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1.
Mol Med ; 21: 389-99, 2015 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-25938659

RESUMEN

Fabry disease, an X-linked glycosphingolipid storage disorder, is caused by the deficient activity of α-galactosidase A (α-Gal A). This results in the lysosomal accumulation in various cell types of its glycolipid substrates, including globotriaosylceramide (GL-3) and lysoglobotriaosylceramide (globotriaosyl lysosphingolipid, lyso-GL-3), leading to kidney, heart, and cerebrovascular disease. To complement and potentially augment the current standard of care, biweekly infusions of recombinant α-Gal A, the merits of substrate reduction therapy (SRT) by selectively inhibiting glucosylceramide synthase (GCS) were examined. Here, we report the development of a novel, orally available GCS inhibitor (Genz-682452) with pharmacological and safety profiles that have potential for treating Fabry disease. Treating Fabry mice with Genz-682452 resulted in reduced tissue levels of GL-3 and lyso-GL-3 and a delayed loss of the thermal nociceptive response. Greatest improvements were realized when the therapeutic intervention was administered to younger mice before they developed overt pathology. Importantly, as the pharmacologic profiles of α-Gal A and Genz-682452 are different, treating animals with both drugs conferred the greatest efficacy. For example, because Genz-682452, but not α-Gal A, can traverse the blood-brain barrier, levels of accumulated glycosphingolipids were reduced in the brain of Genz-682452-treated but not α-Gal A-treated mice. These results suggest that combining substrate reduction and enzyme replacement may confer both complementary and additive therapeutic benefits in Fabry disease.


Asunto(s)
Carbamatos/administración & dosificación , Enfermedad de Fabry/tratamiento farmacológico , Glucosiltransferasas/metabolismo , Glucolípidos/metabolismo , Quinuclidinas/administración & dosificación , Esfingolípidos/metabolismo , Trihexosilceramidas/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Modelos Animales de Enfermedad , Enfermedad de Fabry/metabolismo , Enfermedad de Fabry/patología , Glucosiltransferasas/antagonistas & inhibidores , Humanos , Ratones , alfa-Galactosidasa/administración & dosificación , alfa-Galactosidasa/metabolismo
2.
J Gene Med ; 14(7): 491-500, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22711445

RESUMEN

BACKGROUND: The nuclear membrane of differentiated airway epithelial cells is a significant barrier for nonviral vectors. Trans-cyclohexane-1,2-diol (TCHD) is an amphipathic alcohol that has been shown to collapse nuclear pore cores and allow the uptake of macromolecules that would otherwise be too large for nuclear entry. Previous studies have shown that TCHD can increase lipid-mediated transfection in vitro. METHODS: We aimed to reproduce these in vitro studies using the cationic lipid GL67A, which we are currently assessing in cystic fibrosis trials and, more importantly, we assessed the effects of TCHD on transfection efficiency in differentiated airway epithelium ex vivo and in mouse lung in vivo using three different drug delivery protocols (nebulisation and bolus administration of TCHD to the mouse lung, as well as perfusion of TCHD to the nasal epithelium, which prolongs contact time between the airway epithelium and drug). RESULTS: TCHD (0.5-2%) dose-dependently increased Lipofectamine 2000 and GL67A-mediated transfection of 293T cells by up to 2 logs. Encouragingly, exposure to 8% TCHD (but not 0.5% or 2.0%) increased gene expression in fully differentiated human air liquid interface cultures by approximately 20-fold, although this was accompanied by significant cell damage. However, none of the TCHD treated mice in any of the three protocols had higher gene expression compared to no TCHD controls. CONCLUSIONS: Although TCHD significantly increases gene transfer in cell lines and differentiated airway epithelium ex vivo, this effect is lost in vivo and further highlights that promising in vitro findings often cannot be translated into in vivo applications.


Asunto(s)
Ciclohexanos/farmacología , Ciclohexanoles/farmacología , Técnicas de Transferencia de Gen , Poro Nuclear/efectos de los fármacos , Sistema Respiratorio/efectos de los fármacos , Animales , Células Cultivadas , Ciclohexanos/administración & dosificación , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Epitelio/efectos de los fármacos , Femenino , Terapia Genética , Vectores Genéticos , Humanos , Lípidos/farmacología , Pulmón/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/efectos de los fármacos , Transfección
3.
Mol Genet Metab ; 105(4): 621-8, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22366055

RESUMEN

Niemann Pick type C (NPC) disease is a progressive neurodegenerative disease caused by mutations in NPC1 or NPC2, the gene products of which are involved in cholesterol transport in late endosomes. NPC is characterized by an accumulation of cholesterol, sphingomyelin and glycosphingolipids in the visceral organs, primarily the liver and spleen. In the brain, there is a redistribution of unesterified cholesterol and a concomitant accumulation of glycosphingolipids. It has been suggested that reducing the aberrant lysosomal storage of glycosphingolipids in the brain by a substrate reduction therapy (SRT) approach may prove beneficial. Inhibiting glucosylceramide synthase (GCS) using the iminosugar-based inhibitor miglustat (NB-DNJ) has been reported to increase the survival of NPC mice. Here, we tested the effects of Genz-529468, a more potent iminosugar-based inhibitor of GCS, in the NPC mouse. Oral administration of Genz-529468 or NB-DNJ to NPC mice improved their motor function, reduced CNS inflammation, and increased their longevity. However, Genz-529468 offered a wider therapeutic window and better therapeutic index than NB-DNJ. Analysis of the glycolipids in the CNS of the iminosugar-treated NPC mouse revealed that the glucosylceramide (GL1) but not the ganglioside levels were highly elevated. This increase in GL1 was likely caused by the off-target inhibition of the murine non-lysosomal glucosylceramidase, Gba2. Hence, the basis for the observed effects of these inhibitors in NPC mice might be related to their inhibition of Gba2 or another unintended target rather than a result of substrate reduction.


Asunto(s)
Encéfalo/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Glucosiltransferasas/antagonistas & inhibidores , Iminoazúcares/uso terapéutico , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/mortalidad , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Glucosilceramidas/metabolismo , Glicoesfingolípidos/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Enfermedad de Niemann-Pick Tipo C/enzimología , Tasa de Supervivencia
4.
Mol Ther ; 19(11): 1999-2011, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21712814

RESUMEN

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.


Asunto(s)
Dependovirus/genética , Vectores Genéticos/administración & dosificación , Tolerancia Inmunológica , Inmunidad Humoral , Hígado/metabolismo , alfa-Galactosidasa/genética , Andrógenos/farmacología , Animales , Metilación de ADN , Desaminación , Dependovirus/inmunología , Dosificación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/inmunología , Humanos , Inyecciones , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Transcripción Genética , alfa-Galactosidasa/inmunología , alfa-Galactosidasa/metabolismo
5.
Mol Ther ; 18(12): 2146-54, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20664526

RESUMEN

The efficacy of recombinant enzyme therapy for genetic diseases is limited in some patients by the generation of a humoral immune response to the therapeutic protein. Inducing immune tolerance to the protein prior to treatment has the potential to increase therapeutic efficacy. Using an AAV8 vector encoding human acid α-glucosidase (hGAA), we have evaluated direct intrathymic injection for inducing tolerance. We have also compared the final tolerogenic states achieved by intrathymic and intravenous injection. Intrathymic vector delivery induced tolerance equivalent to that generated by intravenous delivery, but at a 25-fold lower dose, the thymic hGAA expression level was 10,000-fold lower than the liver expression necessary for systemic tolerance induction. Splenic regulatory T cells (Tregs) were apparent after delivery by both routes, but with different phenotypes. Intrathymic delivery resulted in Tregs with higher FoxP3, TGFß, and IL-10 mRNA levels. These differences may account for the differences noted in splenic T cells, where only intravenous delivery appeared to inhibit their activation. Our results imply that different mechanisms may be operating to generate immune tolerance by intrathymic and intravenous delivery of an AAV vector, and suggest that the intrathymic route may hold promise for decreasing the humoral immune response to therapeutic proteins in genetic disease indications.


Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética , Tolerancia Inmunológica/genética , Linfocitos T Reguladores/inmunología , Timo , alfa-Glucosidasas/genética , Adenoviridae/genética , Humanos , Inyecciones Intravenosas , Activación de Linfocitos , Linfocitos T Reguladores/citología , alfa-Glucosidasas/administración & dosificación , alfa-Glucosidasas/farmacología
6.
Mol Ther ; 18(9): 1584-91, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20551907

RESUMEN

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.


Asunto(s)
Folistatina/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , Glucógeno/metabolismo , Músculo Esquelético/metabolismo , Animales , Índice de Masa Corporal , Dependovirus/genética , Modelos Animales de Enfermedad , Folistatina/genética , Vectores Genéticos/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , alfa-Glucosidasas/genética , alfa-Glucosidasas/metabolismo
7.
Mol Ther ; 18(11): 1983-94, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20736932

RESUMEN

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.


Asunto(s)
Dependovirus/genética , Terapia Genética , Hepatocitos/inmunología , Enfermedades por Almacenamiento Lisosomal/terapia , Transgenes/fisiología , alfa-Galactosidasa/sangre , Animales , Anticuerpos Neutralizantes/inmunología , Western Blotting , Vectores Genéticos/administración & dosificación , Células HeLa , Hepatocitos/metabolismo , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/inmunología , Macaca fascicularis , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Plasmaféresis , Biosíntesis de Proteínas , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , alfa-Galactosidasa/genética
8.
Am J Respir Crit Care Med ; 181(10): 1098-105, 2010 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-20133924

RESUMEN

RATIONALE: Premature newborns frequently require manual ventilation for resuscitation during which lung injury occurs. Although surfactant protein (SP)-D regulates pulmonary inflammation, SP-D levels are low in the preterm lung. Commercial surfactants for treatment of respiratory distress syndrome do not contain SP-D. OBJECTIVES: To determine whether addition of recombinant human SP-D (rhSP-D) to commercial surfactant influences lung inflammation in ventilated premature newborn lambs. METHODS: Prematurely delivered lambs (130 d gestation age) were resuscitated with 100% O(2) and peak inspiratory pressure 40 cm H(2)O for 20 minutes and then treated with Survanta or Survanta containing rhSP-D. Ventilation was then changed to regulate tidal volume at 8 to 9 ml/kg. At 5 hours of age lambs were killed for sample collection. MEASUREMENTS AND MAIN RESULTS: Sequential blood gas and tidal volume were similar in lambs treated with or without rhSP-D, indicating that lung immaturity and ventilatory stress used to support premature lambs were comparable between the two groups. Ventilation caused pulmonary inflammation in lambs treated with surfactant alone. In contrast, surfactant containing rhSP-D decreased neutrophil numbers in bronchoalveolar lavage fluid and decreased neutrophil elastase activity in lung tissue. IL-8 mRNA and IL-8 protein were significantly decreased in the +rhSP-D group lamb lungs, to 20% of those in controls. The addition of rhSP-D also rendered Survanta more resistant to plasma protein inhibition of surfactant function. CONCLUSIONS: Treatment with rhSP-D-containing surfactant inhibited lung inflammation and enhanced the resistance of surfactant to inhibition, supporting its potential usefulness for prevention of lung injury in the preterm newborn.


Asunto(s)
Neumonía/prevención & control , Proteína D Asociada a Surfactante Pulmonar/farmacología , Lesión Pulmonar Inducida por Ventilación Mecánica/prevención & control , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Pulmón/fisiopatología , Masculino , Neumonía/etiología , Neumonía/fisiopatología , Proteínas Recombinantes/farmacología , Respiración Artificial/efectos adversos , Ovinos
9.
Am J Respir Cell Mol Biol ; 43(1): 46-54, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19648474

RESUMEN

A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials.


Asunto(s)
Técnicas de Transferencia de Gen , Nariz/patología , Animales , Adhesión Bacteriana , Fibrosis Quística/genética , Citomegalovirus/genética , Elementos de Facilitación Genéticos , Femenino , Terapia Genética/métodos , Liposomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Plásmidos/metabolismo , Regiones Promotoras Genéticas
10.
J Gene Med ; 12(1): 55-63, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19937989

RESUMEN

BACKGROUND: When assessing the efficacy of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy, we routinely evaluate gene transfer in the mouse nose and measure transfection efficiency by assessing transgene-specific mRNA using the real-time (TaqMan) quantitative reverse transcriptase-polymerase chain reaction. TaqMan is traditionally used to quantify expression in whole tissue homogenates, which in the nose would contain many cells types, including respiratory and olfactory epithelium. Only the respiratory epithelium is a satisfactory model for human airway epithelium and therefore CFTR gene transfer should be specifically assessed in respiratory epithelial cells (RECs). METHODS: We have compared laser microdissection, pronase digestion and nasal brushing for: (i) the ability to enrich RECs from the wild-type mouse nose and (ii) the length of time to perform the procedure. Using TaqMan, we subsequently assessed gene transfer in enriched RECs after nasal perfusion of GL67A/pCF1-CFTR complexes in a CF mouse model. RESULTS: Laser microdissection successfully isolated RECs; however, time-consuming sample preparation made this technique unsuitable for high-throughput studies. Pronase digestion was sufficiently rapid but only yielded 19% (range = 13%) RECs (n = 6). The nasal brushing method was superior, yielding 92% (range = 15%) RECs (n = 8) and was equally effective in CF knockout mice (91%, range = 14%, n = 10). Importantly, gene transfer was detectable in brushed RECs from 70% of perfused mice and the number of vector-specific transcripts was comparable to 3.5% of endogenous wild-type Cftr levels. CONCLUSIONS: Isolation of RECs by brushing allows accurate assessment of GTA transfection efficiency in an experimental system that is relevant for CF gene therapy.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulación de la Expresión Génica , Cavidad Nasal/patología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transgenes/genética , Animales , Separación Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteínas de Unión a Ácidos Grasos/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Humanos , Rayos Láser , Ratones , Ratones Endogámicos C57BL , Microdisección , Cavidad Nasal/metabolismo , Tabique Nasal/metabolismo , Tabique Nasal/patología , Pronasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
11.
Mol Genet Metab ; 100(4): 309-15, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20554235

RESUMEN

Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1-2years of age to a more slowly progressive course that causes significant morbidity and early mortality in children and adults. Recombinant human GAA (rhGAA) improves clinical outcomes with variable results. Adjunct therapy that increases the effectiveness of rhGAA may benefit some Pompe patients. Co-administration of the mTORC1 inhibitor rapamycin with rhGAA in a GAA knockout mouse reduced muscle glycogen content more than rhGAA or rapamycin alone. These results suggest mTORC1 inhibition may benefit GSDs that involve glycogen accumulation in muscle.


Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , Glucógeno/biosíntesis , Factores de Transcripción/antagonistas & inhibidores , Envejecimiento/efectos de los fármacos , Envejecimiento/patología , Animales , Relación Dosis-Respuesta a Droga , Terapia de Reemplazo Enzimático , Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Glucógeno Sintasa/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Miocardio/metabolismo , Miocardio/patología , Fosforilación/efectos de los fármacos , Proteínas , Proteínas Recombinantes/uso terapéutico , Sirolimus/análogos & derivados , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismo , alfa-Glucosidasas/metabolismo , alfa-Glucosidasas/uso terapéutico
12.
J Inherit Metab Dis ; 33(3): 281-9, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20336375

RESUMEN

Gaucher disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (acid beta-glucosidase), with consequent cellular accumulation of glucosylceramide (GL-1). The disease is managed by intravenous administrations of recombinant glucocerebrosidase (imiglucerase), although symptomatic patients with mild to moderate type 1 Gaucher disease for whom enzyme replacement therapy (ERT) is not an option may also be treated by substrate reduction therapy (SRT) with miglustat. To determine whether the sequential use of both ERT and SRT may provide additional benefits, we compared the relative pharmacodynamic efficacies of separate and sequential therapies in a murine model of Gaucher disease (D409V/null). As expected, ERT with recombinant glucocerebrosidase was effective in reducing the burden of GL-1 storage in the liver, spleen, and lung of 3-month-old Gaucher mice. SRT using a novel inhibitor of glucosylceramide synthase (Genz-112638) was also effective, albeit to a lesser degree than ERT. Animals administered recombinant glucocerebrosidase and then Genz-112638 showed the lowest levels of GL-1 in all the visceral organs and a reduced number of Gaucher cells in the liver. This was likely because the additional deployment of SRT following enzyme therapy slowed the rate of reaccumulation of GL-1 in the affected organs. Hence, in patients whose disease has been stabilized by intravenously administered recombinant glucocerebrosidase, orally administered SRT with Genz-112638 could potentially be used as a convenient maintenance therapy. In patients naïve to treatment, ERT followed by SRT could potentially accelerate clearance of the offending substrate.


Asunto(s)
Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/terapia , Glucosilceramidas/metabolismo , Lisosomas/enzimología , Animales , Modelos Animales de Enfermedad , Terapia de Reemplazo Enzimático/métodos , Femenino , Glucosilceramidasa/metabolismo , Glucosilceramidasa/uso terapéutico , Inmunohistoquímica , Masculino , Ratones , Pirrolidinas/farmacología , Proteínas Recombinantes/metabolismo , Distribución Tisular
13.
Acta Biochim Biophys Sin (Shanghai) ; 42(1): 45-51, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20043046

RESUMEN

The aim of the study was to assess if low-frequency ultrasound (US), in the range of 30-35 kHz, increases non-viral gene transfer to the mouse lung. US is greatly attenuated in the lung due to large energy losses at the air/tissue interfaces. The advantages of low-frequency US, compared with high-frequency US are: (i) increased cavitation (responsible for the formation of transient pores in the cell membrane) and (ii) reduced energy losses during lung penetration. Cationic lipid GL67/plasmid DNA (pDNA), polyethylenimine (PEI)/pDNA and naked pDNA were delivered via intranasal instillation and the animals were then exposed to US (sonoporation) at 0.07 or 0.1 MPa for 10 min. Under these conditions, US did not enhance GL67 or PEI-mediated transfection. It did, however, increase naked pDNA gene transfer by approximately 4 folds. Importantly, this was achieved in the absence of microbubbles, which are crucial for the commonly used high-frequency (1 MHz) sonoporation but may not be able to withstand nebulization in a clinically relevant setup. Lung hemorrhage was also assessed and shown to increase with US pressure in a dose-dependent manner. We have thus, established that low-frequency US can enhance lung gene transfer with naked pDNA and this enhancement is more effective than the previously reported 1 MHz US.


Asunto(s)
Pulmón/virología , Polietileneimina/química , Transfección/métodos , Animales , Técnicas de Transferencia de Gen , Pulmón/química , Ratones , Transfección/estadística & datos numéricos , Ultrasonido
14.
J Appl Physiol (1985) ; 106(5): 1545-52, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19265061

RESUMEN

Surfactant protein (SP)-D plays an important role in host defense and pulmonary surfactant homeostasis. In SP-D-deficient (Sftpd(-/-)) mice, the abnormal large surfactant forms seen at the ultrastructural level are taken up inefficiently by type II cells, resulting in an over threefold increase in the surfactant pool size. The mechanisms by which SP-D influences surfactant ultrastructure are unknown. We hypothesized that SP-D binds to surfactant immediately after being secreted and influences surfactant ultrastructure conversion. In newborn and adult sheep lungs, immunogold-labeled SP-D was associated with both lamellated membranous lipid structures of newly secreted surfactant and with small aggregate surfactant but not with tubular myelin. Since SP-D preferentially binds to phosphatidylinositol (PI) in vitro, the postnatal changes in PI were assessed. PI content in the bronchoalveolar lavage fluid increased after birth and peaked at 2-5 days of age, a time of rapid conversion of surfactant forms that is associated with the peak of surfactant lipid pool size. SP-D selectively interacted with PI-rich liposomes in vitro, causing their lysis. Similarly, the abnormal surfactant ultrastructure in Sftpd(-/-) mice was corrected by the addition of SP-D or melittin, and both peptides caused lysis of lipid vesicles. The normal conversion of surfactant ultrastructure requires SP-D that preferentially interacts with PI-rich, newly secreted surfactant, causing lysis of surfactant lipid membranes, converting the lipid forms into smaller surfactant lamellated structures that are critical for surfactant uptake by type II cells and normal surfactant homeostasis. SP-D regulates the dramatic decreases in the surfactant pool size that occurs in the newborn period.


Asunto(s)
Pulmón/metabolismo , Lípidos de la Membrana/metabolismo , Fosfatidilcolinas/metabolismo , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Surfactantes Pulmonares/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Líquido del Lavado Bronquioalveolar/química , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Pulmón/efectos de los fármacos , Pulmón/crecimiento & desarrollo , Meliteno/metabolismo , Meliteno/farmacología , Ratones , Ratones Noqueados , Fosfatidilcolinas/análisis , Proteína D Asociada a Surfactante Pulmonar/deficiencia , Proteína D Asociada a Surfactante Pulmonar/farmacología , Proteínas Recombinantes , Ovinos
15.
Mol Ther ; 16(8): 1400-8, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18545223

RESUMEN

Peripheral neuropathy is a particularly debilitating complication of both type 1 and type 2 diabetes characterized by sensory and motor neuron damage and decreased circulating levels of insulin-like growth factor 1 (IGF-1). Quite often, an early hyperalgesia is followed by hypoalgesia and muscle weakness. Hypoalgesia can lead to significant morbidity for which there is no current treatment. Hyperglycemic, streptozotocin (STZ)-induced rodent models reproduce these symptoms. We investigated whether increasing systemic IGF-1 could improve neuronal function in hyper- and hypoalgesic STZ-treated mice. Increased circulating levels of IGF-1 were achieved by delivering a plasmid or adeno-associated viral (AAV) vector bearing mouse IGF-1 to the liver. Treating mice in the hyperalgesia stage prevented later hypoalgesia. Treating mice in the hypoalgesia stage reversed existing hypoalgesia. This latter effect could be seen by merely restoring IGF-1 serum levels to normalcy, which was possible to achieve by IGF-1 gene therapy or insulin treatment. Sensory nerve functional correction was seen to be correlated with attenuated Schwann cell vacuolization and demyelination in peripheral sensory nerve fibers. A further increase in serum IGF-1 levels with gene therapy also improved motor function, consistent with the observed prevention of both muscle atrophy and peripheral motor nerve fiber demyelination. These results suggest that the restoration of systemic levels of IGF-1 may prove to be a highly effective therapeutic modality for treating diabetic peripheral neuropathy.


Asunto(s)
Neuropatías Diabéticas/terapia , Terapia Genética/métodos , Hiperalgesia/terapia , Factor I del Crecimiento Similar a la Insulina/fisiología , Animales , Peso Corporal/fisiología , Dependovirus/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/fisiopatología , Neuropatías Diabéticas/etiología , Neuropatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Vectores Genéticos/genética , Hiperalgesia/inducido químicamente , Hiperalgesia/fisiopatología , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Neuronas Motoras/fisiología
16.
Respir Res ; 9: 65, 2008 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-18811961

RESUMEN

BACKGROUND: Surfactant protein D (SP-D) plays an important role in innate defense against influenza A viruses (IAVs) and other pathogens. METHODS: We tested antiviral activities of recombinant human SP-D against a panel of IAV strains that vary in glycosylation sites on their hemagglutinin (HA). For these experiments a recombinant version of human SP-D of the Met11, Ala160 genotype was used after it was characterized biochemically and structurally. RESULTS: Oligosaccharides at amino acid 165 on the HA in the H3N2 subtype and 104 in the H1N1 subtype are absent in collectin-resistant strains developed in vitro and are important for mediating antiviral activity of SP-D; however, other glycans on the HA of these viral subtypes also are involved in inhibition by SP-D. H3N2 strains obtained shortly after introduction into the human population were largely resistant to SP-D, despite having the glycan at 165. H3N2 strains have become steadily more sensitive to SP-D over time in the human population, in association with addition of other glycans to the head region of the HA. In contrast, H1N1 strains were most sensitive in the 1970s-1980s and more recent strains have become less sensitive, despite retaining the glycan at 104. Two H5N1 strains were also resistant to inhibition by SP-D. By comparing sites of glycan attachment on sensitive vs. resistant strains, specific glycan sites on the head domain of the HA are implicated as important for inhibition by SP-D. Molecular modeling of the glycan attachment sites on HA and the carbohydrate recognition domain of SPD are consistent with these observations. CONCLUSION: Inhibition by SP-D correlates with presence of several glycan attachment sites on the HA. Pandemic and avian strains appear to lack susceptibility to SP-D and this could be a contributory factor to their virulence.


Asunto(s)
Hemaglutininas Virales/metabolismo , Virus de la Influenza A/fisiología , Proteína D Asociada a Surfactante Pulmonar/administración & dosificación , Proteína D Asociada a Surfactante Pulmonar/química , Inactivación de Virus/efectos de los fármacos , Antivirales/administración & dosificación , Antivirales/química , Glicosilación/efectos de los fármacos , Humanos , Virus de la Influenza A/efectos de los fármacos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química
17.
Biomaterials ; 29(10): 1533-40, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18155138

RESUMEN

Imaging of in vivo gene expression using luciferase expression in various organs has been used for several years. In contrast to other organs, in vivo imaging of the lung, particularly after non-viral gene transfer has not been extensively studied. The aim of this study was to address several questions: (1) Does in vivo light emission correlate with standard tissue homogenate-based luciferase detection in a dose-dependent manner? Recombinant Sendai virus (SeV) transduces airway epithelial cells very efficiently and was used to address this question, (2) Is the sensitivity of the assay sufficient to detect non-viral gene transfer? We treated mice with SeV-Lux vector using our standard "sniffing" protocol, a method that predominantly results in lung deposition. Dose-related in vivo light emission was visible in all animals. Importantly, there was a significant correlation (r>0.90, p<0.0001) between the in vivo and ex vivo assays in both the left and right lung. We next transfected the nasal epithelium via nasal perfusion or the lungs ("sniffing") of mice with a luciferase plasmid (pCIKLux) complexed to the cationic lipid GL67 (n=25-27/group) and imaged luciferase expression in vivo 24h after transfection. Gene expression was detectable in both organs. Correlation between the in vivo and ex vivo assays was significant (r=0.52, p<0.005) in the left, but not the right lung. The correlation in the nose was weaker (r=0.45, p<0.05). To our knowledge these studies show for the first time that this non-invasive method of assessing pulmonary gene transfer is viable for evaluating non-viral gene transfer agents.


Asunto(s)
Mediciones Luminiscentes/métodos , Sistema Respiratorio/metabolismo , Transfección/métodos , Animales , Femenino , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/metabolismo , Virus Sendai/genética
18.
Chest ; 132(5): 1447-54, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17925426

RESUMEN

BACKGROUND: Acute lung injury is a common cause of morbidity and mortality following pulmonary or systemic infections. Surfactant protein-D is a member of the collectin family of proteins, which play important roles in innate host defense of the lung. In this study, the effect of exogenous recombinant human SP-D (rhSP-D) on protection of the adult mouse lung from lipopolysaccharide (LPS)-induced and lipoteichoic acid (LTA)-induced injury was assessed. METHODS: The effect of rhSP-D on LPS-induced and LTA-induced lung inflammation and injury was assessed with and without exogenous pulmonary surfactant in Sftpd+/+ and Sftpd-/- mice. A total of 204 mice (6 mice per group) were used for the present study. RESULTS: Sftpd-/- mice were more susceptible to intratracheal LPS than were Sftpd+/+ mice. rhSP-D decreased neutrophilic infiltrates induced by LPS and LTA in the lungs of both Sftpd+/+ and Sftpd-/- mice. The addition of exogenous pulmonary surfactant to rhSP-D further decreased LPS-induced and LTA-induced pulmonary inflammation in Sftpd-/- and Sftpd+/+ mice. CONCLUSIONS: Intratracheal rhSP-D inhibited inflammation induced by intratracheal LPS and LTA instillation in the lung. The antiinflammatory effects of rhSP-D were enhanced by the addition of pulmonary surfactant, providing a potential therapy for the treatment of lung inflammation.


Asunto(s)
Inflamación/inmunología , Pulmón/metabolismo , Proteína D Asociada a Surfactante Pulmonar/inmunología , Surfactantes Pulmonares/inmunología , Enfermedad Aguda , Análisis de Varianza , Animales , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Instilación de Medicamentos , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/farmacología , Ratones , Ácidos Teicoicos/administración & dosificación , Ácidos Teicoicos/farmacología
19.
Respir Res ; 7: 26, 2006 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-16480492

RESUMEN

BACKGROUND: The cationic lipid Genzyme lipid (GL) 67 is the current "gold-standard" for in vivo lung gene transfer. Here, we assessed, if GL67 mediated uptake of siRNAs and asODNs into airway epithelium in vivo. METHODS: Anti-lacZ and ENaC (epithelial sodium channel) siRNA and asODN were complexed to GL67 and administered to the mouse airway epithelium in vivo Transfection efficiency and efficacy were assessed using real-time RT-PCR as well as through protein expression and functional studies. In parallel in vitro experiments were carried out to select the most efficient oligonucleotides. RESULTS: In vitro, GL67 efficiently complexed asODNs and siRNAs, and both were stable in exhaled breath condensate. Importantly, during in vitro selection of functional siRNA and asODN we noted that asODNs accumulated rapidly in the nuclei of transfected cells, whereas siRNAs remained in the cytoplasm, a pattern consistent with their presumed site of action. Following in vivo lung transfection siRNAs were only visible in alveolar macrophages, whereas asODN also transfected alveolar epithelial cells, but no significant uptake into conducting airway epithelial cells was seen. SiRNAs and asODNs targeted to beta-galactosidase reduced betagal mRNA levels in the airway epithelium of K18-lacZ mice by 30% and 60%, respectively. However, this was insufficient to reduce protein expression. In an attempt to increase transfection efficiency of the airway epithelium, we increased contact time of siRNA and asODN using the in vivo mouse nose model. Although highly variable and inefficient, transfection of airway epithelium with asODN, but not siRNA, was now seen. As asODNs more effectively transfected nasal airway epithelial cells, we assessed the effect of asODN against ENaC, a potential therapeutic target in cystic fibrosis; no decrease in ENaC mRNA levels or function was detected. CONCLUSION: This study suggests that although siRNAs and asODNs can be developed to inhibit gene expression in culture systems and certain organs in vivo, barriers to nucleic acid transfer in airway epithelial cells seen with large DNA molecules may also affect the efficiency of in vivo uptake of small nucleic acid molecules.


Asunto(s)
Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Marcación de Gen/métodos , Lípidos/química , Oligonucleótidos Antisentido/genética , ARN Interferente Pequeño/genética , Transfección/métodos , Animales , Células Cultivadas , Células Epiteliales , Silenciador del Gen , Humanos , Ratones , Células 3T3 NIH , Oligonucleótidos Antisentido/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Mucosa Respiratoria
20.
Hum Gene Ther ; 13(17): 2065-77, 2002 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-12490001

RESUMEN

Rapid systemic injection of naked plasmid DNA (pDNA) in a large volume into a mouse tail vein has been shown to result in a high level of gene expression in the liver. However, the potential therapeutic benefit to humans embodied in hydrodynamic transfection of the liver cannot be realized until a clinically viable method for gene delivery is developed. In light of this fact, we have devised and evaluated several methods for delivering pDNA to the isolated rabbit liver using minimally invasive catheter-based techniques. Using a lobar technique, pDNA was delivered hydrodynamically to an isolated hepatic lobe using a balloon occlusion balloon catheter to occlude a selected hepatic vein. A whole organ technique was used wherein the entire hepatic venous system was isolated and the pDNA solution injected hydrodynamically into the vena cava between two balloons used to block hepatic venous outflow. Lobar delivery of a plasmid encoding a secreted alkaline phosphatase (SEAP) reporter gene resulted in significant levels of transgene product in the serum. A nonsecreted transgene product, chloramphenicol acetyltransferase (CAT), showed the highest levels of expression in the injected lobe distal to the injection site. Compared to lobar delivery, whole organ delivery yielded much higher serum levels of SEAP expression and a significantly broader hepatic parenchymal distribution of CAT expression. These preliminary studies suggest that catheter-mediated hydrodynamic delivery of pDNA to the isolated liver may provide a method for human gene therapy that is both therapeutically significant and clinically practicable.


Asunto(s)
Cateterismo , Técnicas de Transferencia de Gen , Hígado/metabolismo , Plásmidos/administración & dosificación , Plásmidos/genética , Transducción Genética/métodos , Animales , Relación Dosis-Respuesta a Droga , Expresión Génica , Terapia Genética/métodos , Técnicas In Vitro , Inyecciones Intravenosas , Masculino , Ratones , Conejos , Transgenes
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