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Brain volumes and cognitive function in MPS IIIA: Cross-sectional study
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Abstracts / Molecular Genetics and Metabolism 102 (2011) S3–S47
human GAA protein (rhGAA) prolongs life however is complicated by high titer antibody (Ab) responses to rhGAA occasionally threatening anaphylactic shock. In an ongoing study, one patient (Subject-A) was given human anti-CD20 initially for B-cell depletion, followed by daily dosing of T-cell depleting agent (Mycophenolate-mofetil) and rhGAA (twice/month). Antibody titers against rhGAA remained at baseline for 4-months, after which levels increased to very high-titer, concomitant with a rise in CD20+ and CD19+ B-cells. The subject subsequently died at 12-months due to respiratory failure. Subjects-B and C received anti-CD20 treatment once every 3-months and daily rapamycin plus rhGAA (twice/month). Anti-hGAA levels in both subjects remained at baseline for > 17-months after initiating treatment, although Subject-C developed a detectable antibody response at 18-months (possibly due to a 2-month gap in rapamycin dosing after MRSA infection). Interestingly, Subject-C developed a population of CD19+ T-cells. IgA and IgM levels stayed below normal, indicating absent or reduced B-cell function. Subject-A had normal levels of IgA, while IgM levels increased to normal by 8-months indicating a fresh population of B-cells. Increased CD3+ levels were observed while NT-pro-BNP, Creatine Kinase levels and LV-massindex showed improvement in all 3 patients. Immune suppression with anti-CD20, Rapamycin and rhGAA can improve survival. B-cell depletion with anti-CD20 may not be sufficient for tolerance induction. A transient regulatory protocol should be developed to avoid constant immune suppression. doi:10.1016/j.ymgme.2010.11.107
Brain volumes and cognitive function in MPS IIIA: Cross-sectional study Igor Nestrasil, Alia Ahmed, Kyle Rudser, Kate Delaney, Elsa Shapiro, University of Minnesota, Minneapolis, MN, USA Background: MPSIII Sanfilippo Syndrome, is one of the few neurodegenerative diseases of childhood that is primarily a neurological disease. Measuring brain volume decreases over time in untreated patients in conjunction with cognitive decline will provide a comparison for future treatment options. Goals: To identify structural specificity of the brain loss evaluated by quantitative MRI linked to cognitive decline. Methods: Cross-sectional quantitative MRI data were collected for 14 children with documented Sanfilippo A syndrome. Volumetric analysis was performed by the automated segmentation and manual tracing of amygdala and hippocampus. All the volumes used in data analysis were adjusted to the intracranial volume. Developmental quotient (DQ) was calculated as the cognitive outcome. Results: Mean age of the enrolled subjects was 5.9 years and the mean DQ was 44. DQ declined with age (p < .01), and robustly with the grey matter loss (p < .001) and with an increase in the compound volume comprising ventricles and cerebrospinal fluid (CSF) segments (p < .001). Grey matter loss was related to the increasing age (p = .02). Left amygdalar volume loss was associated with DQ decline. Discussion: We show that grey matter volume loss with enlarging ventricular and CSF compartments are the leading MRI patterns linked to the cognitive decline in MPSIII. The pathomechanism causing neuronal death is still unknown and no treatment is available for these diseases. We demonstrate a quantitative description of disease progression that is a first step to establish an evaluation tool for future therapies.
doi:10.1016/j.ymgme.2010.11.108
Determination of psychosine in dried blood spots of Krabbe diseased patients Joseph Orsinia, Michele Cagganab, Monica Martinb, Kate Zhangc, WeiLien Chuangc, Joshua Pachecoc, David Wengerd, aNew York State Dept. of Health, Albany, NY, USA, bWadsworth Center, Albany, New York, USA, cGenzyme Corporation, Framingham, MA, USA, dJefferson Medical College, Phildelphia, PA, USA Purpose: New York State has screened over 1 million newborns for Krabbe disease. During the course of screening, we have identified four newborns with infantile Krabbe disease. In addition, several newborns with very low galactocerebrosidase activity have been followed and remain asymptomatic. Thus, a different biomarker is needed to determine if the disease will manifest. The purpose of this study was to determine if psychosine, a breakdown product of myelin degradation could be used as a surrogate marker for progression. As a first step, we examined psychosine concentrations in these twoscreened cohorts. Methods: We applied an advanced HPLC-MS-MS method to determine the psychosine concentrations in dried blood spots (DBS) collected from screened babies with infantile Krabbe disease, and babies who are asymptomatic, but have low GALC activity. DBS from previously used Krabbe patients and normal newborns were also studied. Results: Specimens from the Krabbe disease cohort all had psychosine concentrations (range ~ 7-50 g/mL) above the values measured in normal newborns (<3 g/mL). Psychosine concentrations in the non-symptomatic high-risk category were variable. Conclusions: Psychosine can be quantified in DBS and the concentrations are minimally a factor of 2 times higher in diseased individuals and generally an order of magnitude greater in infantile patients as compared to normal newborns. Further longitudinal studies are needed to determine if psychosine can be used as a predictor of progression in screen positive newborns who do not present with the infantile form of the disease. doi:10.1016/j.ymgme.2010.11.109
MGMT-based Selection of Lentivector Transduced Cells: Application to Fabry Disease Natalia Pacienzaa, Nobuo Mizuea, Matthew Scaifea, Jeffrey Medina,b, University Health Network, Toronto, Ontario, Canada, bDepartment of Medical Biophysics and the Institute of Medical Sciences, University of Toronto, Canada a
Fabry disease is caused by a deficiency of α-galactosidase A (α-gal A) activity. Although enzyme replacement therapy (ERT) is available, this treatment is costly and does not solve the basic genetic defect of the disorder. Genetically-modified hematopoietic stem cells (HSCs) are attractive targets for durable transgene expression in Fabry disease, however low transduction efficiencies of this population may be an obstacle to attainment of successful therapy. The goal of this study is to develop and test a novel lentivector (LV) able to confer drug tolerance to transduced Fabry patient cells by overexpression of the O6-methylguanine-DNA-methyltransferase gene (MGMT). To this end, we have generated a LV carrying a mutated, but functional, form of the selectable marker (MGMT-P140K) followed by an EMCV-IRES element and a codon-optimized copy of the human α-gal A cDNA. Expression and functionality of this cassette was tested in the human erythroleukemic cell line (K562) as well as in Fabry patient B cells and murine Fabry lin- bone marrow cells by intracellular MGMT staining and α-gal A activity assays, before and after selection with O6-
Abstracts / Molecular Genetics and Metabolism 102 (2011) S3–S47
human GAA protein (rhGAA) prolongs life however is complicated by high titer antibody (Ab) responses to rhGAA occasionally threatening anaphylactic shock. In an ongoing study, one patient (Subject-A) was given human anti-CD20 initially for B-cell depletion, followed by daily dosing of T-cell depleting agent (Mycophenolate-mofetil) and rhGAA (twice/month). Antibody titers against rhGAA remained at baseline for 4-months, after which levels increased to very high-titer, concomitant with a rise in CD20+ and CD19+ B-cells. The subject subsequently died at 12-months due to respiratory failure. Subjects-B and C received anti-CD20 treatment once every 3-months and daily rapamycin plus rhGAA (twice/month). Anti-hGAA levels in both subjects remained at baseline for > 17-months after initiating treatment, although Subject-C developed a detectable antibody response at 18-months (possibly due to a 2-month gap in rapamycin dosing after MRSA infection). Interestingly, Subject-C developed a population of CD19+ T-cells. IgA and IgM levels stayed below normal, indicating absent or reduced B-cell function. Subject-A had normal levels of IgA, while IgM levels increased to normal by 8-months indicating a fresh population of B-cells. Increased CD3+ levels were observed while NT-pro-BNP, Creatine Kinase levels and LV-massindex showed improvement in all 3 patients. Immune suppression with anti-CD20, Rapamycin and rhGAA can improve survival. B-cell depletion with anti-CD20 may not be sufficient for tolerance induction. A transient regulatory protocol should be developed to avoid constant immune suppression. doi:10.1016/j.ymgme.2010.11.107
Brain volumes and cognitive function in MPS IIIA: Cross-sectional study Igor Nestrasil, Alia Ahmed, Kyle Rudser, Kate Delaney, Elsa Shapiro, University of Minnesota, Minneapolis, MN, USA Background: MPSIII Sanfilippo Syndrome, is one of the few neurodegenerative diseases of childhood that is primarily a neurological disease. Measuring brain volume decreases over time in untreated patients in conjunction with cognitive decline will provide a comparison for future treatment options. Goals: To identify structural specificity of the brain loss evaluated by quantitative MRI linked to cognitive decline. Methods: Cross-sectional quantitative MRI data were collected for 14 children with documented Sanfilippo A syndrome. Volumetric analysis was performed by the automated segmentation and manual tracing of amygdala and hippocampus. All the volumes used in data analysis were adjusted to the intracranial volume. Developmental quotient (DQ) was calculated as the cognitive outcome. Results: Mean age of the enrolled subjects was 5.9 years and the mean DQ was 44. DQ declined with age (p < .01), and robustly with the grey matter loss (p < .001) and with an increase in the compound volume comprising ventricles and cerebrospinal fluid (CSF) segments (p < .001). Grey matter loss was related to the increasing age (p = .02). Left amygdalar volume loss was associated with DQ decline. Discussion: We show that grey matter volume loss with enlarging ventricular and CSF compartments are the leading MRI patterns linked to the cognitive decline in MPSIII. The pathomechanism causing neuronal death is still unknown and no treatment is available for these diseases. We demonstrate a quantitative description of disease progression that is a first step to establish an evaluation tool for future therapies.
doi:10.1016/j.ymgme.2010.11.108
Determination of psychosine in dried blood spots of Krabbe diseased patients Joseph Orsinia, Michele Cagganab, Monica Martinb, Kate Zhangc, WeiLien Chuangc, Joshua Pachecoc, David Wengerd, aNew York State Dept. of Health, Albany, NY, USA, bWadsworth Center, Albany, New York, USA, cGenzyme Corporation, Framingham, MA, USA, dJefferson Medical College, Phildelphia, PA, USA Purpose: New York State has screened over 1 million newborns for Krabbe disease. During the course of screening, we have identified four newborns with infantile Krabbe disease. In addition, several newborns with very low galactocerebrosidase activity have been followed and remain asymptomatic. Thus, a different biomarker is needed to determine if the disease will manifest. The purpose of this study was to determine if psychosine, a breakdown product of myelin degradation could be used as a surrogate marker for progression. As a first step, we examined psychosine concentrations in these twoscreened cohorts. Methods: We applied an advanced HPLC-MS-MS method to determine the psychosine concentrations in dried blood spots (DBS) collected from screened babies with infantile Krabbe disease, and babies who are asymptomatic, but have low GALC activity. DBS from previously used Krabbe patients and normal newborns were also studied. Results: Specimens from the Krabbe disease cohort all had psychosine concentrations (range ~ 7-50 g/mL) above the values measured in normal newborns (<3 g/mL). Psychosine concentrations in the non-symptomatic high-risk category were variable. Conclusions: Psychosine can be quantified in DBS and the concentrations are minimally a factor of 2 times higher in diseased individuals and generally an order of magnitude greater in infantile patients as compared to normal newborns. Further longitudinal studies are needed to determine if psychosine can be used as a predictor of progression in screen positive newborns who do not present with the infantile form of the disease. doi:10.1016/j.ymgme.2010.11.109
MGMT-based Selection of Lentivector Transduced Cells: Application to Fabry Disease Natalia Pacienzaa, Nobuo Mizuea, Matthew Scaifea, Jeffrey Medina,b, University Health Network, Toronto, Ontario, Canada, bDepartment of Medical Biophysics and the Institute of Medical Sciences, University of Toronto, Canada a
Fabry disease is caused by a deficiency of α-galactosidase A (α-gal A) activity. Although enzyme replacement therapy (ERT) is available, this treatment is costly and does not solve the basic genetic defect of the disorder. Genetically-modified hematopoietic stem cells (HSCs) are attractive targets for durable transgene expression in Fabry disease, however low transduction efficiencies of this population may be an obstacle to attainment of successful therapy. The goal of this study is to develop and test a novel lentivector (LV) able to confer drug tolerance to transduced Fabry patient cells by overexpression of the O6-methylguanine-DNA-methyltransferase gene (MGMT). To this end, we have generated a LV carrying a mutated, but functional, form of the selectable marker (MGMT-P140K) followed by an EMCV-IRES element and a codon-optimized copy of the human α-gal A cDNA. Expression and functionality of this cassette was tested in the human erythroleukemic cell line (K562) as well as in Fabry patient B cells and murine Fabry lin- bone marrow cells by intracellular MGMT staining and α-gal A activity assays, before and after selection with O6-