deficits remained largely refractory to treatment. In the current study, we replaced AAV2/5 with an AAV2/9 vector. This single change significantly improved several endpoints primarily associated with motor function. However, nearly all (14/16) of the combination-treated Twitcher mice and all (19/19) of the combination-treated wild-type mice developed hepatocellular carcinoma (HCC). 10 out of 10 tumors analyzed had AAV integrations within the Rian locus. Several animals had additional integrations within or near genes that regulate cell growth or death, are known or potential tumor suppressors, or are associated with poor prognosis in human HCC. Finally, the substrate reduction drug L-cycloserine significantly decreased the level of the pro-apoptotic ceramide 18:0. These data demonstrate the value of AAV-based combination therapy for Krabbe disease. However, they also suggest that other therapies or co-morbidities must be taken into account before AAV-mediated gene therapy is considered for human therapeutic trials.

Recombinant human alpha- N -acetylglucosaminidase-insulin-like growth factor-2 (rhNAGLU-IGF2) is an investigational enzyme replacement therapy for Sanfilippo B, a lysosomal storage disease. Because recombinant human NAGLU (rhNAGLU) is poorly mannose 6-phosphorylated, we generated a fusion protein of NAGLU with IGF2 to permit its binding to the cation-independent mannose 6-phosphate receptor. We previously administered rhNAGLU-IGF2 intracerebroventricularly to Sanfilippo B mice, and demonstrated therapeutic restoration of NAGLU, normalization of lysosomal storage, and improvement in markers of neurodegeneration and inflammation. Here, we studied repeated intracerebroventricular rhNAGLU-IGF2 delivery in both murine and canine Sanfilippo B to determine potential effects on their behavioral phenotypes and survival. Treated mice showed improvement in disease markers such as heparan sulfate glycosaminoglycans, beta-hexosaminidase, microglial activation, and lysosomal-associated membrane protein-1. Sanfilippo B mice treated with rhNAGLU-IGF2 displayed partial normalization of their stretch attend postures, a defined fear pose in mice (p<0.001). We found an improved rotarod performance in Sanfilippo B mice treated with rhNAGLU-IGF2 compared to vehicle-treated Sanfilippo B mice (p=0.002). We also found a 61% increase in survival in Sanfilippo B mice treated with rhNAGLU-IGF2 (mean 53w, median 48w) compared to vehicle-treated Sanfilippo B mice (mean 33w, median 37w; p<0.001). In canine Sanfilippo B, we found that rhNAGLU-IGF2 administered into cerebrospinal fluid normalized HS and beta-hexosaminidase activity in gray and white matter brain regions. Proteomic analysis of cerebral cortex showed restoration of protein expression levels in pathways relevant to cognitive, synaptic, and lysosomal functions. These data suggest that treatment with rhNAGLU-IGF2 may improve the phenotype of Sanfilippo B disease.

Obesity is a risk factor for complications in singleton and twin pregnancies; however, there are limited data regarding maternal body mass index (BMI) in the setting of twin-twin transfusion syndrome (TTTS). We hypothesized that increased BMI in TTTS is associated with adverse perinatal outcomes and vascular pathology. A retrospective study of twin reversed arterial perfusion (n= 4), selective intrauterine growth restriction (n= 10) and TTTS (n= 33) was conducted. Treatment included fetoscopic laser photocoagulation (FLP) (n= 35) or Solomon technique (n= 12). Ex vivo placental intravascular injections, immunohistochemistry, and perinatal outcomes were compared by maternal BMI. In pregnancy complicated by TTTS, 16/33 women were obese (BMI > 30 kg/m(2)) and 11/33 were overweight (BMI 25-29.9 kg/m(2)). Women who were overweight or obese had an increased rate of premature rupture of membranes (PPROM), cesarean delivery, and/or concomitant comorbidities when compared to the normal weight group. Duration of neonatal intensive care unit (NICU) admission was longer in neonates of overweight/obese women versus normal weight. Placental examination of FLP sites in the obese group showed larger infarcts, increased adipose triglyceride lipase, and a proangiogenic phenotype. Increased BMI is common in our TTTS cohort and it is associated with higher rate of co-morbidity, PPROM, prolonged NICU stay, and an imbalance of placental metabolic and vascular mediators.

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a fatal demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramidase (GALC). GALC deficiency leads to the accumulation of the cytotoxic glycolipid, galactosylsphingosine (psychosine). Complementary evidence suggested that psychosine is synthesized via an anabolic pathway. Here, we show instead that psychosine is generated catabolically through the deacylation of galactosylceramide by acid ceramidase (ACDase). This reaction uncouples GALC deficiency from psychosine accumulation, allowing us to test the long-standing "psychosine hypothesis." We demonstrate that genetic loss of ACDase activity (Farber disease) in the GALC-deficient mouse model of human GLD (twitcher) eliminates psychosine accumulation and cures GLD. These data suggest that ACDase could be a target for substrate reduction therapy (SRT) in Krabbe patients. We show that pharmacological inhibition of ACDase activity with carmofur significantly decreases psychosine accumulation in cells from a Krabbe patient and prolongs the life span of the twitcher (Twi) mouse. Previous SRT experiments in the Twi mouse utilized L-cycloserine, which inhibits an enzyme several steps upstream of psychosine synthesis, thus altering the balance of other important lipids. Drugs that directly inhibit ACDase may have a more acceptable safety profile due to their mechanistic proximity to psychosine biogenesis. In total, these data clarify our understanding of psychosine synthesis, confirm the long-held psychosine hypothesis, and provide the impetus to discover safe and effective inhibitors of ACDase to treat Krabbe disease.

Granular cell tumor is a benign tumor of likely neural or neuroectodermal origin that occurs most commonly in the subcutaneous tissues of the trunk, breast, and extremities of adults. Congenital gingival lesions comprise the majority of the pediatric granular cell tumors. Granular cell tumors are generally small and asymptomatic, and while 1 in 10 patients has multiple tumors, recurrence and malignancy are very rare. Mediastinal granular cell tumors have been reported, most occurring in young adult or middle-aged women. We present a case of a 16-year-old asymptomatic boy with a large mediastinal granular cell tumor incidentally identified after a motor vehicle accident, and we review the intraoperative, microscopic, and ultrastructural features of this tumor. Both the patient's age and anatomical location are unusual for this tumor, which presented technical and diagnostic challenges to the patient care team.

Quadruple synchronous primary neoplasms are exceedingly rare with only one case reported in the English literature. We herein report a case of synchronous quadruple primary neoplasms in a 70-year-old Arabic male with a history of prostate cancer who presented to our hospital for work-up of a brain mass found at an outside hospital. Subsequent (18)Fluorodeoxyglucose (FDG) positron emission tomography demonstrated a 5.9-cm temporoparietal mass and three additional lesions, each with increased maximum standardized uptake value (SUVmax). Histologic examination, immunohistochemistry and cytogenetic analyses of the lesional tissue revealed four primary neoplastic lesions: primary glioblastoma, inguinal schwannoma, well-differentiated neuroendocrine tumor of the terminal ileum and an appendiceal sessile serrated adenoma/polyp. This case is unique among previous reports as our patient presented with four primary neoplasms synchronously. To the best of our knowledge, this combination of synchronous multiple primary neoplasms has not been reported in the English literature.

Background: Increased insulin resistance (IR) was shown to be associated with an increased risk of colorectal cancer (CRC).It is unknown if the onset of CRC impacts insulin resistance after diagnosis.Objective: To determine the association between IR and recently diagnosed CRC in a US representative cohort.Methods: We conducted a cross-sectional study using the National Health and Nutrition Examination Survey (NHANES, 1999(NHANES, -2010)).IR was determined using the homeostasis model assessment (HOMA-IR) and was classified as the upper quartile of HOMA-IR ( ≥ 3.52) in this study.We excluded participants who were less than age 20, pregnant, on insulin or insulin lowering medications, or who had colorectal cancer diagnosis older than 4 years from the time of survey.Results: There were a total of 88,373,262 patients in our weighted cohort, 24,250,441 (27.4%) of which had IR.Basic demographics were significantly different between patients with and without IR as shown in Table 1.Patients with IR were more likely to consume alcohol and dietary fat but less likely to smoke tobacco (p<0.05).IR was associated with higher Body Mass Index (BMI), Waist Circumference and elevated C-reactive protein (CRP); however patients with IR reported more intentional weight change and a significant drop in their weight over the year prior to the survey (Table 2).There were 148,040 patients with CRC who were equally distributed between the groups with and without IR (P> 0.05).After adjusting for age, race and gender, the prevalence of recently diagnosed CRC remained similar in both groups (OR 1.00, 95% CI 0.48, 2.06).Conclusion: There is no conclusive association between insulin resistance and the prevalence of recently diagnosed colorectal cancer in this US nationally representative cohort of adults.Further prospective studies are required to investigate whether CRC-related weight loss leads to improved insulin resistance in this population and how that can alter risk of subsequent CRC.Table 1 Univariate analysis of participants with and without insulin resistance.Table 2 Univariate analysis of metabolic markers in participants with and without insulin resistance.

Mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo Syndrome type B is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU) activity. We previously showed that intracranial adeno-associated virus (AAV)-based gene therapy results in partial improvements of several aspects of the disease. In an attempt to further correct the disease, MPS IIIB mice were treated at 2–4 days of age with intracranial AAV2/5-NAGLU (IC-AAV), intravenous lentiviral-NAGLU (IV-LENTI) or the combination of both (BOTH). The BOTH group had the most complete biochemical and histological improvements of any treatment group. Compared with untreated MPS IIIB animals, all treatments resulted in significant improvements in motor function (rotarod) and hearing (auditory-evoked brainstem response). In addition, each treatment group had a significantly increased median life span compared with the untreated group (322 days). The combination arm had the greatest increase (612 days), followed by IC-AAV (463 days) and IV-LENTI (358 days). Finally, the BOTH group had nearly normal circadian rhythm measures with improvement in time to activity onset. In summary, targeting both the systemic and central nervous system disease of MPS IIIB early in life appears to be the most efficacious approach for this inherited metabolic disorder.

Globoid-cell Leukodystrophy (GLD; Krabbes disease) is a rapidly progressing inherited demyelinating disease caused by a deficiency of the lysosomal enzyme Galactosylceramidase (GALC). Deficiency of GALC leads to altered catabolism of galactosylceramide and the cytotoxic lipid, galactosylsphingosine (psychosine). This leads to a rapidly progressive fatal disease with spasticity, cognitive disability and seizures. The murine model of GLD (Twitcher; GALC-/-) lacks the same enzyme and has similar clinical features. The deficiency of GALC leads to oligodendrocyte death, profound neuroinflammation, and the influx of activated macrophages into the CNS. We showed previously that keratinocyte chemoattractant factor (KC) is highly elevated in the CNS of untreated Twitcher mice and significantly decreases after receiving a relatively effective therapy (bone marrow transplantation combined with gene therapy). The action of KC is mediated through the CXCR2 receptor and is a potent chemoattractant for macrophages and microglia. KC is also involved in oligodendrocyte migration and proliferation. Based on the commonalities between the disease presentation and the functions of KC, we hypothesized that KC and/or CXCR2 contribute to the pathogenesis of GLD. Interestingly, the course of the disease is not significantly altered in KC- or CXCR2-deficient Twitcher mice. There is also no alteration in inflammation or demyelination patterns in these mice. Furthermore, transplantation of CXCR2-deficient bone marrow does not alter the progression of the disease as it does in other models of demyelination. This study highlights the role of multiple redundant cytokines and growth factors in the pathogenesis of GLD.

Despite the encouraging results with enzyme replacement therapy for several lysosomal storage diseases (LSDs), successful delivery of enzyme to brain to correct central nervous system (CNS) storage has been an elusive target (Grubb et al, 2010). In that regard, the paper by Sorrentino et al (2013) in this issue is a major breakthrough. It is likely to generate widespread interest and have a large impact on research in the field. Why is this work so important? Most of the LSDs have some element of CNS involvement. In many of them, like Sanfilippo disease type IIIA, CNS involvement is the dominant feature (Rozaklis et al, 2011). Correction of this aspect of LSDs that profoundly affects brain and leads to progressive neurological deterioration has posed the greatest challenge. Successfully meeting that challenge is a big deal! Why is this work so important? Most of the LSDs have some element of CNS involvement. In many of them, like Sanfilippo disease type IIIA, CNS involvement is the dominant feature. Correction of this aspect of LSDs that profoundly affects brain and leads to progressive neurological deterioration has posed the greatest challenge. Successfully meeting that challenge is a big deal! Access of corrective enzyme to lysosomes in cells of most tissues relies on receptor-mediated endocytosis, by the ubiquitously expressed mannose-6-phosphate (Man-6-P) receptor and mannose receptors on cells of the macrophage lineage. Cells in the CNS have limited access to enzymes targeting these receptors. Indeed, the blood–brain barrier (BBB) effectively blocks access of such proteins except in the perinatal period. In the mouse, access to Man-6-P receptors on the brain capillaries is limited to the first 2 weeks of life, after which the brain becomes very resistant to infused native enzyme (Urayama et al, 2008). For this reason, effective clearance of established CNS storage by infused enzymes has been quite limited. More invasive approaches involving direct injection of enzyme into the cerebrospinal fluid intrathecally, or into the brain itself, have shown promise (Fraldi et al, 2007). In fact, clinical trials are underway to evaluate intrathecal therapy. In urgent situations like spinal cord compression, such aggressive measures are acceptable. However, more widespread application in humans may be impractical. Some success in delivering enzyme to brain with conventional enzyme therapy, but in higher than conventional doses, has been obtained in several animal models (Vogler et al, 2005). In most cases, the correction was limited. These studies suggested that prolonged exposure to circulating enzyme, whether achieved by repeatedly infusing large doses, or by chemically modifying the enzyme to delay its clearance, enhanced the likelihood of neuronal correction. What appeared to be a significant advance in extending the circulating lifetime of β-glucuronidase by chemical modification and enhancing CNS correction in the adult mouse model of mucopolysaccharidosis (MPS) VII (Grubb et al, 2010) proved not to be useful in the murine models of MPS IIIA (Rozaklis et al, 2011) and juvenile neuronal ceroid lipofuscinosis (Batten disease) (Meng et al, 2012). These diseases and many others like them still need a breakthrough. Sorrentino et al (2013) improved on their own prior work and that of others using several clever strategies. They chose the well-characterized murine model for MPS IIIA (Sanfilippo type IIIA). This naturally occurring mouse model has progressive neurological disease due to deficiency of sulphamidase (SGSH) which results in inability to degrade heparan sulphate in the CNS. The pathophysiology and predictable course had been very well defined by Rozaklis et al (2011), who showed that the CNS storage was resistant to even large doses of infused native or chemically modified SGSH. Sorrentino et al (2013) reasoned they might deliver enzyme by transcytosis, targeting a receptor that delivers an essential nutrient across the BBB. Transcytosis involves endocytosis at one cell surface (e.g. apical) followed rapidly by exocytosis at the opposite cell surface (e.g. basolateral) without delivery to lysosomes. Candidate receptors include the transferrin receptor, the insulin-like growth factor receptor, and the LDL receptor (LDLR), each of which has been targeted in other work to deliver chimeric proteins to brain. Usually an antibody to the relevant receptor was used as a ‘Trojan horse’ to carry the desired protein across brain capillary endothelial cells (Zhou et al, 2012). Spencer and Verma (2007) had successfully targeted the LDLR to deliver a virally expressed lysosomal enzyme to brain, providing proof of principle. Although correction of storage was not studied, they subsequently used this approach to deliver the protease neprilysin to brain in a transgenic Alzheimer mouse model. They showed not only efficacy in reducing brain accumulation of amyloid-beta peptide, but also improvement in CNS function (Spencer et al, 2008). Following a similar strategy, Sorrentino et al (2013) produced a chimeric SGSH enzyme with a C-terminal extension comprising the LDLR binding domain from apolipoprotein B. As a novel twist, they substituted the signal sequence from another acid hydrolase, iduronate sulphatase (IDS), which they had found to be secreted at an unusually high rate by transfected cells. Finally, they used an adeno-associated virus vector (AAV2/8) which preferentially targets liver and incorporated the thyroxine-binding globulin promoter to achieve persistent high-level expression in liver. The goal was to make the liver a factory to secrete a continuous supply of therapeutic enzyme. Remarkably, everything appears to have worked as predicted. They demonstrated that the AAV2/8 vector targeted the liver and that high-level expression was seen for over seven months. The predicted enhancement of secretion conferred by the IDS signal sequence was confirmed They demonstrated that the AAV2/8 vector targeted the liver and that high-level expression was seen for over seven months. The predicted enhancement of secretion conferred by the IDS signal sequence was confirmed. (serum levels seven- to ninefold higher). The secreted enzyme was shown to be targeted to lysosomes of mouse embryo fibroblasts in vitro not only by the Man-6-P receptor, but also by an endocytosis receptor which appeared to recognize the apo B domain in the chimeric enzyme, as predicted. Delivery to brain and uptake by both neurons and astrocytes was demonstrated convincingly. In addition, neuropathology and glycosaminoglycan (GAG) storage in brain were corrected. Delivery to brain and uptake by both neurons and astrocytes was demonstrated convincingly. In addition, neuropathology and glycosaminoglycan (GAG) storage in brain were corrected. Biomarkers for increased inflammation and aberrant autophagy that typically accompany GAG storage in brain were normalized. Finally, behavioural abnormalities were also corrected. The females, which typically become hyperactive in this model, calmed down; the males, which typically become sluggish, perked up with treatment. The appropriate controls showed that the apo B tag was required for these therapeutic responses. AAV2/8 expressing the enzyme with or without the IDS signal sequence but without the apo B tag was not effective. All in all, it's quite a complete and remarkable story. Still, some questions remain. Are we certain that the apo B targets receptors on the BBB and promotes transcytosis across the BBB? Although this seems a plausible interpretation, could it be targeting some cells in the macrophage lineage that are recruited to brain and deliver enzyme by this route? Such cell-mediated delivery is thought to explain the CNS correction following haematopoietic stem cell replacement (Biffi et al, 2006). Is sustained high-level expression required, or could this exciting success be translated to conventional enzyme replacement therapy, where weekly or biweekly infusions are typical and brain capillaries are exposed to high levels only briefly? Whatever the mechanism of this extraordinary CNS correction, it is likely that many laboratories will try to replicate these studies with AAV vectors expressing enzymes missing in other lysosomal diseases affecting brain (Krabbe disease, Batten disease, Tay-Sachs disease and many others). In all of these, delivery of enzymes across the BBB to correct CNS pathology and prevent the devastating cognitive impairment is the major unmet need.

Low-grade fibromyxoid sarcoma (previously known as Evans tumor) is a rare soft tissue neoplasm characterized by a deceptively bland appearance despite the potential for late metastasis or recurrence. We describe a 13-year-old patient with a popliteal fossa mass initially thought to be benign that, because of array-comparative genomic hybridization findings and subsequent immunohistochemistry, was diagnosed as low-grade fibromyxoid sarcoma. The array-comparative genomic hybridization demonstrated a loss of 11p11.2p15.5 and a gain of 16p11.2p13.3 with breakpoints involving the CREB3L1 (cAMP responsive element-binding protein 3-like 1) and FUS (fused in sarcoma) genes, respectively. Subsequent fluorescence in situ hybridization analysis of a dual-labeled break-apart FUS probe on interphase cells was positive. Our case highlights the importance of using genetic information obtained via array-comparative genomic hybridization to classify accurately pediatric soft tissue tumors.

Globoid cell leukodystrophy (GLD, Krabbe Disease) is a lysosomal storage disease, resulting from the genetic deficiency of galactosylceramidase (GALC). This disease is marked by accumulation of the cytotoxic lipid psychosine (Psy). Psychosine is known to induce oxidative stress in cultured cells, and this stress can be ameliorated through co-treatment with the antioxidant N-acetyl cysteine (NAC). Oxidative stress has also been observed in vivo in the mouse model of GLD, the Twitcher mouse (Twi). We hypothesized that treating oxidative stress with NAC; either alone or in combination with bone marrow transplant (BMT) would improve the course of disease. All breeding cages were maintained on water containing NAC. Once born, the pups received IP boluses of NAC three times per week, and were maintained on NAC-containing water. A separate cohort of animals received the same regimen of NAC in addition to a BMT on post-natal days 2–3. Although NAC lowers the level of oxidized proteins in the brains of Twi mice, and dramatically improves immunohistochemical markers of disease, neither treatment results in any clinical improvements in the Twi mouse. Our data suggest that oxidative stress may be sufficiently down-stream in the pathogenic cascade initiated by Psy accumulation as to be difficult or impossible to treat with standard pharmacologic agents. It is possible that NAC may synergize with other therapies or combinations of therapies. A better understanding of the initiating effects of Psy toxicity and oxidative damage may uncover treatable therapeutic targets.

Enzyme replacement therapy has been used successfully in many lysosomal storage diseases. However, correction of brain storage has been limited by the inability of infused enzyme to cross the blood-brain barrier (BBB). We recently reported that PerT-GUS, a form of β-glucuronidase (GUS) chemically modified to eliminate its uptake and clearance by carbohydrate-dependent receptors, crossed the BBB and cleared neuronal storage in an immunotolerant model of murine mucopolysaccharidosis (MPS) type VII. In this respect, the chemically modified enzyme was superior to native β-glucuronidase. Chemically modified enzyme was also delivered more effectively to heart, kidney, and muscle. However, liver and spleen, which express high levels of carbohydrate receptors, received nearly fourfold lower levels of PerT-GUS compared with native GUS. A recent report on PerT-treated sulfamidase in murine MPS IIIA confirmed enhanced delivery to other tissues but failed to observe clearance of storage in neurons. To confirm and extend our original observations, we compared the efficacy of 12 weekly i.v. infusions of PerT-GUS versus native GUS on (i) delivery of enzyme to brain; (ii) improvement in histopathology; and (iii) correction of secondary elevations of other lysosomal enzymes. Such correction is a recognized biomarker for correction of neuronal storage. PerT-GUS was superior to native GUS in all three categories. These results provide additional evidence that long-circulating enzyme, chemically modified to escape carbohydrate-mediated clearance, may offer advantages in treating MPS VII. The relevance of this approach to treat other lysosomal storage diseases that affect brain awaits confirmation.

Intrathecal (IT) recombinant human α-l-iduronidase (rhIDU) has been shown to reduce mean brain glycosaminoglycans (GAGs) to normal levels in mucopolysaccharidosis I (MPS I) dogs. In this study, we examined storage in neuroanatomical regions of the MPS I dog brain, including frontal lobe, cerebellum, basal ganglia, thalamus, hippocampal formation, and brainstem, to determine the response of these functional regions to treatment with IT rhIDU. GAG storage in untreated MPS I dogs was significantly different from normal dogs in all examined sections. GAG levels in normal dogs varied by region: frontal lobe (mean: 2.36 ± 0.54 μg/mg protein), cerebellum (2.67 ± 0.33), basal ganglia and thalamus (3.51 ± 0.60), hippocampus (3.30 ± 0.40), and brainstem (3.73 ± 1.10). Following IT treatment, there was a reduction in GAG storage in each region in all treatment groups, except for the brainstem. Percent reduction in GAG levels from untreated to treated MPS I dogs in the deeper regions of the brain was 30% for basal ganglia and thalamus and 30% for hippocampus, and storage reduction was greater in superficial regions, with 61% reduction in the frontal lobe and 54% in the cerebellum compared with untreated MPS I dogs. Secondary lipid storage in neurons was also reduced in frontal lobe, but not in the other brain regions examined. Response to therapy appeared to be greater in more superficial regions of the brain, particularly in the frontal lobe cortex.

Globoid-cell leukodystrophy (GLD) is an inherited demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC). A previous study in the murine model of GLD (twitcher) demonstrated a dramatic synergy between CNS-directed adeno-associated virus 2/5 (AAV2/5) gene therapy and myeloreductive bone marrow transplantation (BMT). However, the mechanism by which these two disparate therapeutic approaches synergize is not clear. In addition, the therapeutic efficacy may have been limited since the CNS-directed gene therapy was restricted to the forebrain and thalamus. In the current study, intrathecal and intracerebellar injections were added to the therapeutic regimen and the mechanism of synergy between BMT and gene therapy was determined. Although AAV2/5 alone provided supraphysiological levels of GALC activity and reduced psychosine levels in both the brain and spinal cord, it significantly increased CNS inflammation. Bone marrow transplantation alone provided essentially no GALC activity to the CNS and did not reduce psychosine levels. When AAV2/5 is combined with BMT, there are sustained improvements in motor function and the median life span is increased to 123 d (range, 92–282 d) compared with 41 d in the untreated twitcher mice. Interestingly, addition of BMT virtually eliminates both the disease and AAV2/5-associated inflammatory response. These data suggest that the efficacy of AAV2/5-mediated gene therapy is limited by the associated inflammatory response and BMT synergizes with AAV2/5 by modulating inflammation.

Chen A, Vogler C, McEntee M, Hanson S, Ellinwood MN, Jens J, Snella E, Passage M, Le S, Guerra C, Dickson P. Glycosaminoglycan storage in neuroanatomical regions of mucopolysaccharidosis I dogs following intrathecal recombinant human iduronidase. APMIS 2011; 119: 513–21. Intrathecal (IT) recombinant human α- l -iduronidase (rhIDU) has been shown to reduce mean brain glycosaminoglycans (GAGs) to normal levels in mucopolysaccharidosis I (MPS I) dogs. In this study, we examined storage in neuroanatomical regions of the MPS I dog brain, including frontal lobe, cerebellum, basal ganglia, thalamus, hippocampal formation, and brainstem, to determine the response of these functional regions to treatment with IT rhIDU. GAG storage in untreated MPS I dogs was significantly different from normal dogs in all examined sections. GAG levels in normal dogs varied by region: frontal lobe (mean: 2.36 ± 0.54 μg/mg protein), cerebellum (2.67 ± 0.33), basal ganglia and thalamus (3.51 ± 0.60), hippocampus (3.30 ± 0.40), and brainstem (3.73 ± 1.10). Following IT treatment, there was a reduction in GAG storage in each region in all treatment groups, except for the brainstem. Percent reduction in GAG levels from untreated to treated MPS I dogs in the deeper regions of the brain was 30% for basal ganglia and thalamus and 30% for hippocampus, and storage reduction was greater in superficial regions, with 61% reduction in the frontal lobe and 54% in the cerebellum compared with untreated MPS I dogs. Secondary lipid storage in neurons was also reduced in frontal lobe, but not in the other brain regions examined. Response to therapy appeared to be greater in more superficial regions of the brain, particularly in the frontal lobe cortex.