Lentivirus-mediated gene therapy has curative potential for a variety of disorders, however, insertional oncogenesis still remains a concern. One approach to increase safety of such treatment modalities is to include a ‘cell fate control safety cassette’ in lentiviral vectors (LVs), enabling pharmacological control over the survival of gene-modified cells (GMCs). Two novel LVs with engineered expression of truncated cell surface molecules (CD19 or LNGFR) fused to a ‘cell fate control’ gene (TmpkF105YR200A) were constructed. Results demonstrated these safety cassettes could be used to control the survival of GMCs in a murine xenogeneic leukemia models. For treatment of Fabry disease, a bicistronic LV containing the fusion safety element and therapeutic α-galactosidase A was constructed. Transduction with this vector restored enzyme activity in Fabry patient’s fibroblasts. These collective results demonstrate that this approach is sufficient to eradicate GMCs, and when combined with a corrective cDNA can provide therapeutic benefit for Fabry disease.

Lentivirus-mediated gene therapy has curative potential for a variety of disorders, however, insertional oncogenesis still remains a concern. One approach to increase safety of such treatment modalities is to include a ‘cell fate control safety cassette’ in lentiviral vectors (LVs), enabling pharmacological control over the survival of gene-modified cells (GMCs). Two novel LVs with engineered expression of truncated cell surface molecules (CD19 or LNGFR) fused to a ‘cell fate control’ gene (TmpkF105YR200A) were constructed. Results demonstrated these safety cassettes could be used to control the survival of GMCs in a murine xenogeneic leukemia models. For treatment of Fabry disease, a bicistronic LV containing the fusion safety element and therapeutic α-galactosidase A was constructed. Transduction with this vector restored enzyme activity in Fabry patient’s fibroblasts. These collective results demonstrate that this approach is sufficient to eradicate GMCs, and when combined with a corrective cDNA can provide therapeutic benefit for Fabry disease.

No description available.

No description available.

Human α-galactosidase (α-GAL; EC 3.2.1.22) is a lysosomal enzyme that hydrolyzes of terminal alpha-linked galactosyl residue of glycosphingolipids. Deficiencies in α-GAL leads to Fabry disease, which is characterized by the build-up of globotriaosylceramide and other neutral substrates in cells, ultimately leading to a multi-systemic organ failure in patients. Hundreds of distinct mutations have been found in the α-GAL gene of Fabry disease patients. One current treatment for Fabry disease is Enzyme Replacement Therapy (ERT), which restores the missing α-GAL function. An alternative treatment, called Pharmacological Chaperone Therapy (PCT), utilizes a small molecule substrate analogue, 1-deoxygalactonojirimycin (DGJ). In order to better understand molecular basis of Fabry disease, this work addresses structural and mechanistic studies of the α-GAL glycoprotein. First, we have determined crystal structures of each stage in the catalytic mechanism of the α-GAL enzymatic reaction. These studies reveal a novel strained conformation of the sugar when it is covalently bound to the enzyme. Second, we examine the molecular mechanism of chaperoning by pharmacological chaperones. A combination of biochemical and biophysical approaches reveals that the high potency of the DGJ chaperone is due to an interaction with α-GAL residue D170. Third, we have investigated mutant α-GAL proteins for their response to pharmacological chaperones, leading to a set of structure-based rules for predicting the effect of pharmacological chaperone on every Fabry disease patient. Fourth, we use rational design approaches to interconvert the specificity of α-GAL into that of a related enzyme, α-N-acetylgalactosaminidase (α-NAGAL). Structural and enzymatic experiments show that the engineered enzyme contains new substrate specificity, as predicted by the design. The structural and mechanistic details we present in this thesis provide better understanding of the catalysis of the human α-galactosidase enzyme as well as define the molecular basis for pharmacological chaperone therapy in Fabry patients. Since α-GAL is one of the best studied lysosomal storage disease, it might be used as a model to better understand other lysosomal storage diseases and as well as other diseases related to misfolded proteins, including Alzheimer's and Parkinson's diseases.

No description available.

INTRODUÇÃO: A Doença de Fabry (DF) é uma doença lisossomal de herança ligada ao X, causada pela deficiência da enzima alfa-galactosidase A (alfa-Gal A), que leva ao acúmulo gradual de glicoesfingolipídeos, em especial a globotriaosilceramida, nos lisossomos do endotélio vascular de tecidos cardíaco, renal, cerebral, olhos e pele. Os principais sintomas iniciais são: dores neuropáticas de extremidades, hipoidrose, dores abdominais recorrentes, angioqueratomas e córnea verticillata. As complicações, que aparecem a partir da terceira década de vida, incluem morte prematura por insuficiência renal, cardíaca e alterações cerebrovasculares. Angioqueratomas são uma das manifestações mais precoces da DF. OBJETIVOS: detectar os portadores da DF a partir de casos de angioqueratoma diagnosticados através de exames anatomopatológicos de biópsia cutânea; descrever o quadro clínico dos afetados e portadoras heterozigotas; realizar aconselhamento genético. MÉTODOS: Uma revisão sistemática de biópsias de pele de 2003 a 2012 foi feita em quatro hospitais universitários. Os pacientes foram convocados para anamnese, exame físico e coleta de história familial. A dosagem enzimática de alfa-Gal A por papel filtro e em leucócitos em homens e a análise molecular por PCR e sequenciamento do gene GLA em homens e mulheres foram realizados naqueles com suspeita de DF. RESULTADOS: Foram localizados 125 registros de angioqueratomas, sendo possível convocar 52 pacientes. Destes, 45 (M:21 e F:24) compareceram para serem examinados. O diagnóstico de DF foi confirmado 3/45 (6,6%), todos do sexo masculino, pela baixa atividade enzimática da alfa-Gal A. A idade dos pacientes eram 16, 21 e 32 anos. Foram identificados outros 13 familiares (cinco homens e oito mulheres) destas 3 famílias. Os principais achados clínicos nos oito homens afetados foram: dores de extremidades (62,5%), angioqueratomas (87,5%), alterações renais (87,5%), cardíacas (12,5%) e presença de córnea verticillata(37,5%) Nas oito mulheres os achados clínicos foram: dores de extremidades (75%), angioqueratomas (12,5%), alterações renais (37,5%) cardíacas (12,5%) e presença de córnea verticillata(0%). A frequência desses achados mostrou-se semelhante à descrita na literatura. As mutações patogênicas no gene GLA, herdadas da mãe, foram identificadas nas 3 famílias e já haviam sido descritas anteriormente. CONCLUSÕES: A DF foi identificada em 3/45 pacientes (6,6%) pelos registros histopatológicos. Portanto, o angioqueratoma é um marcador útil para a detecção da doença. A dosagem enzimática de alfa-Gal A e/ou estudo molecular foram fundamentais para a confirmação diagnóstica da DF. Foi possível identificar outros 13 familiares afetados (5 homens, 8 mulheres), o que reforça a importância do aconselhamento genético. Todos os pacientes apresentavam outros achados clínicos da DF além dos angioqueratomas, os quais, no entanto, não haviam sido diagnosticados. O diagnóstico da DF ainda é tardio e há necessidade de reconhecimento dos médicos sobre as manifestações dessa doença para possibilitar um diagnóstico precoce / INTRODUTION: Fabry disease (FD) is an X-linked lysosomal disorder, caused by the deficiency of the enzyme alfa-galactosidase A (alfa-Gal A), which leads to gradual accumulation of glycosphingolipids, especially globotriaosylceramide, in lysosomes of the vascular endothelium of the cardiac tissue, kidney, brain, eyes and skin. The main initial symptoms are: neuropathic pain in the extremities, hypohidrosis, recurrent abdominal pain, angiokeratomas and cornea verticillata. Complications, which appear from the third decade of life on, include premature death from renal, cardiac and cerebrovascular abnormalities. Angiokeratomas are one of the earliest manifestations of FD. OBJECTIVES: to identify relatives with FD from cases of angiokeratoma diagnosed by pathological examinations of skin biopsy; to describe the clinical features of the affected individuals and heterozygotes; to perform genetic counseling. METHODS: A systematic review of skin biopsies from 2003 to 2012 was done in four university hospitals. Patients were submitted to anamnesis, physical examination and family history collection. The enzymatic assay of alfa-Gal A using dried blood spot in filter paper and leukocytes in men and molecular analysis by PCR and sequencing of the GLA gene in men and women were performed in patients with suspected FD. RESULTS: We found 125 biopsy records of angiokeratomas, and were able to contact 52 patients. Of these, 45 (M: 21 and F: 24) were evaluated. The diagnosis of FD was confirmed in 3/45 (6.6%), all male, with low enzymatic activity of alfa-Gal A. The patients\' ages were 16, 21 and 32 years. We identified 13 other family members (five men and eight women) of these three families. The main clinical findings in the eight affected men were pain in the extremities (62,5%), angiokeratomas (87,5%), renal abnormalities (87,5%), cardiac abnormalities (12,5%) and cornea verticillata (37,5%). In the eight female patients the clinical findings were: pain in the extremities (75%), angiokeratomas (12,5%), renal abnormalities (37,5%) cardiac abnormalities (12,5%) and cornea verticillata (0%). The frequency of these findings was similar to that described in the literature. The pathogenic mutations in the GLA gene, maternally inherited, were identified in three families and had been described previously. CONCLUSIONS: FD was identified in 3/45 patients (6.6%) by histopathologic records. Therefore, angiokeratoma is a useful marker for the detection of disease. The enzymatic measurement of alfa-Gal A and / or molecular studies were essential to confirm the diagnosis of FD. It was possible to identify 13 other affected family members (5 men, 8 women), which reinforces the importance of genetic counseling. All patients, in addition to angiokeratomas, had other clinical manifestations of FD; however these had not been diagnosed before. The diagnosis of FD is still late and doctors need to be aware of the manifestations of this disease to enable early diagnosis

INTRODUÇÃO: A Doença de Fabry (DF) é uma doença lisossomal de herança ligada ao X, causada pela deficiência da enzima alfa-galactosidase A (alfa-Gal A), que leva ao acúmulo gradual de glicoesfingolipídeos, em especial a globotriaosilceramida, nos lisossomos do endotélio vascular de tecidos cardíaco, renal, cerebral, olhos e pele. Os principais sintomas iniciais são: dores neuropáticas de extremidades, hipoidrose, dores abdominais recorrentes, angioqueratomas e córnea verticillata. As complicações, que aparecem a partir da terceira década de vida, incluem morte prematura por insuficiência renal, cardíaca e alterações cerebrovasculares. Angioqueratomas são uma das manifestações mais precoces da DF. OBJETIVOS: detectar os portadores da DF a partir de casos de angioqueratoma diagnosticados através de exames anatomopatológicos de biópsia cutânea; descrever o quadro clínico dos afetados e portadoras heterozigotas; realizar aconselhamento genético. MÉTODOS: Uma revisão sistemática de biópsias de pele de 2003 a 2012 foi feita em quatro hospitais universitários. Os pacientes foram convocados para anamnese, exame físico e coleta de história familial. A dosagem enzimática de alfa-Gal A por papel filtro e em leucócitos em homens e a análise molecular por PCR e sequenciamento do gene GLA em homens e mulheres foram realizados naqueles com suspeita de DF. RESULTADOS: Foram localizados 125 registros de angioqueratomas, sendo possível convocar 52 pacientes. Destes, 45 (M:21 e F:24) compareceram para serem examinados. O diagnóstico de DF foi confirmado 3/45 (6,6%), todos do sexo masculino, pela baixa atividade enzimática da alfa-Gal A. A idade dos pacientes eram 16, 21 e 32 anos. Foram identificados outros 13 familiares (cinco homens e oito mulheres) destas 3 famílias. Os principais achados clínicos nos oito homens afetados foram: dores de extremidades (62,5%), angioqueratomas (87,5%), alterações renais (87,5%), cardíacas (12,5%) e presença de córnea verticillata(37,5%) Nas oito mulheres os achados clínicos foram: dores de extremidades (75%), angioqueratomas (12,5%), alterações renais (37,5%) cardíacas (12,5%) e presença de córnea verticillata(0%). A frequência desses achados mostrou-se semelhante à descrita na literatura. As mutações patogênicas no gene GLA, herdadas da mãe, foram identificadas nas 3 famílias e já haviam sido descritas anteriormente. CONCLUSÕES: A DF foi identificada em 3/45 pacientes (6,6%) pelos registros histopatológicos. Portanto, o angioqueratoma é um marcador útil para a detecção da doença. A dosagem enzimática de alfa-Gal A e/ou estudo molecular foram fundamentais para a confirmação diagnóstica da DF. Foi possível identificar outros 13 familiares afetados (5 homens, 8 mulheres), o que reforça a importância do aconselhamento genético. Todos os pacientes apresentavam outros achados clínicos da DF além dos angioqueratomas, os quais, no entanto, não haviam sido diagnosticados. O diagnóstico da DF ainda é tardio e há necessidade de reconhecimento dos médicos sobre as manifestações dessa doença para possibilitar um diagnóstico precoce / INTRODUTION: Fabry disease (FD) is an X-linked lysosomal disorder, caused by the deficiency of the enzyme alfa-galactosidase A (alfa-Gal A), which leads to gradual accumulation of glycosphingolipids, especially globotriaosylceramide, in lysosomes of the vascular endothelium of the cardiac tissue, kidney, brain, eyes and skin. The main initial symptoms are: neuropathic pain in the extremities, hypohidrosis, recurrent abdominal pain, angiokeratomas and cornea verticillata. Complications, which appear from the third decade of life on, include premature death from renal, cardiac and cerebrovascular abnormalities. Angiokeratomas are one of the earliest manifestations of FD. OBJECTIVES: to identify relatives with FD from cases of angiokeratoma diagnosed by pathological examinations of skin biopsy; to describe the clinical features of the affected individuals and heterozygotes; to perform genetic counseling. METHODS: A systematic review of skin biopsies from 2003 to 2012 was done in four university hospitals. Patients were submitted to anamnesis, physical examination and family history collection. The enzymatic assay of alfa-Gal A using dried blood spot in filter paper and leukocytes in men and molecular analysis by PCR and sequencing of the GLA gene in men and women were performed in patients with suspected FD. RESULTS: We found 125 biopsy records of angiokeratomas, and were able to contact 52 patients. Of these, 45 (M: 21 and F: 24) were evaluated. The diagnosis of FD was confirmed in 3/45 (6.6%), all male, with low enzymatic activity of alfa-Gal A. The patients\' ages were 16, 21 and 32 years. We identified 13 other family members (five men and eight women) of these three families. The main clinical findings in the eight affected men were pain in the extremities (62,5%), angiokeratomas (87,5%), renal abnormalities (87,5%), cardiac abnormalities (12,5%) and cornea verticillata (37,5%). In the eight female patients the clinical findings were: pain in the extremities (75%), angiokeratomas (12,5%), renal abnormalities (37,5%) cardiac abnormalities (12,5%) and cornea verticillata (0%). The frequency of these findings was similar to that described in the literature. The pathogenic mutations in the GLA gene, maternally inherited, were identified in three families and had been described previously. CONCLUSIONS: FD was identified in 3/45 patients (6.6%) by histopathologic records. Therefore, angiokeratoma is a useful marker for the detection of disease. The enzymatic measurement of alfa-Gal A and / or molecular studies were essential to confirm the diagnosis of FD. It was possible to identify 13 other affected family members (5 men, 8 women), which reinforces the importance of genetic counseling. All patients, in addition to angiokeratomas, had other clinical manifestations of FD; however these had not been diagnosed before. The diagnosis of FD is still late and doctors need to be aware of the manifestations of this disease to enable early diagnosis

ABC drug transporter, MDR1, is a drug flippase that moves a variety of hydrophobic molecules from the inner to the outer leaflet of the plasma membrane. We have previously reported that MDR1 can function as a glycolipid flippase, being one of the mechanisms responsible for the translocation of glucosylceramide into the Golgi for neutral, but not acidic, glycosphingolipids (GSLs) synthesis. The interplay between GSLs and MDR1 could provide a whole new spectrum of innovative therapeutic options. We found that cell surface MDR1 partially co-localized with globotriaosyl ceramide (Gb3) in MDR1 transfected cells. Inhibition of GSL biosynthesis results in the loss of drug resistance and of cell surface MDR1. We speculated that an association of MDR1 and cell surface GSLs, in particular Gb3, may be functional at the cell surface, as MDR1 partitions into plasma membrane lipid rafts regulating MDR1 function. We therefore tested adamantyl Gb3 (adaGb3), a water soluble analog of Gb3, on MDR1 functions. AdaGb3 was able to inhibit MDR1-mediated rhodamine 123 drug efflux from MDR1 expressing cells, like cyclosporin A (CsA), a classical MDR1 inhibitor. AdaGb3 was also able to reverse vinblastine drug resistance in cell culture, whereas adamantyl galactosylceramide had no effect on drug resistance. The strong MDR1 reversal effects of adaGb3, as well as its favourable in vivo features make it a possible choice for inhibition of MDR1 to increase bioavailability of drugs across the intestinal epithelium (De Rosa et al., 2008). Thus, specific GSL analogs provide a new approach to MDR reversal. We have previously shown that MDR1 inhibitor CsA depletes Fabry cell lines of Gb3, the characteristic GSL accumulated in this disease, by preventing its de novo synthesis, and can also deplete Gaucher lymphoid cell lines of accumulated GlcCer (Mattocks et al., 2006). Liver and heart sections of Fabry mice treated with third generation MDR1 inhibitors showed significantly less Gb3 than liver and heart sections of untreated Fabry mice. Thus, MDR1 inhibition offers a potential alternative therapeutic approach not only for Fabry disease given the extraordinary cost of conventional enzyme replacement therapy, but also for other neutral GSL storage diseases, such as Gaucher disease.

ABC drug transporter, MDR1, is a drug flippase that moves a variety of hydrophobic molecules from the inner to the outer leaflet of the plasma membrane. We have previously reported that MDR1 can function as a glycolipid flippase, being one of the mechanisms responsible for the translocation of glucosylceramide into the Golgi for neutral, but not acidic, glycosphingolipids (GSLs) synthesis. The interplay between GSLs and MDR1 could provide a whole new spectrum of innovative therapeutic options. We found that cell surface MDR1 partially co-localized with globotriaosyl ceramide (Gb3) in MDR1 transfected cells. Inhibition of GSL biosynthesis results in the loss of drug resistance and of cell surface MDR1. We speculated that an association of MDR1 and cell surface GSLs, in particular Gb3, may be functional at the cell surface, as MDR1 partitions into plasma membrane lipid rafts regulating MDR1 function. We therefore tested adamantyl Gb3 (adaGb3), a water soluble analog of Gb3, on MDR1 functions. AdaGb3 was able to inhibit MDR1-mediated rhodamine 123 drug efflux from MDR1 expressing cells, like cyclosporin A (CsA), a classical MDR1 inhibitor. AdaGb3 was also able to reverse vinblastine drug resistance in cell culture, whereas adamantyl galactosylceramide had no effect on drug resistance. The strong MDR1 reversal effects of adaGb3, as well as its favourable in vivo features make it a possible choice for inhibition of MDR1 to increase bioavailability of drugs across the intestinal epithelium (De Rosa et al., 2008). Thus, specific GSL analogs provide a new approach to MDR reversal. We have previously shown that MDR1 inhibitor CsA depletes Fabry cell lines of Gb3, the characteristic GSL accumulated in this disease, by preventing its de novo synthesis, and can also deplete Gaucher lymphoid cell lines of accumulated GlcCer (Mattocks et al., 2006). Liver and heart sections of Fabry mice treated with third generation MDR1 inhibitors showed significantly less Gb3 than liver and heart sections of untreated Fabry mice. Thus, MDR1 inhibition offers a potential alternative therapeutic approach not only for Fabry disease given the extraordinary cost of conventional enzyme replacement therapy, but also for other neutral GSL storage diseases, such as Gaucher disease.