RESUMO
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.
Assuntos
Doença de Fabry/complicações , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Ereção Peniana , Pênis/enzimologia , Priapismo/etiologia , Animais , Modelos Animais de Doenças , Terapia de Reposição de Enzimas/métodos , Doença de Fabry/enzimologia , Doença de Fabry/fisiopatologia , Doença de Fabry/terapia , Terapia Genética/métodos , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Pênis/fisiopatologia , Priapismo/enzimologia , Priapismo/fisiopatologia , Priapismo/terapia , Fluxo Sanguíneo Regional , Transdução de Sinais , Regulação para Cima , alfa-Galactosidase/biossíntese , alfa-Galactosidase/genéticaRESUMO
Sandhoff disease, a GM2 gangliosidosis caused by a deficiency in ß-hexosaminidase, is characterized by progressive neurodegeneration. Although loss of neurons in association with lysosomal storage of glycosphingolipids occurs in patients with this disease, the molecular pathways that lead to the accompanying neurological defects are unclear. Using an authentic murine model of GM2 gangliosidosis, we examined the pattern of neuronal loss in the central nervous system and investigated the effects of gene transfer using recombinant adeno-associated viral vectors expressing ß-hexosaminidase subunits (rAAV2/1-Hex). In 4-month-old Sandhoff mice with neurological deficits, cells staining positively for the apoptotic signature in the TUNEL reaction were found in the ventroposterior medial and ventroposterior lateral (VPM/VPL) nuclei of the thalamus. There was progressive loss of neuronal density in this region with age. Comparable loss of neuronal density was identified in the lateral vestibular nucleus of the brainstem and a small but statistically significant loss was present in the ventral spinal cord. Loss of neurons was not detected in other regions that were analysed. Administration of rAAV2/1-Hex into the brain of Sandhoff mice prevented the decline in neuronal density in the VPM/VPL. Preservation of neurons in the VPM/VPL was variable at the humane endpoint in treated animals, but correlated directly with increased lifespan. Loss of neurons was localized to only a few regions in the Sandhoff brain and was prevented by rAAV-mediated transfer of ß-hexosaminidase gene function at considerable distances from the site of vector administration.
Assuntos
Encéfalo/metabolismo , Encéfalo/patologia , Dependovirus/genética , Vetores Genéticos/genética , Neurônios/patologia , Doença de Sandhoff/terapia , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Marcação In Situ das Extremidades Cortadas , Camundongos , Neurônios/metabolismo , Doença de Sandhoff/enzimologia , Doença de Sandhoff/metabolismo , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
One treatment approach for lysosomal storage diseases (LSDs) is the systemic infusion of recombinant enzyme. Although this enzyme replacement is therapeutic for the viscera, many LSDs have central nervous system (CNS) components that are not adequately treated by systemic enzyme infusion. Direct intracerebroventricular (ICV) infusion of a high concentration of recombinant human acid sphingomyelinase (rhASM) into the CNS over a prolonged time frame (hours) has shown therapeutic efficacy in a mouse model of Niemann-Pick A (NP/A) disease. To evaluate whether such an approach would translate to a larger brain, rhASM was infused into the lateral ventricles of both rats and Rhesus macaques, and the resulting distribution of enzyme characterized qualitatively and quantitatively. In both species, ICV infusion of rhASM resulted in parenchymal distribution of enzyme at levels that were therapeutic in the NP/A mouse model. Enzyme distribution was global in nature and exhibited a relatively steep gradient from the cerebrospinal fluid compartment to the inner parenchyma. Additional optimization of an ICV delivery approach may provide a therapeutic option for LSDs with neurologic involvement.
Assuntos
Encéfalo/metabolismo , Proteínas Recombinantes/farmacocinética , Esfingomielina Fosfodiesterase/farmacocinética , Animais , Encéfalo/enzimologia , Feminino , Infusões Intraventriculares , Macaca mulatta , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Esfingomielina Fosfodiesterase/administração & dosagemRESUMO
In mice, liver-restricted expression of lysosomal enzymes from adeno-associated viral serotype 8 (AAV8) vectors results in reduced antibodies to the expressed proteins. To ask whether this result might translate to patients, nonhuman primates (NHPs) were injected systemically with AAV8 encoding α-galactosidase A (α-gal). As in mice, sustained expression in monkeys attenuated antibody responses to α-gal. However, this effect was not robust, and sustained α-gal levels were 1-2 logs lower than those achieved in male mice at the same vector dose. Because our mouse studies had shown that antibody levels were directly related to expression levels, several strategies were evaluated to increase expression in monkeys. Unlike mice, expression in monkeys did not respond to androgens. Local delivery to the liver, immune suppression, a self-complementary vector and pharmacologic approaches similarly failed to increase expression. While equivalent vector copies reached mouse and primate liver and there were no apparent differences in vector form, methylation or deamination, transgene expression was limited at the mRNA level in monkeys. These results suggest that compared to mice, transcription from an AAV8 vector in monkeys can be significantly reduced. They also suggest some current limits on achieving clinically useful antibody reduction and therapeutic benefit for lysosomal storage diseases using a systemic AAV8-based approach.
Assuntos
Dependovirus/genética , Vetores Genéticos/administração & dosagem , Tolerância Imunológica , Imunidade Humoral , Fígado/metabolismo , alfa-Galactosidase/genética , Androgênios/farmacologia , Animais , Metilação de DNA , Desaminação , Dependovirus/imunologia , Dosagem de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/imunologia , Humanos , Injeções , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transcrição Gênica , alfa-Galactosidase/imunologia , alfa-Galactosidase/metabolismoRESUMO
Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.
Assuntos
Dependovirus/genética , Terapia Genética , Hepatócitos/imunologia , Doenças por Armazenamento dos Lisossomos/terapia , Transgenes/fisiologia , alfa-Galactosidase/sangue , Animais , Anticorpos Neutralizantes/imunologia , Western Blotting , Vetores Genéticos/administração & dosagem , Células HeLa , Hepatócitos/metabolismo , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/imunologia , Macaca fascicularis , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Plasmaferese , Biossíntese de Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , alfa-Galactosidase/genéticaRESUMO
Due to the lack of acid alpha-glucosidase (GAA) activity, Pompe mice develop glycogen storage pathology and progressive skeletal muscle dysfunction with age. Applying either gene or enzyme therapy to reconstitute GAA levels in older, symptomatic Pompe mice effectively reduces glycogen storage in skeletal muscle but provides only modest improvements in motor function. As strategies to stimulate muscle hypertrophy, such as by myostatin inhibition, have been shown to improve muscle pathology and strength in mouse models of muscular dystrophy, we sought to determine whether these benefits might be similarly realized in Pompe mice. Administration of a recombinant adeno-associated virus serotype 8 vector encoding follistatin, an inhibitor of myostatin, increased muscle mass and strength but only in Pompe mice that were treated before 10 months of age. Younger Pompe mice showed significant muscle fiber hypertrophy in response to treatment with follistatin, but maximal gains in muscle strength were achieved only when concomitant GAA administration reduced glycogen storage in the affected muscles. Despite increased grip strength, follistatin treatment failed to improve rotarod performance. These findings highlight the importance of treating Pompe skeletal muscle before pathology becomes irreversible, and suggest that adjunctive therapies may not be effective without first clearing skeletal muscle glycogen storage with GAA.
Assuntos
Folistatina/metabolismo , Doença de Depósito de Glicogênio Tipo II/terapia , Glicogênio/metabolismo , Músculo Esquelético/metabolismo , Animais , Índice de Massa Corporal , Dependovirus/genética , Modelos Animais de Doenças , Folistatina/genética , Vetores Genéticos/genética , Doença de Depósito de Glicogênio Tipo II/genética , Doença de Depósito de Glicogênio Tipo II/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismoRESUMO
Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid alpha-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.
Assuntos
Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Manosefosfatos/química , Oligossacarídeos/química , Engenharia de Proteínas/métodos , alfa-Glucosidases/metabolismo , alfa-Glucosidases/uso terapêutico , Animais , Modelos Animais de Doenças , Glicogênio/metabolismo , Doença de Depósito de Glicogênio Tipo II/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Ligação Proteica , Receptor IGF Tipo 2/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/genética , alfa-Glucosidases/farmacologiaRESUMO
Systemic administration of recombinant acid sphingomyelinase (rhASM) into ASM deficient mice (ASMKO) results in hydrolysis of the abnormal storage of sphingomyelin in lysosomes of the liver, spleen and lung. However, the efficiency with which the substrate is cleared from the lung, particularly the alveolar macrophages, appears to be lower than from the other visceral tissues. To determine if delivery of rhASM into the air spaces of the lung could enhance clearance of pulmonary sphingomyelin, enzyme was administered to ASMKO mice by intranasal instillation. Treatment resulted in a significant and dose-dependent reduction in sphingomyelin levels in the lung. Concomitant with this reduction in substrate levels was a decrease in the amounts of the pro-inflammatory cytokine, MIP-1alpha, in the bronchoalveolar lavage fluids and an improvement in lung pathology. Maximal reduction of lung sphingomyelin levels was observed at 7 days post-treatment. However, reaccumulation of the substrate was noted starting at day 14 suggesting that repeated treatments will be necessary to effect a sustained reduction in sphingomyelin levels. In addition to reducing the storage abnormality in the lung, intranasal delivery of rhASM also resulted in clearance of the substrate from the liver and spleen. Hence, pulmonary administration of rhASM may represent an alternative route of delivery to address the visceral pathology associated with ASM deficiency.
Assuntos
Pulmão/metabolismo , Lisossomos/metabolismo , Doenças de Niemann-Pick/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Esfingomielina Fosfodiesterase/administração & dosagem , Esfingomielina Fosfodiesterase/uso terapêutico , Esfingomielinas/metabolismo , Administração Intranasal , Animais , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Feminino , Humanos , Cinética , Fígado/metabolismo , Fígado/patologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças de Niemann-Pick/enzimologia , Doenças de Niemann-Pick/metabolismo , Doenças de Niemann-Pick/patologia , Proteínas Recombinantes/administração & dosagem , Esfingomielina Fosfodiesterase/genética , Baço/metabolismo , Baço/patologiaRESUMO
The availability of a murine model of Pompe disease has enabled an evaluation of the relative merits of various therapeutic paradigms, including gene therapy. We report here that administration of a recombinant adeno-associated virus serotype 8 (AAV8) vector (AAV8/DC190-GAA) encoding human acid alpha-glucosidase (GAA) into presymptomatic Pompe mice resulted in nearly complete correction of the lysosomal storage of glycogen in all the affected muscles. A relatively high dose of AAV8/DC190-GAA was necessary to attain a threshold level of GAA for inducing immunotolerance to the expressed enzyme and for correction of muscle function, coordination, and strength. Administration of AAV8/DC190-GAA into older Pompe mice with overt disease manifestations was also effective at correcting the lysosomal storage abnormality. However, these older mice exhibited only marginal improvements in motor function and no improvement in muscle strength. Examination of histologic sections showed evidence of skeletal muscle degeneration and fibrosis in aged Pompe mice whose symptoms were abated or rescued by early but not late treatment with AAV8/DC190-GAA. These results suggest that AAV8-mediated hepatic expression of GAA was effective at addressing the biochemical and functional deficits in Pompe mice. However, early therapeutic intervention is required to maintain significant muscle function and should be an important consideration in the management and treatment of Pompe disease.
Assuntos
Dependovirus , Vetores Genéticos , Doença de Depósito de Glicogênio Tipo II/fisiopatologia , Doença de Depósito de Glicogênio Tipo II/terapia , Fígado/enzimologia , alfa-Glucosidases/genética , Animais , Modelos Animais de Doenças , Doença de Depósito de Glicogênio Tipo II/complicações , Humanos , Glicogênio Hepático/genética , Glicogênio Hepático/metabolismo , Camundongos , Camundongos Mutantes , Atividade Motora , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Doenças Musculares/etiologia , Doenças Musculares/fisiopatologia , Doenças Musculares/terapia , alfa-Glucosidases/sangueRESUMO
Niemann-Pick disease (NPD) is caused by the loss of acid sphingomyelinase (ASM) activity, which results in widespread accumulation of undegraded lipids in cells of the viscera and CNS. In this study, we tested the effect of combination brain and systemic injections of recombinant adeno-associated viral vectors encoding human ASM (hASM) in a mouse model of NPD. Animals treated by combination therapy exhibited high levels of hASM in the viscera and brain, which resulted in near-complete correction of storage throughout the body. This global reversal of pathology translated to normal weight gain and superior recovery of motor and cognitive functions compared to animals treated by either brain or systemic injection alone. Furthermore, animals in the combination group did not generate antibodies to hASM, demonstrating the first application of systemic-mediated tolerization to improve the efficacy of brain injections. All of the animals treated by combination therapy survived in good health to an investigator-selected 54 weeks, whereas the median lifespans of the systemic-alone, brain-alone, or untreated ASM knockout groups were 47, 48, and 34 weeks, respectively. These data demonstrate that combination therapy is a promising therapeutic modality for treating NPD and suggest a potential strategy for treating disease indications that cause both visceral and CNS pathologies.
Assuntos
Encéfalo/enzimologia , Encéfalo/patologia , Dependovirus/genética , Doenças de Niemann-Pick/genética , Doenças de Niemann-Pick/terapia , Animais , Regulação Enzimológica da Expressão Gênica , Terapia Genética , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Knockout , Doenças de Niemann-Pick/enzimologia , Doenças de Niemann-Pick/patologia , Esfingomielina Fosfodiesterase/deficiência , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Taxa de SobrevidaRESUMO
The advent of novel adeno-associated virus (AAV) serotype vectors with higher transduction activity has encouraged a re-evaluation of the merits of this delivery platform for a variety of diseases. We report here that administration of a recombinant AAV8-based serotype vector encoding human α-galactosidase A into Fabry mice facilitated more rapid and significantly higher levels of production of the enzyme than an AAV2 vector. This translated into improved clearance of globotriaosylceramide, the glycosphingolipid that accumulates in the lysosomes of affected Fabry cells, and to correction of the peripheral neuropathy shown associated with this disease. The higher levels of α-galactosidase A expression also allowed for a more rapid induction of immunotolerance to the enzyme. Recombinant AAV8 vectors that facilitated hepatic-restricted expression of high levels of α-galactosidase A conferred immunotolerance to the expressed enzyme as early as 30 days post-treatment. Animals expressing lower levels of the hydrolase, such as those treated with an AAV2-based vector or with lower doses of the AAV8-based vector, were also able to develop immunotolerance, but only after a more extended time period. Adoptive transfer of T cells isolated from the spleens of immunotolerized mice suppressed the formation of antibodies in naïve recipient animals, suggesting the possible role of regulatory T cells in effecting this state.
RESUMO
The advent of novel adeno-associated virus (AAV) serotype vectors with higher transduction activity has encouraged a re-evaluation of the merits of this delivery platform for a variety of diseases. We report here that administration of a recombinant AAV8-based serotype vector encoding human alpha-galactosidase A into Fabry mice facilitated more rapid and significantly higher levels of production of the enzyme than an AAV2 vector. This translated into improved clearance of globotriaosylceramide, the glycosphingolipid that accumulates in the lysosomes of affected Fabry cells, and to correction of the peripheral neuropathy shown associated with this disease. The higher levels of alpha-galactosidase A expression also allowed for a more rapid induction of immunotolerance to the enzyme. Recombinant AAV8 vectors that facilitated hepatic-restricted expression of high levels of alpha-galactosidase A conferred immunotolerance to the expressed enzyme as early as 30 days post-treatment. Animals expressing lower levels of the hydrolase, such as those treated with an AAV2-based vector or with lower doses of the AAV8-based vector, were also able to develop immunotolerance, but only after a more extended time period. Adoptive transfer of T cells isolated from the spleens of immunotolerized mice suppressed the formation of antibodies in naïve recipient animals, suggesting the possible role of regulatory T cells in effecting this state.
Assuntos
Dependovirus/genética , Doença de Fabry/enzimologia , Expressão Gênica/genética , Fígado/metabolismo , alfa-Galactosidase/metabolismo , Animais , Anticorpos/imunologia , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Doença de Fabry/genética , Doença de Fabry/patologia , Doença de Fabry/terapia , Regulação Enzimológica da Expressão Gênica , Terapia Genética , Vetores Genéticos/genética , Humanos , Tolerância Imunológica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Doenças do Sistema Nervoso Periférico/enzimologia , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/terapia , alfa-Galactosidase/genética , alfa-Galactosidase/imunologiaRESUMO
Acid sphingomyelinase deficiency is a lysosomal storage disorder in which the defective lysosomal hydrolase fails to degrade sphingomyelin. The resulting accumulation of substrate in the lysosomes of histiocytic cells leads to hepatosplenomegaly and severe pulmonary inflammation. Administration of a recombinant AAV1 vector encoding human acid sphingomyelinase to acid sphingomyelinase knockout (ASMKO) mice effectively reduced the accumulated substrate in all of the affected visceral organs. However, more complete and rapid clearance of sphingomyelin was observed when an AAV8-based serotype vector was used in lieu of AAV1. Importantly, AAV8-mediated hepatic expression of higher and sustained levels of the enzyme also corrected the abnormal cellularity, cell differentials, and levels of the chemokine MIP-1alpha in the bronchoalveolar lavage fluids of the ASMKO mice. Treatment also reversed the morphological aberrations associated with the alveolar macrophages of ASMKO mice and restored their phagocytic activity. No antibodies to the expressed enzyme were detected when the viral vectors were used in conjunction with a transcription cassette harboring a liver-restricted enhancer/promoter. Together, these data support the continued development of AAV8-mediated hepatic gene transfer as an approach to treat the visceral manifestations observed in individuals with acid sphingomyelinase deficiency.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Fígado/enzimologia , Doenças de Niemann-Pick/terapia , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Elementos Facilitadores Genéticos , Técnicas de Transferência de Genes , Terapia Genética/instrumentação , Humanos , Cinética , Fígado/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Regiões Promotoras Genéticas , Esfingomielinas/metabolismo , Fatores de TempoRESUMO
The successful application of gene therapy for the treatment of genetic diseases such as Fabry is reliant on the development of vectors that are safe and that facilitate sustained expression of therapeutic levels of the transgene product. Here, we report that intravenous administration of a recombinant AAV2 vector encoding human alpha-galactosidase A under the transcriptional control of a liver-restricted enhancer/promoter (AAV2/DC190-alphagal) generated significantly higher levels of expression in BALB/c and Fabry mice than could be realized using the ubiquitous CMV promoter (AAV2/CMVHI-alphagal). Moreover, AAV2/DC190-alphagal-mediated hepatic expression of alpha-galactosidase A was sustained for 12 months in BALB/c mice and was associated with a significantly reduced immune response to the expressed enzyme. Subsequent challenge of the AAV2/DC190-alphagal-treated animals with recombinant human alpha-galactosidase A at 6 months failed to elicit the production of anti-alpha-galactosidase A antibodies, suggesting the induction of immune tolerance in these animals. The levels of expression attained with AAV2/DC190-alphagal in the Fabry mice were sufficient to reduce the abnormal accumulation of globotriaosylceramide in the liver, spleen, and heart to basal levels and in the kidney by approximately 40% at 8 weeks. Together, these results demonstrate that AAV2-mediated gene transfer that limits the expression of alpha-galactosidase A to the liver may be a viable strategy for treating Fabry disease.
Assuntos
Dependovirus/genética , Doença de Fabry/terapia , Terapia Genética , Tolerância Imunológica , Fígado/metabolismo , Regiões Promotoras Genéticas/genética , alfa-Galactosidase/uso terapêutico , Animais , DNA Recombinante/genética , Modelos Animais de Doenças , Elementos Facilitadores Genéticos/genética , Doença de Fabry/genética , Engenharia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismoRESUMO
BACKGROUND: Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A, leading to an accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in most tissues of the body. The goal of this study was to determine if systemic delivery of a nonviral vector could correct the enzyme deficiency and reduce the levels of GL-3 in different tissues of a transgenic knockout mouse model of the disease. METHODS: Cationic lipid was complexed with a CpG-depleted plasmid DNA vector and then injected intravenously into Fabry mice. The levels of alpha-galactosidase A and GL-3 in different tissues were assayed at various time points after injection. RESULTS: Expression of alpha-galactosidase A was detected in the different tissues of Fabry mice for up to 3 months after complex administration, but resulted in minimal reductions in GL-3 levels. However, the use of the anti-inflammatory drug dexamethasone and multiple dosing increased alpha-galactosidase A expression and resulted in significant reductions of GL-3 in all the organs with the exception of the kidney. In addition, injecting complex into young Fabry mice partially prevented the normal accumulation of GL-3 in the heart, lung, and liver. CONCLUSIONS: Systemic delivery of a cationic lipid-pDNA complex partially corrected the enzyme deficiency and reduced glycolipid storage in a mouse model of Fabry disease. The results are one of the few demonstrations of long-term efficacy in a genetic disease model using nonviral vectors. However, substantial improvements in expression, especially in critical organs such as the kidney, are required before these vectors can become a viable approach to treat Fabry disease and other lysosomal storage disorders.
Assuntos
Doença de Fabry/genética , Doença de Fabry/terapia , Terapia Genética , Vetores Genéticos , Triexosilceramidas/metabolismo , alfa-Galactosidase/genética , Animais , Modelos Animais de Doenças , Feminino , Expressão Gênica , Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , alfa-Galactosidase/biossínteseRESUMO
Progress towards developing gene therapy for Gaucher disease has been hindered by the lack of an animal model. Here we describe a mouse model of Gaucher disease which has a chemically induced deficiency of glucocerebrosidase and that accumulates elevated levels of glucosylceramide (GL-1) in the lysosomes of Kupffer cells. Administration of mannose-terminated glucocerebrosidase (Cerezyme) resulted in the reduction of GL-1 levels in the livers of these animals. Gene transduction of hepatocytes with a plasmid DNA vector encoding human glucocerebrosidase (pGZB-GC) generated high-level expression and secretion of the enzyme into systemic circulation with consequent normalization of Kupffer cell GL-1 levels. This suggested that the de novo synthesized and unmodified enzyme produced by hepatocyte transduction was also capable of being delivered to the cells that are primarily affected in Gaucher disease. Immunolocalization studies also revealed that preferential transduction and expression of human glucocerebrosidase in the Kupffer cells with subsequent reduction in the GL-1 levels could be attained with a low dose of a recombinant adenoviral vector encoding the human enzyme (Ad2/CMV-GC). This observation raises the possibility of gene therapy for Gaucher disease that involves directly transducing the affected histiocytes using recombinant adenoviral vectors. Together, these data demonstrate the potential for use of in vivo gene therapy vectors for treating Gaucher disease.
Assuntos
Doença de Gaucher/terapia , Terapia Genética/métodos , Adenoviridae/genética , Animais , Modelos Animais de Doenças , Feminino , Doença de Gaucher/induzido quimicamente , Doença de Gaucher/enzimologia , Doença de Gaucher/genética , Expressão Gênica , Vetores Genéticos , Glucosilceramidase/deficiência , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Glucosilceramidase/uso terapêutico , Glucosilceramidas/metabolismo , Humanos , Células de Kupffer/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Endogâmicos F344RESUMO
Systemic administration of recombinant adenoviral vectors for gene therapy of chronic diseases such as Fabry disease can be limited by dose-dependent toxicity. Because administration of a high dose of Ad2/CMVHI-alpha gal encoding human alpha-galactosidase A results in expression of supraphysiological levels of the enzyme, we sought to determine whether lower doses would suffice to correct the enzyme deficiency and lysosomal storage abnormality observed in Fabry mice. Reducing the dose of Ad2/CMVHI-alpha gal by 10-fold (from 10(11) to 10(10) particles/mouse) resulted in a greater than 200-fold loss in transgene expression. In Fabry mice, the reduced expression of alpha-galactosidase A, using the lower dose of Ad2/CMVHI-alpha gal, was associated with less than optimal clearance of the accumulated glycosphingolipid (GL-3) from the affected lysosomes. It was determined that this lack of linearity in dose response was not due to an inability to deliver the recombinant viral vectors to the liver but rather to sequestration, at least in part, of the viral vectors by the Kupffer cells. This lack of correlation between dose and expression levels could be obviated by supplementing the low dose of Ad2/CMVHI-alpha gal with an unrelated adenoviral vector or by depleting the Kupffer cells before administration of Ad2/CMVHI-alpha gal. Prior removal of the Kupffer cells, using clodronate liposomes, facilitated the use of a 100-fold lower dose of Ad2/CMVHI-alpha gal (10(9) particles/mouse) to effect the nearly complete clearance of GL-3 from the affected organs of Fabry mice. These results suggest that practical strategies that minimize the interaction between the recombinant adenoviral vectors and the reticuloendothelial system (RES) may improve the therapeutic window of this vector system. In this regard, we showed that pretreatment of mice with gamma globulins also resulted in significantly enhanced adenovirus-mediated transduction and expression of alpha-galactosidase A in the liver.
Assuntos
Adenoviridae/genética , Doença de Fabry/terapia , Terapia Genética , Vetores Genéticos , Animais , Ácido Clodrônico/farmacologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Células de Kupffer/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Transdução Genética , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , gama-Globulinas/farmacologiaRESUMO
Gene therapy efforts have focused primarily on the use of either the liver or skeletal muscle as depot organs for the production of a variety of therapeutic proteins that act systemically. Here we examined the lung to determine whether it could function as yet another portal for the secretion of proteins into the circulation. Fabry disease is caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A, resulting in the abnormal deposition of the glycosphingolipid globotriaosylceramide (GL-3) in vascular lysosomes. Pulmonary instillation of a recombinant adenoviral vector (Ad2/CMVHI-alpha(gal)) encoding human alpha-galactosidase A into Fabry mice resulted in high-level transduction and expression of the enzyme in the lung. Importantly, enzymatic activity was also detected in the plasma, liver, spleen, heart, and kidneys of the Fabry mice. The detection of enzymatic activity outside of the lung, along with the finding that viral DNA was limited to the lung, indicates that the enzyme crossed the air/blood barrier, entered the systemic circulation, and was internalized by the distal visceral organs. The levels of alpha-galactosidase A attained in these tissues were sufficient to reduce GL-3 to basal levels in the lung, liver, and spleen and to approximately 50% of untreated levels in the heart. Together, these results suggest that the lung may be a viable alternate depot organ for the production and systemic secretion of alpha-galactosidase A for Fabry disease.