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Approved therapies for Fabry disease (FD) include migalastat, an oral pharmacological chaperone, and agalsidase beta and agalsidase alfa, 2 forms of enzyme replacement therapy. Broad tissue distribution may be beneficial for clinical efficacy in FD, which has severe manifestations in multiple organs. Here, migalastat and agalsidase beta biodistribution were assessed in mice and modeled using physiologically based pharmacokinetic (PBPK) analysis, and migalastat biodistribution was subsequently extrapolated to humans. In mice, migalastat concentration was highest in kidneys and the small intestine, 2 FD‐relevant organs. Agalsidase beta was predominantly sequestered in the liver and spleen (organs unaffected in FD). PBPK modeling predicted that migalastat 123 mg every other day resulted in concentrations exceeding the in vitro half‐maximal effective concentration in kidneys, small intestine, skin, heart, and liver in human subjects. However, extrapolation of mouse agalsidase beta concentrations to humans was unsuccessful. In conclusion, migalastat may distribute to tissues that are inaccessible to intravenous agalsidase beta in mice, and extrapolation of mouse migalastat concentrations to humans showed adequate tissue penetration, particularly in FD‐relevant organs.

Fabry disease is caused by deficient activity of α-galactosidase A, an enzyme that hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins, and subsequent accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb(3)), globotriaosylsphingosine (lyso-Gb(3)), and galabiosylceramide. However, there is no known link between these compounds and disease severity. In this study, we compared Gb(3) isoforms (various fatty acids) and lyso-Gb(3) analogs (various sphingosine modifications) in two strains of Fabry disease mouse models: a pure C57BL/6 (B6) background or a B6/129 mixed background, with the latter exhibiting more prominent cardiac and renal hypertrophy and thermosensation deficits. Total Gb(3) and lyso-Gb(3) levels in the heart, kidney, and dorsal root ganglion (DRG) were similar in the two strains. However, levels of the C20-fatty acid isoform of Gb(3) and particular lyso-Gb(3) analogs (+18, +34) were significantly higher in Fabry-B6/129 heart tissue when compared with Fabry-B6. By contrast, there was no difference in Gb(3) and lyso-Gb(3) isoforms/analogs in the kidneys and DRG between the two strains. Furthermore, using immunohistochemistry, we found that Gb(3) massively accumulated in DRG mechanoreceptors, a sensory neuron subpopulation with preserved function in Fabry disease. However, Gb(3) accumulation was not observed in nonpeptidergic nociceptors, the disease-relevant subpopulation that has remarkably increased isolectin-B4 (the marker of nonpeptidergic nociceptors) binding and enlarged cell size. These findings suggest that specific species of Gb(3) or lyso-Gb(3) may play major roles in the pathogenesis of Fabry disease, and that Gb(3) and lyso-Gb(3) are not responsible for the pathology in all tissues or cell types.

Here we present our progress in inducing an ectopic brown adipose tissue (BAT) phenotype in skeletal muscle (SKM) as a potential gene therapy for obesity and its comorbidities. We used ultrasound-targeted microbubble destruction (UTMD), a novel targeted, non-viral approach to gene therapy, to deliver genes in the BAT differentiation pathway into rodent SKM to engineer a thermogenic BAT phenotype with ectopic mUCP-1 overexpression. In parallel, we performed a second protocol using wild-type Ucp-1-null knockout mice to test whether the effects of the gene therapy are UCP-1 dependent. Our main findings were a robust cellular presence of mUCP-1 immunostaining (IHC), significantly higher expression levels of mUCP-1 measured by qRT-PCR, and highest temperature elevation measured by infrared thermography in the treated thigh, achieved in rats after delivering the UTMD-PRDM16/PGC-1a/BMP7/hyPB gene cocktail. Interestingly, the weight loss obtained in the treated rats with the triple gene delivery, never recovered the levels observed in the controls in spite of food intake recovery. Our results establish the feasibility of minimally invasive UTMD gene-based therapy administration in SKM, to induce overexpression of ectopic mUCP-1 after delivery of the thermogenic BAT gene program, and describe systemic effects of this intervention on food intake, weight loss, and thermogenesis.

Fabry disease is a glycosphingolipidosis caused by deficient activity of α-galactosidase A; it is one of a few diseases that are associated with priapism, an abnormal prolonged erection of the penis. The goal of this study was to investigate the pathogenesis of Fabry disease-associated priapism in a mouse model of the disease. We found that Fabry mice develop late-onset priapism. Neuronal nitric oxide synthase (nNOS), which was predominantly present as the 120-kDa N-terminus-truncated form, was significantly upregulated in the penis of 18-month-old Fabry mice compared to wild type controls (~fivefold). Endothelial NOS (eNOS) was also upregulated (~twofold). NO level in penile tissues of Fabry mice was significantly higher than wild type controls at 18 months. Gene transfer-mediated enzyme replacement therapy reversed abnormal nNOS expression in the Fabry mouse penis. The penile nNOS level was restored by antiandrogen treatment, suggesting that hyperactive androgen receptor signaling in Fabry mice may contribute to nNOS upregulation. However, the phosphodiesterase-5A expression level and the adenosine content in the penis, which are known to play roles in the development of priapism in other etiologies, were unchanged in Fabry mice. In conclusion, these data suggested that increased nNOS (and probably eNOS) content and the consequential elevated NO production and high arterial blood flow in the penis may be the underlying mechanism of priapism in Fabry mice. Furthermore, in combination with previous findings, this study suggested that regulation of NOS expression is susceptible to α-galactosidase A deficiency, and this may represent a general pathogenic mechanism of Fabry vasculopathy.

Lysosomal replacement enzymes are essential therapeutic options for rare congenital lysosomal enzyme deficiencies, but enzymes in clinical use are only partially effective due to short circulatory half-life and inefficient biodistribution. Replacement enzymes are primarily taken up by cell surface glycan receptors, and glycan structures influence uptake, biodistribution, and circulation time. It has not been possible to design and systematically study effects of different glycan features. Here we present a comprehensive gene engineering screen in Chinese hamster ovary cells that enables production of lysosomal enzymes with N-glycans custom designed to affect key glycan features guiding cellular uptake and circulation. We demonstrate distinct circulation time and organ distribution of selected glycoforms of α-galactosidase A in a Fabry disease mouse model, and find that an α2-3 sialylated glycoform designed to eliminate uptake by the mannose 6-phosphate and mannose receptors exhibits improved circulation time and targeting to hard-to-reach organs such as heart. The developed design matrix and engineered CHO cell lines enables systematic studies towards improving enzyme replacement therapeutics., Lysosomal replacement enzymes are taken up by cell surface receptors that recognize glycans, the effects of different glycan features are unknown. Here the authors present a gene engineering screen in CHO cells that allows custom N-glycan-decorated enzymes with improved circulation time and organ distribution.

Established adriamycin cardiomyopathy is a lethal disease. When congestive heart failure develops, mortality is approximately 50% in a year. It has been known that ANGPTLs has various functions in lipid metabolism, inflammation, cancer cell invasion, hematopoietic stem activity and diabetes. We hypothesized that ANGPTL8 is capable of maintaining heart function by stimulating adult cardiac progenitor cells to initiate myocardial regeneration. We employed UTMD to deliver piggybac transposon plasmids with the human ANGPTL8 gene to the liver of rats with adriamycin cardiomyopathy. After ANGPTL8 gene liver delivery, overexpression of transgenic human ANGPTL8 was found in rat liver cells and blood. UTMD- ANGPTL8 gene therapy restored LV mass, fractional shortening index, and LV posterior wall diameter to nearly normal. Our results also showed that ANGPTL8 reversed established ADM cardiomyopathy. This was associated with activation of ISL-1 positive cardiac progenitor cells in the epicardium. A time-course experiment shown that ISL-1 cardiac progenitor cells proliferated and formed a niche in the epicardial layer and then migrated into sub-epicardium. The observed myocardial regeneration accompanying reversal of adriamycin cardiomyopathy was associated with upregulation of PirB expression on the cell membrane of cardiac muscle cells or progenitor cells stimulated by ANGPTL8.

Enzyme replacement therapy (ERT) is an effective treatment for several lysosomal storage disorders (LSDs). Intravenously infused enzymes are taken up by tissues through either the mannose 6-phosphate receptor (M6PR) or the mannose receptor (MR). It is generally believed that M6PR-mediated endocytosis is a key mechanism for ERT in treating LSDs that affect the non-macrophage cells of visceral organs. However, the therapeutic efficacy of MR-mediated delivery of mannose-terminated enzymes in these diseases has not been fully evaluated. We tested the effectiveness of a non-phosphorylated α-galactosidase A produced from moss (referred to as moss-aGal) in vitro and in a mouse model of Fabry disease. Endocytosis of moss-aGal was MR-dependent. Compared to agalsidase alfa, a phosphorylated form of α-galactosidase A, moss-aGal was more preferentially targeted to the kidney. Cellular localization of moss-aGal and agalsidase alfa in the heart and kidney was essentially identical. A single injection of moss-aGal led to clearance of accumulated substrate in the heart and kidney to an extent comparable to that achieved by agalsidase alfa. This study suggested that mannose-terminated enzymes may be sufficiently effective for some LSDs in which non-macrophage cells are affected, and that M6P residues may not always be a prerequisite for ERT as previously considered. Electronic supplementary material The online version of this article (doi:10.1007/s10545-015-9886-9) contains supplementary material, which is available to authorized users.

Recently GLP-1 was found to have cardioprotective effects independent of those attributable to tight glycemic control. Methods and results We employed ultrasound targeted microbubble destruction (UTMD) to deliver piggybac transposon plasmids encoding the GLP-1 gene with a nuclear localizing signal to rat hearts with adriamycin cardiomyopathy. After a single UTMD treatment, overexpression of transgenic GLP-1 was found in nuclei of rat heart cells with evidence that transfected cardiac cells had undergone proliferation. UTMD-GLP-1 gene therapy restored LV mass, fractional shortening index, and LV posterior wall diameter to nearly normal. Nuclear overexpression of GLP-1 by inducing phosphorylation of FoxO1-S256 and translocation of FoxO1 from the nucleus to the cytoplasm significantly inactivated FoxO1 and activated the expression of cyclin D1 in nuclei of cardiac muscle cells. Reversal of adriamycin cardiomyopathy appeared to be mediated by dedifferentiation and proliferation of nuclear FoxO1-positive cardiac muscle cells with evidence of embryonic stem cell markers (OCT4, Nanog, SOX2 and c-kit), cardiac early differentiation markers (NKX2.5 and ISL-1) and cellular proliferation markers (BrdU and PHH3) after UTMD with GLP-1 gene therapy. Conclusions Intranuclear myocardial delivery of the GLP-1gene can reverse established adriamycin cardiomyopathy by stimulating myocardial regeneration.

ANGPTL8 is a circulatory hormone secreted from liver and adipose tissue that promotes pancreatic beta cell proliferation and interferes with triacylglycerol metabolism in mice. The clinical significance of its effects on inducing beta cell proliferation is limited because it causes severe hypertriacylglycerolaemia. We employed ultrasound-targeted microbubble destruction (UTMD) to deliver human ANGPTL8 gene plasmids to the pancreas, liver and skeletal muscle of normal adult rats. Human ANGPTL8 was consistently detected in the circulation 1 month after UTMD. ANGPTL8 gene delivery promoted the proliferation of adult and aged beta cells, expanded the beta cell mass, improved glucose tolerance and increased the fasting blood insulin level after UTMD treatment without causing severe hypertriacylglycerolaemia. ANGPTL8 gene therapy significantly alleviated but did not totally reverse STZ-induced diabetes in a rat model. ANGPTL8 induced adult and aged beta cell regeneration in a rat model.

Knockout mouse model of Fabry disease (Ohshima, 1997) is widely used, and has been proven an invaluable tool to investigate disease pathogenesis and therapies. However, the importance of genetic background of this mouse line has been underestimated, and in many publications WT control mice were chosen incorrectly. Our aim was to examine the effects of genetic background on biochemical and functional abnormalities of Fabry mice. We characterized Fabry mice in B6/129 mixed background (~75% B6) and Fabry mice on a purely C57BL/6 strain (10 generations backcrossing), compared with WT mice on an identical genetic background. Both Fabry-B6/129 and Fabry-B6 developed progressive cardiac and renal hypertrophy and impaired thermosensation. However, Fabry-B6/129 exhibited earlier onset and more severe phenotypes than Fabry-B6. Cardiac and/or renal hypertrophy appeared at age of 6 months in Fabry-B6/129, but at 12 months in Fabry-B6. At 12 months, kidney-to-body weight ratio increased by 50% in Fabry-B6/129 (vs. WT), but only 15% in Fabry-B6. The onset of the thermosensation abnormality was at age 2 months in Fabry-B6/129 and 7 months in Fabry-B6. Cardiac expression of hypertrophy marker, atrial natriuretic peptide (ANP), was upregulated in Fabry-B6/129, but not in Fabry-B6. Despite these differences, globotriaosylceramide (Gb3) and lyso-Gb3 levels in the heart, kidney and dorsal root ganglia were similar between Fabry-B6/129 and Fabry-B6. However, Gb3 level in liver (organ unaffected in Fabry disease) was twofold higher in Fabry-B6/129 compared to Fabry-B6. Additionally, several parameters including body weight, organ weight, kidney Gb3 and cardiac ANP level were different between WT-B6/129 and WT-B6. In conclusion: 1) genetic background has a significant impact on the severity and onset of phenotypes in Fabry mice 2) the lack of correlation between phenotype and Gb3/lyso-Gb3 levels in disease-relevant organs suggests that the effect of genetic background on the phenotype is determined by other genetic and epigenetic factors.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent accumulation of glycosphingolipids (mainly globotriaosylceramide, Gb3), leading to multisystem organ dysfunction. Oxidative stress and nitric oxide synthase (NOS) uncoupling are thought to contribute to Fabry cardiovascular diseases. We hypothesized that decreased tetrahydrobiopterin (BH4) plays a role in the pathogenesis of Fabry disease. We found that BH4 was decreased in the heart and kidney but not in the liver and aorta of Fabry mice. BH4 was also decreased in the plasma of female Fabry patients, which was not corrected by enzyme replacement therapy (ERT). Gb3 levels were inversely correlated with BH4 levels in animal tissues and cultured patient cells. To investigate the role of BH4 deficiency in disease phenotypes, 12-month-old Fabry mice were treated with gene transfer-mediated ERT or substrate reduction therapy (SRT) for 6 months. In the Fabry mice receiving SRT but not ERT, BH4 deficiency was restored, concomitant with ameliorated cardiac and renal hypertrophy. Additionally, glutathione levels were decreased in Fabry mouse tissues in a sex-dependent manner. Renal BH4 levels were closely correlated with glutathione levels and inversely correlated with cardiac and kidney weight. In conclusion, this study showed that BH4 deficiency occurs in Fabry disease and may contribute to the pathogenesis of the disease through oxidative stress associated with a reduced antioxidant capacity of cells and NOS uncoupling. This study also suggested dissimilar efficacy of ERT and SRT in correcting pre-existing pathologies in Fabry disease.

The aim of our study was to measure globotriaosylceramide (Gb(3)) and lyso-Gb(3) levels by tandem mass spectrometry in the urine and kidney in Fabry (gla knockout) mice and wild-type controls. We found that urine Gb(3) of male and female Fabry mice was higher than wild-type mice of the same sex but also significantly higher in male mice compared with females of the same genotype. In kidney tissue, sex and genotype-dependent differences in Gb(3) levels paralleled those in the urine. Isoforms C16, C22:1, and C24OHA were particularly higher in males compared with females in both wild-type and Fabry mice. Similarly, kidney lyso-Gb(3) concentrations were significantly higher in 12-month-old male Fabry mice than in their homozygous female counterparts. However, lyso-Gb(3) was undetectable in wild-type mice of both sexes. α-Galactosidase A activity and mRNA levels in kidney were significantly lower in male wild-type mice compared with female mice. This study shows the sex differences in kidney and urine Gb(3) and kidney lyso-Gb(3) levels in both wild-type and Fabry mice, and it suggests that these male-female differences should be taken into consideration when using murine models for Fabry disease.

Roots play a key role in ecosystem functioning as they transfer water and nutrients from soil to plants. Traditional methods for measuring roots are labor-intensive and destructive in nature, which limits quantitative and repeatable assessments in long-term research. Ground-penetrating radar (GPR) provides a non-destructive method to measure plant roots. Based on the superiority of GPR with 2 GHz frequency, we developed a new, practical method to estimate root biomass. First, average root matter density was measured by collecting a small number of root samples. Second, under controlled, experimental conditions in a sandy area, a root diameter estimation model base on GPR was developed from which root diameter was estimated. Third, root volume was calculated using the estimated root diameter and assuming the shape of roots to be cylindrical. Finally, root biomass was estimated by averaging root matter density and root volume. Results of this study suggest the following: (1) the density of coarse roots with diameters greater than 0.5 cm is relatively uniform; (2) a new wave shape parameter, ΔT, extracted from profile data of 2 GHz frequency antenna is independent of root depth, thus enabling the construction of a root diameter estimation model with high accuracy; and (3) results of a field experiment demonstrated the GPR-based method to be feasible and effective in estimating biomass of coarse roots. These findings are helpful for improving GPR-based root diameter and biomass estimation models and suggest the potential of GPR data in studying root systems.

In western China seismic wave fields are very complicated and have low signal to noise ratio. In this paper, we focus on complex wave field research by forward modeling and indicate that density should not be ignored in wave field simulation if the subsurface physical properties are quite different. We use the acoustic wave equation with density in the staggered finite-difference method to simulate the wave fields. For this purpose a complicated geologic structural model with rugged surfaces, near-surface low-velocity layers, and high-velocity outcropping layers was designed. Based on the instantaneous wave field distribution, we analyzed the mechanism forming complex wave fields. The influence of low velocity layers on the wave field is very strong. A strong waveguide occurs between the top and base of a low velocity layer, producing multiples which penetrate into the earth and form strong complex wave fields in addition to reflections from subsurface interfaces. For verifying the correctness of the simulated wave fields, prestack depth migration was performed using different algorithms from the forward modeling. The structure revealed by the stacked migration profile is same as the known structure.

Most lysosomal storage diseases (LSDs) are life-threatening genetic diseases. The pathogenesis of these diseases is poorly understood. Induced pluripotent stem (iPS) cell technology offers new opportunities for both mechanistic studies and development of stem cell– based therapies. Here we report the generation of disease-specific iPS cells from mouse models of Fabry disease, globoid cell leukodystrophy (GLD), and mucopolysaccharidosis VII (MPSVII). These mouse model–derived iPS cells showed defects in disease-specific enzyme activities and significant accumulation of substrates for these enzymes. In the lineage-directed differentiation studies, Fabry-iPS and GLD-iPS cells were efficiently differentiated into disease-relevant cell types, such as cardiomyocytes and neural stem cells, which might be useful in mechanistic and therapeutic studies. Notably, MPSVII-iPS cells demonstrated a markedly impaired ability to form embryoid bodies (EBs) in vitro. MPSVII-EBs exibited elevated levels of hyaluronan and its receptor CD44, and markedly reduced expression levels of E-cadherin and cell-proliferating marker. Partial correction of enzyme deficiency in MSPVII-iPS cells led to improved EB formation and reversal of aberrant protein expression. These data indicate a potential mechanism for the partial lethality of MPSVII mice in utero, and suggest a possible abnormality of embryonic development in MPSVII patients. Thus, our study demonstrates the unique promise of iPS cells for studying the pathogenesis and treatment of LSDs.

Pompe disease results from the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA), leading to accumulated glycogen in the heart and the skeletal muscles, which causes cardiomyopathy and muscle weakness. In this study, we tested the feasibility of gene therapy for Pompe disease using a lentivirus vector (LV). Newborn GAA knockout mice were treated with intravenous injection of LV encoding human GAA (hGAA) through the facial superficial temporal vein. The transgene expression in the tissues was analyzed up to 24 weeks after treatment. Our results showed that the recombinant LV was efficient not only in increasing the GAA activity in tissues but also in decreasing their glycogen content. The examination of histological sections showed clearence of the glycogen storage in skeletal and cardiac muscles 16 and 24 weeks after a single vector injection. Levels of expressed hGAA could be detected in serum of treated animals until 24 weeks. No significant immune reaction to transgene was detected in most treated animals. Therefore, we show that LV-mediated delivery system was effective in correcting the biochemical abnormalities and that this gene transfer system might be suitable for further studies on delivering GAA to Pompe disease mouse models.

Cross-hole electromagnetic (EM) inversion has been increasingly used to study the variations in rock properties between boreholes, particularly the properties and spatial distribution of the fluids in a subterranean reservoir. In this study, a practical method for simulating and inverting cross-hole EM data to reconstruct a resistivity distribution is presented. The iterative regularized inversion method incorporates a priori information and variable roughness measures, assuming that the medium in the target area is two dimensional and the injected source is a point dipole source. A forward solver for two-and-half-dimensional (2.5D) EM modelling was developed using the finite element method (FEM) for evaluating the forward problem and the 'pseudo-forward' problem needed to construct the sensitivity matrix. The resulting linear system of equations in the forward problem is solved efficiently by the modified incomplete Cholesky biconjugate gradient iterative method. The partial derivatives forming the sensitivity matrix are computed by the adjoint equation based on the reciprocity principle. The regularized inversion scheme, which can be constrained using the available a priori information from well logging, is used to determine the inter-well resistivity distribution from synthetic EM data and the field data acquired in Gudao oil field in east China. The synthetic and field data examples indicate that our scheme is robust and stable in the presence of random noise and therefore can be used for general cross-hole EM interpretation.

In this study, we present a practical technique of transforming cross-hole EM data into the inter-well resistivity distribution. The a priori information constraint is incorporated into an iterative regularized inversion procedure and a variable roughness is added into the inversion process. Finite element approximation based on a two and a half-dimensional (2.5D) model has been developed for the forward problem and the “pseudo-forward” problem needed for constructing the sensitivity matrix and synthetic data set. The regularized least-squares inversion scheme, constrained with the a priori information obtained from well logs, was adopted to reconstruct the inter-well resistivity profile from two synthetic electromagnetic data sets and field data acquired in the Gudao Oil Field, East China. The partial derivatives of the sensitivity matrix were computed by the adjoint equation based on the reciprocity principle. Inversion results of the synthetic and field data examples suggest that our method is robust and stable in the presence of random noise in the field data and can be used for cross-hole EM field data interpretation.

Fabry disease is an inborn error of glycosphingolipid catabolism resulting from a deficiency of lysosomal enzyme alpha-galactosidase A. The major clinical manifestations of the disease, such as stroke, cardiac dysfunction, and renal impairment, are thought to be caused by vasculopathy due to progressive accumulation of globotriaosylceramide in vascular endothelial cells. The pathogenesis of the vasculopathy has not been elucidated. Since in vitro studies using primary endothelial cells are hampered by the limited lifespan of these cells, the availability of cultured endothelial cells with an extended lifespan is critical for the study of the vasculopathy of Fabry disease. We therefore generated an endothelial cell line from a Fabry hemizygote by introduction of human telomerase reverse transcriptase gene. The cell line has markedly extended lifespan compared to parental primary cells. The cells stably express many key markers of endothelial cells such as von Willebrand factor, CD31, CD34, and endothelial nitric oxide synthase (eNOS) and retain functional characteristics such as uptake of acetylated low-density lipoprotein, responsiveness to angiogenic growth factors, up-regulation of eNOS production upon extracellular stimuli, and formation of tube-like structures on Matrigel basement membrane matrix. The cells show significantly reduced activity of alpha-galactosidase A compared with primary endothelial cells from normal individuals and accumulate globotriaosylceramide in lysosomes. This cell line will provide a useful in vitro model of Fabry disease and will facilitate systematic studies to investigate pathogenic mechanisms and explore new therapeutic approaches for Fabry disease.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent intracellular accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3). Vascular endothelial cells may play important roles in disease pathogenesis, and are one of the main target cell types in therapeutic interventions. In this study, we generated immortalized aortic endothelial cell lines from a mouse model of Fabry disease. These cells retained endothelial cell-specific markers and functions. Gb3 expression level in one of these clones (referred to as FMEC2) was highly susceptible to culture media, and appeared to be regulated by glucosylceramide synthase. Results also showed that Gb3 could be upregulated by hydrocortisone. FMEC2 express the mannose 6-phosphate receptor and sortilin but not the mannose receptor. Uptake studies suggested that sortilin plays a role in the binding and internalization of mammalian cell-produced α-galactosidase A. Moss-aGal (a plant-made enzyme) was endocytosed by FMEC2 via a receptor other than the aforementioned receptors. In conclusion, this study suggests that glucosylceramide synthase and hydrocortisone may play important roles in modulating Gb3 levels in Fabry mouse aortic endothelial cells, and that endocytosis of recombinant α-galactosidase A involves a combination of multiple receptors depending on the properties of the enzyme.

Fabry disease is caused by deficient activity of lysosomal enzyme α-galactosidase A. The enzyme deficiency results in intracellular accumulation of glycosphingolipids, leading to a variety of clinical manifestations including hypertrophic cardiomyopathy and renal insufficiency. The mechanism through which glycosphingolipid accumulation causes these manifestations remains unclear. Current treatment, especially when initiated at later stage of the disease, does not produce completely satisfactory results. Elucidation of the pathogenesis of Fabry disease is therefore crucial to developing new treatments. We found increased activity of androgen receptor (AR) signaling in Fabry disease. We subsequently also found that blockade of AR signaling either through castration or AR-antagonist prevented and reversed cardiac and kidney hypertrophic phenotype in a mouse model of Fabry disease. Our findings implicate abnormal AR pathway in the pathogenesis of Fabry disease and suggest blocking AR signaling as a novel therapeutic approach.

Globoid cell leukodystrophy (GLD, Krabbe disease) is a severe demyelinating disease caused by a genetic defect of β-galactocerebrosidase (GALC). To date treatment to GLD is limited to hematopoietic stem cell transplantation. Experimental approaches by means of gene therapy in twitcher mouse, an authentic murine model of human GLD, showed significant but only marginal improvements of the disease. To clarify whether the introduction of GALC could provide beneficial effects on the oligodendrocytes in GLD, we transduced twitcher oligodendrocytes by stereotactically injecting recombinant retrovirus encoding GALC-myc-tag fusion gene into the forebrain subventricular zone of neonatal twitcher mouse. In vivo effects of exogenous GALC on twitcher oligodendrocytes were studied histologically by combined immunostaining for the myc-epitope and the oligodendroglial specific marker, π form of glutathione- S -transferase, at around 40 days of age. We show here that GALC transduction led to dramatic morphological improvement of the twitcher oligodendrocytes comparing with those in untreated twitcher controls. This study provided direct in vivo evidence that GALC transduction could prevent or correct aberrant morphology of oligodendrocytes in GLD which may be closely related to the dysfunction and/or degeneration of oligodendrocytes and the demyelination in this disease.

The use of stem cells has enabled the successful generation of simple organs. However, anatomically complicated organs such as the kidney have proven more refractory to stem-cell-based regenerative techniques. Given the limits of allogenic organ transplantation, an ultimate therapeutic solution is to establish self-organs from autologous stem cells and transplant them as syngrafts back into donor patients. To this end, we have striven to establish an in vitro organ factory to build up complex organ structures from autologous adult stem cells by using the kidney as a target organ. Cultivation of human mesenchymal stem cells in growing rodent embryos enables their differentiation within a spatially and temporally appropriate developmental milieu, facilitating the first step of nephrogenesis. We show that a combination of whole-embryo culture, followed by organ culture, encourages exogenous human mesenchymal stem cells to differentiate and contribute to functional complex structures of the new kidney.

Background Brain-directed prenatal gene therapy may benefit some lysosomal storage diseases that affect the central nervous system (CNS) before birth. Our previous study showed that intrauterine introduction of recombinant adenoviruses into cerebral ventricles results in efficient gene transfer to the CNS in the mouse. However, transgene expression decreased with time due to the non-integrative property of adenoviral vectors. In this study, in order to obtain permanent gene transduction, we investigated the feasibility of retrovirus-mediated in utero gene transduction. Methods Concentrated retrovirus encoding the LacZ gene was injected into the cerebral ventricles of the embryos of normal and twitcher mice (a murine model of Krabbe disease) at embryonic day 12. The distribution and maintenance of the transgene expression in the recipient brain were analyzed histochemically, biochemically and by the quantitative polymerase chain reaction method pre- and postnatally. Results Efficient and highly persistent gene transduction to the brain was achieved both in normal and the twitcher mouse. Transduced neurons, astrocytes and oligodendrocytes were distributed throughout the brain. The transduced LacZ gene, its transcript and protein expression in the brain were maintained for 14 months without decrement. In addition, gene transduction to multiple tissues other than the brain was also detected at low levels. Conclusions This study suggests that brain-directed in utero gene transfer using retrovirus vector may be beneficial to the treatment of lysosomal storage diseases with severe brain damage early in life, such as Krabbe disease. Copyright © 2005 John Wiley & Sons, Ltd.

Current therapies for lysosomal storage diseases (LSDs), enzyme replacement therapy and bone marrow transplantation are effective for visceral organ pathology of LSD, but their effectiveness for brain involvement in LSDs is still a subject of controversy. As an alternative approach, we transplanted genetically modified bone marrow stromal (BMS) cells to lateral ventricle of newborn mucopolysaccharidosis VII (MPS VII) mice. MPS VII is one of LSDs and caused by deficiency of beta-glucuronidase (GUSB), resulting in accumulation of glycosaminoglycans (GAGs) in brain. At 2 weeks after transplantation, the GUSB enzyme-positive cells were identified in olfactory bulb, striatum and cerebral cortex, and the enzymatic activities in various brain areas increased. The GAGs contents in brain were reduced to near normal level at 4 weeks after transplantation. Although GUSB activity declined to homozygous level after 8 weeks, the reduction of GAGs persisted for 16 weeks. Microscopic examination indicated that the lysosomal distention was not found in treated animal brain. Cognitive function in MPS VII animals as evaluated by Morris Water Maze test in treated mice showed a marked improvement over nontreated animals. Brain transplantation of genetically modified BMS cells appears to be a promising approach to treat diffuse CNS involvement of LSDs.

Background In some lysosomal storage diseases, considerable alterations of the central nervous system (CNS) occur prior to birth and neurodegeneration progresses rapidly soon after birth causing early death in patients. No effective treatment is available after birth. Treatment may need to be initiated before birth to prevent the onset or progression of neurological changes and thereby irreversible brain damage. The aim of this study is to investigate the feasibility and effectiveness of brain-directed prenatal gene therapy for lysosomal storage diseases. Methods Recombinant adenovirus encoding the lacZ gene was injected into the lateral ventricles of mouse embryos and the pattern of gene transduction to the CNS was investigated. In the therapeutic experiment, adenovirus expressing β-glucuronidase was injected into the cerebral ventricles of the embryos of mucopolysaccharidosis VII mice and the therapeutic effects on the brain were evaluated. Results Injection of adenoviral vectors to the cerebral ventricles of mouse embryos led to widespread gene transduction throughout the brain and the spinal cord and transgene expression persisted over 10 months in those surviving the procedure. The prenatal transduction of the therapeutic gene to the brain of the mucopolysaccharidosis VII mouse efficiently prevented lysosomal storage in most brain cells before birth until 4 months after birth. Conclusions Brain-directed in utero gene therapy through an intra-ventricular route would be an effective strategy to treat some lysosomal storage diseases with early and severe CNS alterations. Copyright © 2004 John Wiley & Sons, Ltd.

In the present study, we investigated the feasibility of using human neural stem cells (NSCs) in the treatment of diffuse central nervous system (CNS) alterations in a murine model of mucopolysaccharidosis VII (MPS VII), a lysosomal storage disease caused by a genetic defect in the β-glucuronidase gene. An immortalized NSC line derived from human fetal telencephalon was genetically engineered to overexpress β-glucuronidase and transplanted into the cerebral ventricles of neonatal MPS VII mouse. Transplanted human NSCs were found to integrate and migrate in the host brain and to produce large amount of β-glucuronidase. Brain contents of the substrates of β-glucuronidase were reduced to nearly normal levels, and widespread clearing of lysosomal storage was observed in the MPS VII mouse brain at 25 days posttransplantation. The number of engrafted cells decreased markedly after the transplantation, and it appears that the major cause of the cell death was not the immune response of the host but apoptotic cell death of grafted human NSCs. Results showed that human NSCs would serve as a useful gene transfer vehicle for the treatment of diffuse CNS lesions in human lysosomal storage diseases and are potentially applicable in the treatment of patients suffering from neurological disorders. © 2003 Wiley-Liss, Inc.

We examined neuroprotective effects of recombinant adenoviral vectors encoding glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT1), insulin-like growth factor-1 (IGF1), and transforming growth factor-beta2 (TGFbeta2) on lesioned adult rat facial motoneurons. The right facial nerves of adult Fischer 344 male rats were avulsed and removed from the stylomastoid foramen, and adenoviral vectors were injected into the facial canal. Animals avulsed and treated with adenovirus encoding GDNF, BDNF, CNTF, CT1, IGF1 and TGFbeta2 showed intense immunolabeling for these factors in lesioned facial motoneurons, respectively, indicating adenoviral induction of the neurotrophic factors in these neurons. The treatment with adenovirus encoding GDNF, BDNF, or TGFbeta2 after avulsion significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase immunoreactivity and prevented the induction of nitric oxide synthase activity in these neurons. The treatment with adenovirus encoding CNTF, CT1 or IGF1, however, failed to protect these neurons after avulsion. These results indicate that the gene transfer of GDNF and BDNF and TGFbeta2 but not CNTF, CT1 or IGF1 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

The twitcher mouse is a murine model of human globoid cell leukodystrophy (GLD; Krabbe disease) caused by a genetic defect in the activity of galactosylceramidase (GALC). An accumulation of cytotoxic metabolite, galactosylsphingosine (psychosine), in myelin forming cells (oligodendrocytes and Schwann cells) of the twitcher mouse as well as patients with GLD has been suggested to cause dysfunction of these cells and subsequent demyelination in the central and peripheral nervous system. To investigate further the cellular pathomechanism of GLD, we established spontaneously immortalized Schwann cell lines from the twitcher mouse. Long-term cultures of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of 3-week-old twitcher mice were maintained for 6 months, and spontaneously developed colonies were expanded further and characterized. One of the cell lines, designated TwS1, showed distinct Schwann cell phenotypes, was passaged twice a week and maintained for over 10 months without phenotypic alterations. The TwS1 cells had a nonsense mutation in the GALC genome, and showed markedly reduced GALC activity and elevated psychosine levels. Ultrastructurally, varieties of cytoplasmic inclusions were demonstrated in TwS1 cells. When TwS1 cells were infected with a retrovirus vector encoding GALC, GALC activity was markedly increased and psychosine levels were significantly decreased. These immortalized Schwann cells can be useful in studies on the nervous system lesions in GLD.

In chronic inflammation, macrophages and neutrophils, which are derived from bone marrow, play a pivotal role. Therefore, reconstitution of bone marrow with anti-inflammatory stem cells may modify inflammation. In this study, transplantation-based gene therapy was applied to glomerular inflammation for a long-lasting suppression of the glomerular damage seen in chronic nephritis. Bone marrow cells were harvested from male donor mice, which had received 5-fluorouracil 3 days previously, and transduced with an interleukin 1 (IL-1) receptor antagonist (IL-1Ra) or a mock gene using a retrovirus vector. After confirmation that transduced cells possessed the transgene at approximately 0.7 copies per cell and secreted recombinant IL-1Ra, these cells were infused into sublethally irradiated (6 Gy) female recipients once daily for 4 consecutive days. These female recipient mice had the male Y antigen in bone marrow, liver, and spleen, and 10% to 20% of their spleen cells possessed the transgene even 8 weeks after transplantation. Glomerulonephritis was then induced in these mice. Renal function and histology were retarded in the mice whose bone marrow was reconstituted with IL-1Ra–producing cells compared with mock transduced cells. In situ hybridization using a Y painting probe revealed that transplanted donor cells were recruited into the glomerulus upon induction of nephritis, suggesting therapeutic effects were channeled through the secretion of IL-1Ra from these cells. Furthermore, the survival rate after a second challenge with nephrotoxic antibody was significantly improved in the IL-1Ra chimera. These results suggest that reconstitution of bone marrow for continuous supply of anti-inflammatory cells may be a useful strategy for the treatment of chronic inflammation.

Twitcher mouse is a murine model of human globoid cell leukodystrophy (Krabbe disease), which is characterized by a genetic deficiency in galactocerebrosidase (GALC) activity. The nervous system is affected early and severely by demyelination in the white matter. So far, there is no effective treatment for Krabbe disease except bone marrow transplantation (BMT). However, BMT has inherent limitations such as unavailability of donors and graft-versus-host disease. In this study, we injected recombinant adenovirus encoding GALC into the lateral ventricle of twitcher mice at postnatal day 0 (PND 0) and the therapeutic effects were evaluated. Our results showed slight, but significant improvements in motor functions, body weight and twitching and a prolonged life span. In brain, GALC activity was increased to 15% that of normal littermates and psychosine concentration was decreased to 55% that of untreated twitcher mice at PND 15. The number of PAS-positive globoid cells in brain stem was also reduced significantly at PND 35. In contrast, when adenoviruses were injected to the twitcher mice at PND 15, almost no improvements were observed. These results demonstrate that the timing of treatment may be of great importance in Krabbe disease.

Krabbe disease is a devastating neurodegenerative lysosomal storage disorder caused by a deficiency of β-galactocerebrosidase (GALC). Gene therapy is a promising therapeutic approach for Krabbe disease. As the human brain is large and it is difficult to achieve global gene transduction, the efficacy of cross-correction is a critical determinant of the outcome of gene therapy for this disease. We investigated whether HIV Tat protein transduction domain (PTD) can improve the cross-correction of GALC. Tat-PTD significantly increased (~6-fold) cross-correction of GALC through enhanced secretion and uptake in a cell-culture model system. The effects of Tat-PTD were gene and flanking amino acids dependent. Tat-fusion increased the secretion of α-galactosidase A (α-gal A), but this did not improve its cross-correction. Tat-fusion did not change either secretion or uptake of β-glucocerebrosidase (GC). Tat-PTD increased GALC protein synthesis, abolished reactivity of GC to the 8E4 antibody, and likely reduced mannose phosphorylation in all these lysosomal enzymes. This study demonstrated that Tat-PTD can be useful for increasing cross-correction efficiency of lysosomal enzymes. However, Tat-PTD is not a mere adhesive motif but possesses a variety of biological functions. Therefore, the potential beneficial effect of Tat-PTD should be assessed individually on each lysosomal enzyme.Molecular Therapy-Nucleic Acids (2013) 2, e130; doi:10.1038/mtna.2013.57; published online 22 October 2013.