The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Reuben Matalon - One of the best experts on this subject based on the ideXlab platform.
-
Redirecting N-acetylaspartate metabolism in the central nervous system normalizes myelination and rescues Canavan disease
JCI insight, 2017Co-Authors: Dominic J. Gessler, Danning Li, Hongxia Xu, Qin Su, Julio Sanmiguel, Serafettin Tuncer, Constance M. Moore, Jean A. King, Reuben MatalonAbstract:: Canavan disease (CD) is a debilitating and lethal leukodystrophy caused by mutations in the Aspartoacylase (ASPA) gene and the resulting defect in N-acetylaspartate (NAA) metabolism in the CNS and peripheral tissues. Recombinant adeno-associated virus (rAAV) has the ability to cross the blood-brain barrier and widely transduce the CNS. We developed a rAAV-based and optimized gene replacement therapy, which achieves early, complete, and sustained rescue of the lethal disease phenotype in CD mice. Our treatment results in a super-mouse phenotype, increasing motor performance of treated CD mice beyond that of WT control mice. We demonstrate that this rescue is oligodendrocyte independent, and that gene correction in astrocytes is sufficient, suggesting that the establishment of an astrocyte-based alternative metabolic sink for NAA is a key mechanism for efficacious disease rescue and the super-mouse phenotype. Importantly, the use of clinically translatable high-field imaging tools enables the noninvasive monitoring and prediction of therapeutic outcomes for CD and might enable further investigation of NAA-related cognitive function.
-
raav gene therapy in a canavan s disease mouse model reveals immune impairments and an extended pathology beyond the central nervous system
Molecular Therapy, 2016Co-Authors: Seemin Seher Ahmed, Reuben Matalon, Stefan A Schattgen, Ashley E Frakes, Elif M Sikoglu, Thomas G Hampton, Andrew R Denninger, Daniel A Kirschner, Brian K Kaspar, Guangping GaoAbstract:Aspartoacylase (AspA) gene mutations cause the pediatric lethal neurodegenerative Canavan disease (CD). There is emerging promise of successful gene therapy for CD using recombinant adeno-associated viruses (rAAVs). Here, we report an intracerebroventricularly delivered AspA gene therapy regime using three serotypes of rAAVs at a 20-fold reduced dose than previously described in AspA(-/-) mice, a bona-fide mouse model of CD. Interestingly, central nervous system (CNS)-restricted therapy prolonged survival over systemic therapy in CD mice but failed to sustain motor functions seen in systemically treated mice. Importantly, we reveal through histological and functional examination of untreated CD mice that AspA deficiency in peripheral tissues causes morphological and functional abnormalities in this heretofore CNS-defined disorder. We demonstrate for the first time that AspA deficiency, possibly through excessive N-acetyl aspartic acid accumulation, elicits both a peripheral and CNS immune response in CD mice. Our data establish a role for peripheral tissues in CD pathology and serve to aid the development of more efficacious and sustained gene therapy for this disease.
-
496 from gene therapy to gene enhancement optimized pre clinical gene therapy transforms mice with the severest canavan disease phenotype into supermice
Molecular Therapy, 2015Co-Authors: Reuben Matalon, Dominic J. Gessler, Guangping GaoAbstract:Canavan Disease (CD) is a rare and lethal inherited CNS disorder caused by autosomal recessive mutations in the Aspartoacylase gene. To date, there is no effective treatment available. Gene replacement therapy is an attractive approach for this devastating disease, although the physiologic function of AspA and its metabolite N-acetylaspartate (NAA) is still not fully understood.We previously reported that systemic delivery of the human Aspartoacylase (hAspA) gene by recombinant adeno-associated virus (rAAV) to the CNS of CD mice ameliorated symptoms, partially restored motor function and increased life expectancy to the extent of wild-type (WT) mice. We further sought to investigate the mechanism behind this pre-clinical gene therapy for CD and to improve therapeutic potency. We hypothesized that Kozak sequence and codon modification of hAspA cDNA enhance therapeutic expression to transform our 1st generation AspA expression cassette into a more potent 2nd generation pre-clinical remedy and a better tool to study the physiologic role of AspA.We created expression cassettes containing codon-optimized (Opt) hAspA cDNA together with different Kozak sequences (i.e. HKz and FKz). Our in vitro and in vivo data demonstrate stronger hAspA expression and enzyme activity of the FKzhAspA-Opt vector over all other tested constructs as well as decreased NAA in the CNS below WT level by MRS analysis. Interestingly, rAAVFKzhAspA-Opt treated CD mice perform equally on inverted screen and balance beam but significantly (>50%) better than WT at p28, p90 and p180 on rotarod, suggesting that ubiquitously high expressed hAspA exerts some enhancing effect on treated mice. To confirm these findings, we moved to another mouse model of CD (Nur7 mouse). FKzhAspA-Opt treated Nur7 pups show a similar trend as seen in the CD mice on motor function tests. In addition, open-field testing to measure overall activity and anxiety demonstrates that treated Nur7 mice are more mobile with significantly longer travel distance but anxiety levels equal to WT mice. Currently, we are investigating the molecular mechanism underlying this enhanced performance of treated versus WT mice by studying the metabolome, microRNAome and the possibility of achieving therapeutic efficacy with reduced doses of the optimized vector.Overall, our data demonstrate that our 2nd generation gene therapy for CD not only completely restores the disease phenotype but also achieves motor function performance and overall activity superior to WT mice in two different CD mouse models. Using the same AAV serotype with identical tissue and cell tropism as previously published by our group, implies that excessive NAA is highly detrimental to normal physiology and the NAA re duction independent of the hAspA expressing cell type is sufficient for therapeutic success, which might also suggest a positive correlation of higher AspA activity and lower NAA levels with gene therapeutic outcome for this inborn error of NAA metabolism.
-
lack of Aspartoacylase activity disrupts survival and differentiation of neural progenitors and oligodendrocytes in a mouse model of canavan disease
Journal of Neuroscience Research, 2009Co-Authors: S Kumar, Reuben Matalon, Juan Carlos Biancotti, Jean De VellisAbstract:Loss of the oligodendrocyte (OL)-specific enzyme Aspartoacylase (ASPA) from gene mutation results in the sponginess and loss of white matter (WM) in Canavan disease (CD). This study addresses the fate of OLs during the pathophysiology of CD in an adult ASPA knockout (KO) mouse strain. Massive arrays of neural stem/progenitor cells, immunopositive for PSA-NCAM, nestin, vimentin, and NG2, were observed within the severely affected spongy WM of the KO mouse brain. In these mice, G1-->S cell cycle progression was confirmed by an increase in cdk2-kinase activity, a reduction in mitotic inhibitors p21(Cip1) and p27(Kip1), and an increase in bromodeoxyuridine (BrdU) incorporation. Highly acetylated nuclear histones H2B and H3 were detected in adult KO mouse WM, suggesting the existence of noncompact chromatin as seen during early development. Costaining for BrdU- or Ki67-positive cells with markers for neural progenitors confirmed a continuous generation of OL lineage cells in KO WM. We observed a severe reduction in 21.5- and 18.5-kDa myelin basic protein and PLP/DM20 proteolipid proteins combined with a decrease in myelinated fibers and a perinuclear retention of myelin protein staining, indicating impairment in protein trafficking. Death of OLs, neurons, and astrocytes was identified in every region of the KO brain. Immature OLs constituted the largest population of dying cells, particularly in WM. We also report an early expression of full-length ASPA mRNA in normal mouse brain at embryonic day 12.5, when OL progenitors first appear during development. These findings support involvement of ASPA in CNS development and function.
-
absence like and tonic seizures in Aspartoacylase attractin double mutant mice
Experimental Animals, 2007Co-Authors: Hiroshi Gohma, Sankar Surendran, Reuben Matalon, Takashi Kuramoto, Kazuhiro Kitada, Stephen K Tyring, Masashi Sasa, Tadao SerikawaAbstract:The Spontaneously Epileptic Rat (SER), a double-mutant for tremor and zitter mutations, shows spontaneous occurrences of absence-like and tonic seizures. Several lines of evidence suggest that the combined effect of Aspa and Atrn mutations is the most likely cause of the epileptic phenotype of the SER. To address this issue, we produced a new double-mutant mouse line carrying both homozygous Aspa-knockout and Atrn mg-3J mutant alleles. The Aspa/Atrn double-mutant mice exhibited absence-like and tonic seizures that were characterized by the appearance of 5-7 Hz spike-wave-like complexes and low voltage fast waves on EEGs. These results demonstrate directly that the simultaneous loss of the Aspa and Atrn gene functions causes epileptic seizures in the mouse and suggest that both Aspa and Atrn deficiencies might be responsible for epileptic seizures in the SER.
Sankar Surendran - One of the best experts on this subject based on the ideXlab platform.
-
doi:10.4061/2011/426058 Research Article Aspartoacylase Deficiency in the White Matter of Human Immunodeficiency Virus Encephalitis: Novel Mechanism in Axonal Damage
2011Co-Authors: Sankar Surendran, Srinivasagam RajasankarAbstract:Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aspartoacylase/aminoacylase II (ASPA/ACY II) is mainly synthesized in oligodendrocytes to contribute in myelin synthesis. Although axonal damage is seen in the brain with human immunodeficiency virus encephalitis (HIVE), ASPA contribution in the pathology is not known. Immunostaining study showed that ASPA protein is reduced in the white matter of patients with HIVE compared to the control. Western blot study further confirmed ASPA deficiency in the HIVE brain compared to the control. This paper suggests that HIVE condition affects ASPA to contribute in myelin loss/axonal damage seen in the disease. 1
-
Aspartoacylase Deficiency in the White Matter of Human Immunodeficiency Virus Encephalitis: Novel Mechanism in Axonal Damage
Hindawi Limited, 2011Co-Authors: Sankar Surendran, Srinivasagam RajasankarAbstract:Aspartoacylase/aminoacylase II (ASPA/ACY II) is mainly synthesized in oligodendrocytes to contribute in myelin synthesis. Although axonal damage is seen in the brain with human immunodeficiency virus encephalitis (HIVE), ASPA contribution in the pathology is not known. Immunostaining study showed that ASPA protein is reduced in the white matter of patients with HIVE compared to the control. Western blot study further confirmed ASPA deficiency in the HIVE brain compared to the control. This paper suggests that HIVE condition affects ASPA to contribute in myelin loss/axonal damage seen in the disease
-
Upregulation of N-acetylaspartic acid resulting nitric oxide toxicity induces Aspartoacylase mutations and protein interaction to cause pathophysiology seen in Canavan disease.
Medical Hypotheses, 2010Co-Authors: Sankar SurendranAbstract:Summary Aspartoacylase (ASPA) converts N-acetylaspartic acid into aspartate and acetate. In Canavan disease (CD), N-acetylaspartic acid (NAA) is found to be increased and over 65 mutations including IVS4+1 G→T, deletion of introns and exons have been reported in the ASPA gene. These changes lead to severe form or mild form of CD. The present study was aimed to understand mechanism in the cause of mutations in ASPA and pathophysiology seen in patients with CD. We have reported that elevated levels of NAA induce inducible nitric oxide (iNOS) to produce nitric oxide toxicity in CD. Nitric oxide toxicity has been shown to induce several mutations including base change G→T and deletion and enhances protein interaction in several genes. Therefore we hypothesize that upregulation of NAA stimulates NOS and the resulting nitric oxide toxicity induces ASPA mutations and protein interaction to result pathophysiological abnormalities seen in patients with CD.
-
upregulation of n acetylaspartic acid alters inflammation transcription and contractile associated protein levels in the stomach and smooth muscle contractility
Molecular Biology Reports, 2009Co-Authors: Sankar SurendranAbstract:N-acetylaspartic acid (NAA) is converted into aspartate and acetate by Aspartoacylase. Abnormal levels of the enzyme leads to accumulation of NAA and these changes have been observed in Canavan disease and type 2 diabetes. How upregulation of NAA affect the gastrointestine protein levels and the function is not known. Incubation of rat stomach tissue with NAA 1.5 mM, 1.5 μM and 1.5 nM induced inflammatory agents TNFα, p38MAPK, iNOS, PKC, COX2 and ICAM3; transcription factors phospho-NF-kBp65, cjun and cfos; contractile proteins MLCK and phospho MLC; and calcium channel α1C and calcium channel, voltage-dependent, beta 3 subunit compared to their respective control. Incubation of circular smooth muscle cells with the above doses of NAA induced contractility compared to the control. These studies suggest that NAA alters proteins levels and smooth muscle contractility and these changes likely to contribute to gastrointestinal disorder seen in these diseases.
-
absence like and tonic seizures in Aspartoacylase attractin double mutant mice
Experimental Animals, 2007Co-Authors: Hiroshi Gohma, Sankar Surendran, Reuben Matalon, Takashi Kuramoto, Kazuhiro Kitada, Stephen K Tyring, Masashi Sasa, Tadao SerikawaAbstract:The Spontaneously Epileptic Rat (SER), a double-mutant for tremor and zitter mutations, shows spontaneous occurrences of absence-like and tonic seizures. Several lines of evidence suggest that the combined effect of Aspa and Atrn mutations is the most likely cause of the epileptic phenotype of the SER. To address this issue, we produced a new double-mutant mouse line carrying both homozygous Aspa-knockout and Atrn mg-3J mutant alleles. The Aspa/Atrn double-mutant mice exhibited absence-like and tonic seizures that were characterized by the appearance of 5-7 Hz spike-wave-like complexes and low voltage fast waves on EEGs. These results demonstrate directly that the simultaneous loss of the Aspa and Atrn gene functions causes epileptic seizures in the mouse and suggest that both Aspa and Atrn deficiencies might be responsible for epileptic seizures in the SER.
Matthias Klugmann - One of the best experts on this subject based on the ideXlab platform.
-
Aspartoacylase-LacZ Knockin Mice: An Engineered Model of Canavan Disease
2016Co-Authors: Nadine Mersmann, Dmitri Tkachev, Ruth Jelinek, Wolfgang Weber-fahr, Er Sartorius, Matthias KlugmannAbstract:Canavan Disease (CD) is a recessive leukodystrophy caused by loss of function mutations in the gene encoding Aspartoacylase (ASPA), an oligodendrocyte-enriched enzyme that hydrolyses N-acetylaspartate (NAA) to acetate and aspartate. The neurological phenotypes of different rodent models of CD vary considerably. Here we report on a novel targeted aspa mouse mutant expressing the bacterial b-Galactosidase (lacZ) gene under the control of the aspa regulatory elements. X-Gal staining in known ASPA expression domains confirms the integrity of the modified locus in heterozygous aspa lacZ-knockin (aspalacZ/+) mice. In addition, abundant ASPA expression was detected in Schwann cells. Homozygous (aspalacZ/lacZ) mutants are ASPA-deficient, show CD-like histopathology and moderate neurological impairment with behavioural deficits that are more pronounced in aspalacZ/lacZ males than females. Non-invasive ultrahigh field proton magnetic resonance spectroscopy revealed increased levels of NAA, myo-inositol and taurine in the aspalacZ/lacZ brain. Spongy degeneration was prominent in hippocampus, thalamus, brain stem, and cerebellum, whereas white matter of optic nerve and corpus callosum was spared. Intracellular vacuolisation in astrocytes coincides with axonal swellings in cerebellum and brain stem of aspalacZ/lacZ mutants indicating that astroglia may act as an osmolyte buffer in the aspa-deficient CNS. In summary, the aspalacZ mouse is an accurate model of CD and an important tool to identify novel aspects o
-
Loss of Central Auditory Processing in a Mouse Model of Canavan Disease
PloS one, 2014Co-Authors: Georg Von Jonquieres, Claudia B. Klugmann, Kristina E. Froud, Ann C. Y. Wong, Gary D. Housley, Matthias KlugmannAbstract:Canavan Disease (CD) is a leukodystrophy caused by homozygous null mutations in the gene encoding Aspartoacylase (ASPA). ASPA-deficiency is characterized by severe psychomotor retardation, and excessive levels of the ASPA substrate N-acetylaspartate (NAA). ASPA is an oligodendrocyte marker and it is believed that CD has a central etiology. However, ASPA is also expressed by Schwann cells and ASPA-deficiency in the periphery might therefore contribute to the complex CD pathology. In this study, we assessed peripheral and central auditory function in the AspalacZ/lacZ rodent model of CD using auditory brainstem response (ABR). Increased ABR thresholds and the virtual loss of waveform peaks 4 and 5 from AspalacZ/lacZ mice, indicated altered central auditory processing in mutant mice compared with Aspawt/wt controls and altered central auditory processing. Analysis of ABR latencies recorded from AspalacZ/lacZ mice revealed that the speed of nerve conduction was unchanged in the peripheral part of the auditory pathway, and impaired in the CNS. Histological analyses confirmed that ASPA was expressed in oligodendrocytes and Schwann cells of the auditory system. In keeping with our physiological results, the cellular organization of the cochlea, including the organ of Corti, was preserved and the spiral ganglion nerve fibres were normal in ASPA-deficient mice. In contrast, we detected substantial hypomyelination in the central auditory system of AspalacZ/lacZ mice. In summary, our data suggest that the lack of ASPA in the CNS is responsible for the observed hearing deficits, while ASPA-deficiency in the cochlear nerve fibres is tolerated both morphologically and functionally.
-
Aspartoacylase lacz knockin mice an engineered model of canavan disease
PLOS ONE, 2011Co-Authors: Nadine Mersmann, Dmitri Tkachev, Ruth Jelinek, Philipp Thomas Roth, Wiebke Mobius, Torben Ruhwedel, Sabine Ruhle, Wolfgang Weberfahr, Alexander Sartorius, Matthias KlugmannAbstract:Canavan Disease (CD) is a recessive leukodystrophy caused by loss of function mutations in the gene encoding Aspartoacylase (ASPA), an oligodendrocyte-enriched enzyme that hydrolyses N-acetylaspartate (NAA) to acetate and aspartate. The neurological phenotypes of different rodent models of CD vary considerably. Here we report on a novel targeted aspa mouse mutant expressing the bacterial β-Galactosidase (lacZ) gene under the control of the aspa regulatory elements. X-Gal staining in known ASPA expression domains confirms the integrity of the modified locus in heterozygous aspa lacZ-knockin (aspalacZ/+) mice. In addition, abundant ASPA expression was detected in Schwann cells. Homozygous (aspalacZ/lacZ) mutants are ASPA-deficient, show CD-like histopathology and moderate neurological impairment with behavioural deficits that are more pronounced in aspalacZ/lacZ males than females. Non-invasive ultrahigh field proton magnetic resonance spectroscopy revealed increased levels of NAA, myo-inositol and taurine in the aspalacZ/lacZ brain. Spongy degeneration was prominent in hippocampus, thalamus, brain stem, and cerebellum, whereas white matter of optic nerve and corpus callosum was spared. Intracellular vacuolisation in astrocytes coincides with axonal swellings in cerebellum and brain stem of aspalacZ/lacZ mutants indicating that astroglia may act as an osmolyte buffer in the aspa-deficient CNS. In summary, the aspalacZ mouse is an accurate model of CD and an important tool to identify novel aspects of its complex pathology.
-
restoration of Aspartoacylase activity in cns neurons does not ameliorate motor deficits and demyelination in a model of canavan disease
Molecular Therapy, 2005Co-Authors: Matthias Klugmann, Tadao Serikawa, Matthew J During, Claudia B Leichtlein, Wymond C Symes, Deborah YoungAbstract:Canavan disease is an early onset leukodystrophy associated with psychomotor retardation, seizures, and premature death. This disorder is caused by mutations in the gene encoding the enzyme Aspartoacylase (ASPA). Normally, ASPA is enriched in oligodendrocytes and ASPA deficiency results in elevated levels of its substrate molecule, N-acetylaspartate (NAA), brain edema, and dysmyelination. Using adeno-associated virus, we permanently expressed ASPA in CNS neurons of the tremor rat, a genetic model of Canavan disease, and examined the efficacy of the treatment by monitoring NAA metabolism, myelination, motor behavior, and seizures. Assessment of ASPA protein and enzyme activity in whole brain hemispheres showed restoration to normal levels as long as 6 months after treatment. This finding correlated with a reduction of NAA levels, along with a rescue of the seizure phenotype. However, gross brain pathology, such as dilated ventricles and spongiform vacuolization, was unchanged. Moreover, hypomyelination and motor deficits were not resolved by ASPA gene transfer. Our data suggest that NAA-mediated neuronal hyperexcitation but not oligodendrocyte dysfunction can be compensated for by neuronal ASPA expression.
Paola Leone - One of the best experts on this subject based on the ideXlab platform.
-
gata6 regulates Aspartoacylase expression in resident peritoneal macrophages and controls their survival
Journal of Experimental Medicine, 2014Co-Authors: Emmanuel L Gautier, Jeremy S. Francis, Paola Leone, Stoyan Ivanov, Jesse W Williams, Stanley Chingcheng Huang, Genevieve Marcelin, Keke C Fairfax, Peter L Wang, David B WilsonAbstract:The transcription factor Gata6 regulates proliferation and differentiation of epithelial and endocrine cells and cancers. Among hematopoietic cells, Gata6 is expressed selectively in resident peritoneal macrophages. We thus examined whether the loss of Gata6 in the macrophage compartment affected peritoneal macrophages, using Lyz2-Cre x Gata6flox/flox mice to tackle this issue. In Lyz2-Cre x Gata6flox/flox mice, the resident peritoneal macrophage compartment, but not macrophages in other organs, was contracted, with only a third the normal number of macrophages remaining. Heightened rates of death explained the marked decrease in peritoneal macrophage observed. The metabolism of the remaining macrophages was skewed to favor oxidative phosphorylation and alternative activation markers were spontaneously and selectively induced in Gata6-deficient macrophages. Gene expression profiling revealed perturbed metabolic regulators, including Aspartoacylase (Aspa), which facilitates generation of acetyl CoA. Mutant mice lacking functional Aspa phenocopied the higher propensity to death and led to a contraction of resident peritoneal macrophages. Thus, Gata6 regulates differentiation, metabolism, and survival of resident peritoneal macrophages.
-
Long-Term Follow-Up After Gene Therapy for Canavan Disease
Science translational medicine, 2012Co-Authors: Paola Leone, Jeremy S. Francis, Mitra Assadi, Larissa T. Bilaniuk, David Shera, Edwin H. Kolodny, Dah-jyuu Wang, Olga Goldfarb, Scott W.j. Mcphee, H. Warren GoldmanAbstract:Canavan disease is a hereditary leukodystrophy caused by mutations in the Aspartoacylase gene ( ASPA ), leading to loss of enzyme activity and increased concentrations of the substrate N -acetyl-aspartate (NAA) in the brain. Accumulation of NAA results in spongiform degeneration of white matter and severe impairment of psychomotor development. The goal of this prospective cohort study was to assess long-term safety and preliminary efficacy measures after gene therapy with an adeno-associated viral vector carrying the ASPA gene (AAV2- ASPA ). Using noninvasive magnetic resonance imaging and standardized clinical rating scales, we observed Canavan disease in 28 patients, with a subset of 13 patients being treated with AAV2- ASPA . Each patient received 9 × 10 11 vector genomes via intraparenchymal delivery at six brain infusion sites. Safety data collected over a minimum 5-year follow-up period showed a lack of long-term adverse events related to the AAV2 vector. Posttreatment effects were analyzed using a generalized linear mixed model, which showed changes in predefined surrogate markers of disease progression and clinical assessment subscores. AAV2- ASPA gene therapy resulted in a decrease in elevated NAA in the brain and slowed progression of brain atrophy, with some improvement in seizure frequency and with stabilization of overall clinical status.
-
rapid detection of three large novel deletions of the Aspartoacylase gene in non jewish patients with canavan disease
Molecular Genetics and Metabolism, 2006Co-Authors: B. J. Zeng, Paola Leone, Gregory M. Pastores, Zhao-hui Wang, Paola Torres, S S Raghavan, Edwin H. KolodnyAbstract:Abstract Canavan disease (CD), an autosomal recessive neurodegenerative disorder, is caused by mutations in the Aspartoacylase (ASPA) gene. In the present study, the ASPA gene was analyzed in 24 non-Jewish patients with CD from 23 unrelated families. Within this cohort, we found three large novel deletions of approximate 92, 56, and 12.13 kb in length, using both self-ligation of restriction endonuclease-digested DNA fragments with long-distance inverse PCR and multiplex dosage quantitative PCR analysis of genomic DNA. The 92 kb large deletion results in complete absence of the ASPA gene in one homozygous and one compound heterozygous patient, respectively. The 56 kb large deletion causes absence of the majority of the ASPA gene except for exon 1 alone in a compound heterozygous patient. The 12.13 kb deletion involves deletion of the ASPA gene from intron 3 to intron 5 including exons 4 and 5 (I3 to E4E5I5) in a compound heterozygous patient. Patients with the three large deletions clinically manifested severe symptoms at birth, including seizures. Our study showed that the combined use of long-distance inverse PCR and multiplex dosage quantitative PCR analysis of genomic DNA is a helpful and rapid technique to search for large deletions, particularly for detection of large deletions in compound heterozygous patients.
-
Aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for canavan disease
Annals of Neurology, 2000Co-Authors: Reuben Matalon, Paola Leone, Christopher G. Janson, L Bilianuk, Zhiyue J Wang, F Sorgi, L Huang, R. KaulAbstract:With the ultimate goal of developing safe and effective in vivo gene therapy for the treatment of Canavan disease and other neurological disorders, we developed a non-viral lipid-entrapped, polycation-condensed delivery system (LPD) for central nervous system gene transfer, in conjunction with adeno-associated virus (AAV)-based plasmids containing recombinant Aspartoacylase (ASPA). The gene delivery system was tested in healthy rodents and primates, before proceeding to preliminary studies in 2 children with Canavan disease. Toxicity and expression testing was first carried out in human 293 cells, which demonstrated effective transduction of cells and high levels of functional ASPA activity. We performed in vivo toxicity and expression testing of LPD/pAAVaspa and LPD/pAAVlac in rodents, which demonstrated widespread gene expression for more than 10 months after intraventricular delivery, and local expression in deep brain nuclei and white matter tracts for more than 6 months after intraparenchymal injections, with no significant adverse effects. We also performed intraventricular delivery of LPD/pAAVaspa to 2 cynomologous monkeys, with 2 additional monkeys receiving LPD and saline controls. None of the monkeys demonstrated significant adverse effects, and at 1 month the 2 LPD/pAAVaspa monkeys were positive for human ASPA transcript by reverse transcriptase polymerase chain reaction of brain tissue punches. Finally, we performed the first in vivo gene transfer study for a human neurodegenerative disease in 2 children with Canavan disease to assess the in vivo toxicity and efficacy of ASPA gene delivery. Our results suggest that LPD/pAAVaspa is well tolerated in human subjects and is associated with biochemical, radiological, and clinical changes.
-
global cns gene transfer for a childhood neurogenetic enzyme deficiency canavan disease
Current Opinion in Molecular Therapeutics, 1999Co-Authors: Paola Leone, Christopher G. Janson, Scott W.j. Mcphee, Matthew J DuringAbstract:The neurogenetic prototypic disease on which we chose to test our gene therapy strategy is Canavan disease (CD). CD is an autosomal recessive leukodystrophy associated with spongiform degeneration of the brain. At present the disease is uniformly fatal in affected probands. CD is characterized by mutations in the Aspartoacylase (ASPA) gene, resulting in loss of enzyme activity. In this review, recent evidence is summarized on the etiology and possible treatments for CD. In particular, we discuss two gene delivery systems representing recent advances in both viral and liposome technology: a novel cationic liposome-polymer-DNA (LPD) complex, DCChol/DOPE-protamine, as well as recombinant adeno-associated virus (AAV) vectors.
Juergen Geist - One of the best experts on this subject based on the ideXlab platform.
-
impacts of the phenylpyrazole insecticide fipronil on larval fish time series gene transcription responses in fathead minnow pimephales promelas following short term exposure
Science of The Total Environment, 2012Co-Authors: Sebastian Beggel, Inge Werner, Richard E Connon, Juergen GeistAbstract:Abstract The utilization of molecular endpoints in ecotoxicology can provide rapid and valuable information on immediate organismal responses to chemical stressors and is increasingly used for mechanistic interpretation of effects at higher levels of biological organization. This study contributes knowledge on the sublethal effects of a commonly used insecticide, the phenylpyrazole fipronil, on larval fathead minnow ( Pimephales promelas ), utilizing a quantitative transcriptomic approach. Immediately after 24 h of exposure to fipronil concentrations of ≥ 31 μg.L − 1 , highly significant changes in gene transcription were observed for Aspartoacylase, metallothionein, glucocorticoid receptor, cytochrome P450 3A126 and vitellogenin. Different mechanisms of toxicity were apparent over the course of the experiment, with short-term responses indicating neurotoxic effects. After 6 days of recovery, endocrine effects were observed with vitellogenin being up-regulated 90-fold at 61 μg.L − 1 fipronil. Principal component analysis demonstrated a significant increase in gene transcription changes over time and during the recovery period. In conclusion, multiple mechanisms of action were observed in response to fipronil exposure, and unknown delayed effects would have been missed if transcriptomic responses had only been measured at a single time-point. These challenges can be overcome by the inclusion of multiple endpoints and delayed effects in experimental designs.
-
changes in gene transcription and whole organism responses in larval fathead minnow pimephales promelas following short term exposure to the synthetic pyrethroid bifenthrin
Aquatic Toxicology, 2011Co-Authors: Sebastian Beggel, Inge Werner, Richard E Connon, Juergen GeistAbstract:Abstract The combination of molecular and whole-organism endpoints in ecotoxicology provides valuable information about the ecological relevance of sublethal stressor effects in aquatic ecosystems such as those caused by the use of insecticides and translocation of their residues into surface waters. This study contributes knowledge about the sublethal effects of a common use insecticide, the synthetic pyrethroid bifenthrin, on larval fathead minnow (Pimephales promelas). Transcriptomic responses, assessed by quantitative real-time PCR, combined with individual effects on swimming performance were used to estimate the ecological relevance of insecticide impacts. Significant transcriptomic responses were observed at 0.07 μg L−1 bifenthrin (lowest observed effect concentration, LOEC) but mostly followed a biphasic rather than a linear dose–response with increasing concentration. Transcript patterns for genes involved in detoxification, neuromuscular function and energy metabolism were linked to an impairment of swimming performance at ≥0.14 μg L−1 bifenthrin. With increasing treatment concentration, a significant down-regulation was observed for genes coding for cyp3a, Aspartoacylase, and creatine kinase, whereas metallothionein was up-regulated. Additionally, bifenthrin induced endocrine responses as evident from a significant up-regulation of vitellogenin and down-regulation of insuline-like growth factor transcripts. Recovery occurred after 6 days and was dependent on the magnitude of the initial stress. During the recovery period, down-regulation of vitellogenin was observed at lowest exposure concentrations. The data presented here emphasize that links can be made between gene transcription changes and behavioral responses which is of great value for the evaluation and interpretation of biomarker responses.
