RESUMO
Systemic administration of adeno-associated virus (AAV) vectors for spinal cord gene therapy has challenges including toxicity at high doses and pre-existing immunity that reduces efficacy. Intrathecal (IT) delivery of AAV vectors into cerebral spinal fluid can avoid many issues, although distribution of the vector throughout the spinal cord is limited, and vector entry to the periphery sometimes initiates hepatotoxicity. Here we performed biopanning in non-human primates (NHPs) with an IT injected AAV9 peptide display library. We identified top candidates by sequencing inserts of AAV DNA isolated from whole tissue, nuclei, or nuclei from transgene-expressing cells. These barcoded candidates were pooled with AAV9 and compared for biodistribution and transgene expression in spinal cord and liver of IT injected NHPs. Most candidates displayed increased retention in spinal cord compared with AAV9. Greater spread from the lumbar to the thoracic and cervical regions was observed for several capsids. Furthermore, several capsids displayed decreased biodistribution to the liver compared with AAV9, providing a high on-target/low off-target biodistribution. Finally, we tested top candidates in human spinal cord organoids and found them to outperform AAV9 in efficiency of transgene expression in neurons and astrocytes. These capsids have potential to serve as leading-edge delivery vehicles for spinal cord-directed gene therapies.
Assuntos
Dependovirus , Terapia Genética , Vetores Genéticos , Medula Espinal , Dependovirus/genética , Animais , Medula Espinal/metabolismo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Terapia Genética/métodos , Transgenes , Técnicas de Transferência de Genes , Capsídeo/metabolismo , Distribuição Tecidual , Injeções Espinhais , Transdução Genética , Macaca mulatta , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismoRESUMO
Systemic administration of adeno-associated virus (AAV) vectors for spinal cord gene therapy has challenges including toxicity at high doses and pre-existing immunity that reduces efficacy. Intrathecal delivery of AAV vectors into the cerebral spinal fluid (CSF) can avoid many of the issues of systemic delivery, although achieving broad distribution of the vector and transgene expression throughout the spinal cord is challenging and vector entry to the periphery occurs, sometimes initiating hepatotoxicity. Here we performed two rounds of in vivo biopanning in non-human primates (NHPs) with an AAV9 peptide display library injected intrathecally and performed insert sequencing on DNA isolated from either whole tissue (conventional selection), isolated nuclei, or nuclei from transgene-expressing cells. A subsequent barcoded pool of candidates and AAV9 was compared at the DNA (biodistribution) and RNA (expression) level in spinal cord and liver of intrathecally injected NHPs. Most of the candidates displayed enhanced biodistribution compared to AAV9 at all levels of spinal cord ranging from 2 to 265-fold. Nuclear isolation or expression-based selection yielded 4 of 7 candidate capsids with enhanced transgene expression in spinal cord (up to 2.4-fold), while no capsid obtained by conventional selection achieved that level. Furthermore, several capsids displayed lower biodistribution to the liver of up to 1,250-fold, compared to AAV9, providing a remarkable on target/off target biodistribution ratio. These capsids may have potential for gene therapy programs directed at the spinal cord and the selection method described here should be useful in clinically relevant large animal models.
RESUMO
Hunter syndrome is a rare x-linked recessive genetic disorder that affects lysosomal metabolism due to deficiency of iduronate-2-sulfatase (IDS), with subsequent accumulation of glycosaminoglycans heparan and dermatan sulfates (GAG). Enzyme replacement therapy is the only FDA-approved remedy and is an expensive life-time treatment that alleviates some symptoms of the disease without neurocognitive benefit. We previously reported successful treatment in a mouse model of mucopolysaccharidosis type II (MPS II) using adeno-associated viral vector serotype 9 encoding human IDS (AAV9.hIDS) via intracerebroventricular injection. As a less invasive and more straightforward procedure, here we report intravenously administered AAV9.hIDS in a mouse model of MPS II. In animals administered 1.5 × 1012 vg of AAV9.hIDS at 2 months of age, we observed supraphysiological levels of IDS enzyme activity in the circulation (up to 9100-fold higher than wild-type), in the tested peripheral organs (up to 560-fold higher than wild-type), but only 4% to 50% of wild type levels in the CNS. GAG levels were normalized on both sides of the blood-brain-barrier (BBB) in most of tissues tested. Despite low levels of the IDS observed in the CNS, this treatment prevented neurocognitive decline as shown by testing in the Barnes maze and by fear conditioning. This study demonstrates that a single dose of IV-administered AAV9.hIDS may be an effective and non-invasive procedure to treat MPS II that benefits both sides of the BBB, with implications for potential use of IV-administered AAV9 for other neuronopathic lysosomal diseases.
RESUMO
The mucopolysaccharidoses (MPS) are a group of recessively inherited conditions caused by deficiency of lysosomal enzymes essential to the catabolism of glycosaminoglycans (GAG). MPS I is caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA), while MPS II is caused by a lack of iduronate-2-sulfatase (IDS). Lack of these enzymes leads to early mortality and morbidity, often including neurological deficits. Enzyme replacement therapy has markedly improved the quality of life for MPS I and MPS II affected individuals but is not effective in addressing neurologic manifestations. For MPS I, hematopoietic stem cell transplant has shown effectiveness in mitigating the progression of neurologic disease when carried out in early in life, but neurologic function is not restored in patients transplanted later in life. For both MPS I and II, gene therapy has been shown to prevent neurologic deficits in affected mice when administered early, but the effectiveness of treatment after the onset of neurologic disease manifestations has not been characterized. To test if neurocognitive function can be recovered in older animals, human IDUA or IDS-encoding AAV9 vector was administered by intracerebroventricular injection into MPS I and MPS II mice, respectively, after the development of neurologic deficit. Vector sequences were distributed throughout the brains of treated animals, associated with high levels of enzyme activity and normalized GAG storage. Two months after vector infusion, treated mice exhibited spatial navigation and learning skills that were normalized, that is, indistinguishable from those of normal unaffected mice, and significantly improved compared to untreated, affected animals. We conclude that cognitive function was restored by AAV9-mediated, central nervous system (CNS)-directed gene transfer in the murine models of MPS I and MPS II, suggesting that gene transfer may result in neurodevelopment improvements in severe MPS I and MPS II when carried out after the onset of cognitive decline.
Assuntos
Disfunção Cognitiva , Iduronato Sulfatase , Mucopolissacaridose II , Mucopolissacaridose I , Doenças do Sistema Nervoso , Humanos , Animais , Camundongos , Idoso , Qualidade de Vida , Mucopolissacaridose II/genética , Mucopolissacaridose II/terapia , Mucopolissacaridose I/genética , Mucopolissacaridose I/terapia , Sistema Nervoso Central/metabolismo , Iduronidase/genética , Iduronidase/metabolismo , Iduronato Sulfatase/genética , Disfunção Cognitiva/metabolismo , Glicosaminoglicanos/metabolismo , Modelos Animais de DoençasRESUMO
Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). The two current treatments [hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT)], are insufficiently effective in addressing neurologic disease, in part due to the inability of lysosomal enzyme to cross the blood brain barrier. With a goal to more effectively treat neurologic disease, we have investigated the effectiveness of AAV-mediated IDUA gene delivery to the brain using several different routes of administration. Animals were treated by either direct intracerebroventricular (ICV) injection, by intrathecal (IT) infusion into the cerebrospinal fluid, or by intranasal (IN) instillation of AAV9-IDUA vector. AAV9-IDUA was administered to IDUA-deficient mice that were either immunosuppressed with cyclophosphamide (CP), or immunotolerized at birth by weekly injections of human iduronidase. In animals treated by ICV or IT administration, levels of IDUA enzyme ranged from 3- to 1000-fold that of wild type levels in all parts of the microdissected brain. In animals administered vector intranasally, enzyme levels were 100-fold that of wild type in the olfactory bulb, but enzyme expression was close to wild type levels in other parts of the brain. Glycosaminoglycan levels were reduced to normal in ICV and IT treated mice, and in IN treated mice they were normalized in the olfactory bulb, or reduced in other parts of the brain. Immunohistochemical analysis showed extensive IDUA expression in all parts of the brain of ICV treated mice, while IT treated animals showed transduction that was primarily restricted to the hind brain with some sporadic labeling seen in the mid- and fore brain. At 6 months of age, animals were tested for spatial navigation, memory, and neurocognitive function in the Barnes maze; all treated animals were indistinguishable from normal heterozygous control animals, while untreated IDUA deficient animals exhibited significant learning and spatial navigation deficits. We conclude that IT and IN routes are acceptable and alternate routes of administration, respectively, of AAV vector delivery to the brain with effective IDUA expression, while all three routes of administration prevent the emergence of neurocognitive deficiency in a mouse MPS I model.
RESUMO
Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by deficiency of alpha-L-iduronidase (IDUA), resulting in accumulation of heparan and dermatan sulfate glycosaminoglycans (GAGs). Individuals with the most severe form of the disease (Hurler syndrome) suffer from neurodegeneration, intellectual disability, and death by age 10. Current treatments for this disease include allogeneic hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). However, these treatments do not address CNS manifestations of the disease. In this study we compared the ability of intravenously administered AAV serotypes 9 and rh10 (AAV9 and AAVrh10) for delivery and expression of the IDUA gene in the CNS. Adult C57BL/6 MPS I mice were infused intravenously with either AAV9 or AAVrh10 vector encoding the human IDUA gene. Treated animals demonstrated supraphysiological levels and widespread restoration of IDUA enzyme activity in the plasma and all organs including the CNS. High levels of IDUA enzyme activity were observed in the plasma, brain and spinal cord ranging from 10 to 100-fold higher than heterozygote controls, while levels in peripheral organs were also high, ranging from 1000 to 10,000-fold higher than control animals. In general, levels of IDUA expression were slightly higher in peripheral organs for AAVrh10 administered animals although these differences were not significant except for the lung. Levels of IDUA expression between AAV 9 and rh10 were roughly equivalent in the brain. Urinary and tissue GAGs were significantly reduced starting at 3 weeks after vector infusion, with restoration of normal GAG levels by the end of the study in animals treated with either AAV9 or rh10. These results demonstrate that non-invasive intravenous AAV9 or AAVrh10-mediated IDUA gene therapy is a potentially effective treatment for both systemic and CNS manifestations of MPS I, with implications for the treatment of other metabolic and neurological diseases as well.
RESUMO
Sustained suppression of VEGF is needed in many patients with neovascular age-related macular degeneration (NVAMD), and gene transfer of a VEGF-neutralizing protein is a promising approach to achieve it. Initial clinical trials testing this approach have shown encouraging signals, but evidence of robust transgene expression and consistent antiangiogenic and antipermeability activity has been lacking. In this study, we demonstrate expression of an anti-human VEGF antibody fragment (antiVEGFfab) after subretinal injection of AAV8-antiVEGFfab. In transgenic mice expressing human VEGF in retina (rho/VEGF mice), a model of type 3 choroidal neovascularization (NV), eyes injected with ≥1 × 107 gene copies (GC) of AAV8-antiVEGFfab had significantly less mean area of NV than null vector-injected eyes. A dose-dependent response was observed with modest reduction of NV with ≤3 × 107, >50% reduction with ≥1 × 108 GC and almost complete elimination of NV with 3 × 109 or 1 × 1010 GC. In Tet/opsin/VEGF mice, in which doxycycline-induced high expression of VEGF leads to severe vascular leakage and exudative retinal detachment (RD), reduction of total RD by 70%-80% occurred with 3 × 109 or 1 × 1010 GC of AAV8-antiVEGFfab, an effect that was sustained for at least a month. These data strongly support initiating clinical trials testing subretinal injection of AAV8-antiVEGFfab in patients with NVAMD.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Fragmentos Fab das Imunoglobulinas/genética , Degeneração Macular/genética , Degeneração Macular/patologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Animais , Neovascularização de Coroide/genética , Neovascularização de Coroide/patologia , Neovascularização de Coroide/terapia , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/administração & dosagem , Fragmentos Fab das Imunoglobulinas/metabolismo , Degeneração Macular/terapia , Camundongos , Neovascularização Retiniana/terapia , Transdução Genética , Transgenes , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a rare X-linked recessive lysosomal disorder caused by defective iduronate-2-sulfatase (IDS), resulting in accumulation of heparan sulfate and dermatan sulfate glycosaminoglycans (GAGs). Enzyme replacement is the only Food and Drug Administration-approved therapy available for MPS II, but it is expensive and does not improve neurologic outcomes in MPS II patients. This study evaluated the effectiveness of adeno-associated virus (AAV) vector encoding human IDS delivered intracerebroventricularly in a murine model of MPS II. Supraphysiological levels of IDS were observed in the circulation (160-fold higher than wild type) for at least 28 weeks post injection and in most tested peripheral organs (up to 270-fold) at 10 months post injection. In contrast, only low levels of IDS were observed (7-40% of wild type) in all areas of the brain. Sustained IDS expression had a profound effect on normalization of GAG in all tested tissues and on prevention of hepatomegaly. Additionally, sustained IDS expression in the central nervous system (CNS) had a prominent effect in preventing neurocognitive deficit in MPS II mice treated at 2 months of age. This study demonstrates that CNS-directed, AAV9 mediated gene transfer is a potentially effective treatment for Hunter syndrome, as well as other monogenic disorders with neurologic involvement.
Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Iduronato Sulfatase/genética , Mucopolissacaridose II/genética , Mucopolissacaridose II/psicologia , Animais , Sistema Nervoso Central/metabolismo , Cognição , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/administração & dosagem , Glicosaminoglicanos/metabolismo , Humanos , Iduronato Sulfatase/sangue , Iduronato Sulfatase/metabolismo , Masculino , Camundongos , Mucopolissacaridose II/sangue , Mucopolissacaridose II/terapia , Testes Neuropsicológicos , Projetos Piloto , Fatores de Tempo , Distribuição Tecidual , Transdução GenéticaRESUMO
Mucopolysaccharidosis type I (MPS I) is a progressive, multi-systemic, inherited metabolic disease caused by deficiency of α-L-iduronidase (IDUA). Current treatments for this disease are ineffective in treating central nervous system (CNS) disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier. A noninvasive and effective approach was taken in the treatment of CNS disease by intranasal administration of an IDUA-encoding adeno-associated virus serotype 9 (AAV9) vector. Adult IDUA-deficient mice aged 3 months were instilled intranasally with AAV9-IDUA vector. Animals sacrificed 5 months post instillation exhibited IDUA enzyme activity levels that were up to 50-fold that of wild-type mice in the olfactory bulb, with wild-type levels of enzyme restored in all other parts of the brain. Intranasal treatment with AAV9-IDUA also resulted in the reduction of tissue glycosaminoglycan storage materials in the brain. There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb, but there was no evidence of the presence of transduced cells in other portions of the brain. This indicates that reduction of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain. At 8 months of age, neurocognitive testing using the Barnes maze to assess spatial navigation demonstrated that treated IDUA-deficient mice were no different from normal control animals, while untreated IDUA-deficient mice exhibited significant learning and navigation deficits. This novel, noninvasive strategy for intranasal AAV9-IDUA instillation could potentially be used to treat CNS manifestations of human MPS I.
Assuntos
Sistema Nervoso Central/metabolismo , Dependovirus/metabolismo , Técnicas de Transferência de Genes , Iduronidase/genética , Iduronidase/uso terapêutico , Mucopolissacaridose I/patologia , Mucopolissacaridose I/terapia , Doenças do Sistema Nervoso/prevenção & controle , Administração Intranasal , Animais , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiopatologia , Cognição , Proteínas de Fluorescência Verde/metabolismo , Humanos , Iduronidase/metabolismo , Lisossomos/metabolismo , Camundongos , Mucopolissacaridose I/fisiopatologia , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/fisiopatologia , Neurônios/metabolismo , Bulbo Olfatório/metabolismo , Bulbo Olfatório/patologia , Transdução GenéticaRESUMO
Mucopolysaccharidosis type II (MPS II) is a rare X-linked genetic disorder caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS), leading to impaired catabolism of ubiquitous polysaccharides and abnormal accumulation of these undegraded substrates in the lysosome. Like many lysosomal storage diseases, MPS II is characterized by both somatic and central nervous system (CNS) involvement. Intravenous enzyme replacement therapy can improve somatic manifestations of MPS II, but systemic IDS does not cross the blood-brain barrier and therefore cannot address CNS disease. In this study, an adeno-associated virus serotype 9 vector carrying the IDS gene was injected into the cerebrospinal fluid (CSF) of IDS deficient mice, a model of MPS II. Treated mice exhibited dose-dependent IDS expression and resolution of brain storage lesions, as well as improvement in long-term memory in a novel object recognition test. These findings suggest that delivery of adeno-associated virus vectors into CSF could serve as a platform for efficient, long-term enzyme delivery to the CNS, potentially addressing this critical unmet need for patients with MPS II and many related lysosomal enzyme deficiencies.
Assuntos
Doenças do Sistema Nervoso Central/terapia , Dependovirus/genética , Terapia Genética , Vetores Genéticos/administração & dosagem , Glicoproteínas/genética , Iduronidase/genética , Mucopolissacaridose II/fisiopatologia , Animais , Barreira Hematoencefálica , Doenças do Sistema Nervoso Central/genética , Líquido Cefalorraquidiano/metabolismo , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Terapia de Reposição de Enzimas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucopolissacaridose II/líquido cefalorraquidianoRESUMO
Recombinant vectors based on adeno-associated virus serotype 8 (AAV8) have been successfully used in the clinic and hold great promise for liver-directed gene therapy. Preexisting immunity against AAV8 or the development of antibodies against the therapeutic transgene product might negatively affect the outcomes of gene therapy. In the prospect of an AAV8-mediated, liver-directed gene therapy clinical trial for mucopolysaccharidosis VI (MPS VI), a lysosomal storage disorder caused by arylsulfatase B (ARSB) deficiency, we investigated in a multiethnic cohort of MPS VI patients the prevalence of neutralizing antibodies (Nab) to AAV8 and the presence of ARSB cross-reactive immunologic material (CRIM), which will either affect the efficacy of gene transfer or the duration of phenotypic correction. Thirty-six MPS VI subjects included in the study harbored 45 (62.5%) missense, 13 (18%) nonsense, 9 (12.5%) frameshift (2 insertions and 7 deletions), and 5 (7%) splicing ARSB mutations. The detection of ARSB protein in 24 patients out of 34 (71%) was predicted by the type of mutations. Preexisting Nab to AAV8 were undetectable in 19/33 (58%) analyzed patients. Twelve out of 31 patients (39%) tested were both negative for Nab to AAV8 and CRIM-positive. In conclusion, this study allows estimating the number of MPS VI patients eligible for a gene therapy trial by intravenous injections of AAV8.
Assuntos
Anticorpos Neutralizantes/sangue , Dependovirus/imunologia , Terapia Genética/métodos , Mucopolissacaridose VI/imunologia , N-Acetilgalactosamina-4-Sulfatase/sangue , Seleção de Pacientes , Estudos de Coortes , Reações Cruzadas , Análise Mutacional de DNA , Dependovirus/genética , Terapia Genética/normas , Humanos , Itália , Mucopolissacaridose VI/terapia , Mutação/genética , N-Acetilgalactosamina-4-Sulfatase/genética , Países Baixos , TurquiaRESUMO
During the acute-phase reaction, SAA (serum amyloid A) replaces apoA-I (apolipoprotein A-I) as the major HDL (high-density lipoprotein)-associated apolipoprotein. A remarkable portion of SAA exists in a lipid-free/lipid-poor form and promotes ABCA1 (ATP-binding cassette transporter A1)-dependent cellular cholesterol efflux. In contrast with lipid-free apoA-I and apoE, lipid-free SAA was recently reported to mobilize SR-BI (scavenger receptor class B, type I)-dependent cellular cholesterol efflux [Van der Westhuyzen, Cai, de Beer and de Beer (2005) J. Biol. Chem. 280, 35890-35895]. This unique property could strongly affect cellular cholesterol mobilization during inflammation. However, in the present study, we show that overexpression of SR-BI in HEK-293 cells (human embryonic kidney cells) (devoid of ABCA1) failed to mobilize cholesterol to lipid-free or lipid-poor SAA. Only reconstituted vesicles containing phospholipids and SAA promoted SR-BI-mediated cholesterol efflux. Cholesterol efflux from HEK-293 and HEK-293[SR-BI] cells to lipid-free and lipid-poor SAA was minimal, while efficient efflux was observed from fibroblasts and CHO cells (Chinese-hamster ovary cells) both expressing functional ABCA1. Overexpression of SR-BI in CHO cells strongly attenuated cholesterol efflux to lipid-free SAA even in the presence of an SR-BI-blocking IgG. This implies that SR-BI attenuates ABCA1-mediated cholesterol efflux in a way that is not dependent on SR-BI-mediated re-uptake of cholesterol. The present in vitro experiments demonstrate that the lipidation status of SAA is a critical factor governing cholesterol acceptor properties of this amphipathic apolipoprotein. In addition, we demonstrate that SAA mediates cellular cholesterol efflux via the ABCA1 and/or SR-BI pathway in a similar way to apoA-I.
Assuntos
Antígenos CD36/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Proteína Amiloide A Sérica/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Metabolismo dos Lipídeos , Fosfolipídeos/metabolismo , Transdução de SinaisRESUMO
The etiology of human female infertility is often uncertain. The sterility of high-density lipoprotein (HDL) receptor-negative (SR-BI(-/-)) female mice suggests a link between female infertility and abnormal lipoprotein metabolism. SR-BI(-/-) mice exhibit elevated plasma total cholesterol [with normal-sized and abnormally large HDL and high unesterified to total plasma cholesterol (UC:TC) ratio]. We explored the influence of hepatic SR-BI on female fertility by inducing hepatic SR-BI expression in SR-BI(-/-) animals by adenovirus transduction or stable transgenesis. For transgenes, we used both wild-type SR-BI and a double-point mutant, Q402R/Q418R (SR-BI-RR), which is unable to bind to and mediate lipid transfer from wild-type HDL normally, but retains virtually normal lipid transport activities with low-density lipoprotein. Essentially wild-type levels of hepatic SR-BI expression in SR-BI(-/-) mice restored to nearly normal the HDL size distribution and plasma UC:TC ratio, whereas approximately 7- to 40-fold overexpression dramatically lowered plasma TC and increased biliary cholesterol secretion. In contrast, SR-BI-RR overexpression had little effect on SR-BI(+/+) mice, but in SR-BI(-/-) mice, it substantially reduced levels of abnormally large HDL and normalized the UC:TC ratio. In all cases, hepatic transgenic expression restored female fertility. Overexpression in SR-BI(-/-) mice of lecithin:cholesterol acyl transferase, which esterifies plasma HDL cholesterol, did not normalize the UC:TC ratio, probably because the abnormal HDL was a poor substrate, and did not restore fertility. Thus, hepatic SR-BI-mediated lipoprotein metabolism influences murine female fertility, raising the possibility that dyslipidemia might contribute to human female infertility and that targeting lipoprotein metabolism might complement current assisted reproductive technologies.
Assuntos
Fertilidade , Lipoproteínas HDL/metabolismo , Fígado/metabolismo , Receptores Depuradores Classe B/fisiologia , Adenoviridae/genética , Animais , Colesterol/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Receptores Depuradores Classe B/genéticaRESUMO
The hypercholesterolemia characteristic of apolipoprotein (apoE)-deficient mice fed on a regular chow diet is caused by the abnormal accumulation of apoB-48-carrying remnants of chylomicrons and very low density lipoproteins in the plasma. Treatment of apoE-deficient mice with ciprofibrate or other peroxisome proliferator-activated receptor alpha agonists severely aggravates their hypercholesterolemia by interfering with one or more mechanisms of remnant removal from the circulation that do not require mediation by apoE (Fu, T., Kashireddy, P., and Borensztajn, J. (2003) Biochem. J. 373, 941-947). In the present investigation we report that ciprofibrate treatment causes the down-regulation of hepatic scavenger receptor class B, type I (SR-BI) protein expression in the livers of apoE-deficient mice. On cessation of the treatment SR-BI expression returns to its pretreatment levels, coinciding with a reversal of the hypercholesterolemia to base-line concentrations. Restoration of SR-BI expression in ciprofibrate-treated apoE-deficient mice by recombinant adenoviral gene transfer abolishes the ciprofibrate-induced over accumulation of apoB-48-carrying remnants in the plasma. We also report that remnants isolated from the plasma of ciprofibrate-treated apoE-deficient mice bind to murine SR-BI expressed in stably transfected cultured cells. These observations suggest that, in addition to its well established role as high density lipoprotein receptor, SR-BI can also function as a remnant receptor responsible for the clearance of remnants from the circulation of apoE-deficient mice.
Assuntos
Adenoviridae/genética , Apolipoproteínas E/fisiologia , Antígenos CD36/metabolismo , Colesterol/análogos & derivados , Ácido Clofíbrico , Ácido Clofíbrico/análogos & derivados , Hipercolesterolemia/metabolismo , Proteínas de Membrana , Receptores Imunológicos , Receptores de Lipoproteínas , Animais , Apolipoproteínas E/genética , Células CHO , Colesterol/metabolismo , Colesterol/farmacologia , Ácido Clofíbrico/farmacologia , Cricetinae , Regulação para Baixo , Feminino , Ácidos Fíbricos , Técnicas de Transferência de Genes , Vetores Genéticos , Hipolipemiantes/farmacologia , Immunoblotting , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proliferadores de Peroxissomos/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Depuradores , Receptores Depuradores Classe B , Succinimidas/farmacologia , Fatores de Tempo , Fatores de Transcrição/antagonistas & inibidores , TransfecçãoRESUMO
BACKGROUND: Recombinant adenoviruses are an established tool for somatic gene transfer to multiple cell types in animals as well as in tissue culture. However, generation of adenoviruses expressing transgenes that are potentially toxic to the host cell line represents a practical problem. The aim of this study was to construct an adenoviral expression system that prevents transgene expression during the generation and propagation of the virus, and allows efficient gene transfer to lung and liver, major target organs of gene therapy. METHODS: Using the tet-off system we constructed tetracycline (tet) regulatable recombinant adenoviruses expressing the marker gene LacZ (Adtet-off.LacZ) as well as a secretory protein, human group IIA secretory phospholipase A(2) (Adtet-off.hsPLA(2)). Expression (Western blot, activity assay) was tested in vitro (HeLa cells), and in vivo by gene transfer to lung and liver. RESULTS: Without addition of tetracycline we demonstrated expression of LacZ (Adtet-off.LacZ) and sPLA(2) (Adtet-off.hsPLA(2)) in HeLa cells. Providing additional tet-transactivator (tTA) protein either by stable transfection or coinfection with a tTA-expressing adenovirus resulted in a further increase of LacZ and sPLA(2) expression. Transgene expression in vitro was eliminated by the addition of tetracycline to the culture medium. Adtet-off.LacZ and Adtet-off.hsPLA(2) allowed successful gene transfer in vivo to lung and liver. While the expression was highly efficient within the lungs, however, additional tTA was necessary to achieve high-level expression within liver. CONCLUSIONS: Tet-regulatable adenoviral expression systems may facilitate the construction of recombinant adenoviruses encoding potentially toxic transgenes and permit regulated transgene expression.
Assuntos
Adenoviridae/metabolismo , Técnicas de Transferência de Genes , Fígado , Pulmão , Fosfolipases A/genética , Tetraciclina/farmacologia , Transgenes , Adenoviridae/genética , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Marcação de Genes , Vetores Genéticos , Fosfolipases A2 do Grupo II , Células HeLa , Humanos , Óperon Lac/fisiologia , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipases A/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/fisiologiaRESUMO
The nuclear bile acid receptor FXR has been proposed to play a central role in the feedback repression of the gene encoding cholesterol 7 alpha-hydroxylase (CYP7A1), the first and rate-limiting step in the biosynthesis of bile acids. We demonstrate that FXR directly regulates expression of fibroblast growth factor-19 (FGF-19), a secreted growth factor that signals through the FGFR4 cell-surface receptor tyrosine kinase. In turn, FGF-19 strongly suppresses expression of CYP7A1 in primary cultures of human hepatocytes and mouse liver through a c-Jun N-terminal kinase (JNK)-dependent pathway. This signaling cascade defines a novel mechanism for feedback repression of bile acid biosynthesis and underscores the vital role of FXR in the regulation of multiple pathways of cholesterol catabolism in the liver.
Assuntos
Ácidos e Sais Biliares/biossíntese , Colesterol 7-alfa-Hidroxilase/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Hepatócitos/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Antracenos/farmacologia , Linhagem Celular , Células Cultivadas , Ácido Quenodesoxicólico/farmacologia , Colesterol 7-alfa-Hidroxilase/metabolismo , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/genética , Repressão Enzimática , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica , Humanos , Isoxazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores Citoplasmáticos e Nucleares , Proteínas Recombinantes/farmacologia , Elementos de Resposta , Fatores de Transcrição/agonistas , Fatores de Transcrição/genética , TransfecçãoRESUMO
Hepatoma cell lines serve as a suitable model to study hepatic clearance of lipoprotein-associated cholesteryl esters (CEs). The present study aimed at investigating holoparticle-association of and selective CE-uptake from human high density lipoprotein subclass 3 (HDL3) by non-malignant adult (Chang-liver) and non-malignant fetal (WRL-68) epithelial cell lines as well as a hepatocellular carcinoma (HUH-7) cell line. Binding properties of 125I-HDL3 at 4 and 37 degrees C were similar for all three cell lines while degradation rates were highest for Chang-liver cells. Calculating the selective uptake of HDL3-associated CEs as the difference between [3H]CE- and 125I-HDL3 cell-association revealed that the selective lipid uptake and holoparticle-association was similar in Chang-liver while in WRL-68 and HUH-7 cells pronounced capacity for lipid tracer uptake in excess of holoparticle uptake was measured. Using RT-PCR, Northern and Western blot analysis, as well as immunocytochemical technique pronounced expression of scavenger receptor class B, type I (SR-BI) but not SR-BII (a splice variant of SR-BI less efficient for selective CE-uptake than SR-BI) could be identified in HUH-7 and WRL-68 cells. A polyclonal antiserum raised against SR-BI significantly decreased cell-association of [3H]CE-HDL3 in HUH-7 and WRL-68. The present findings suggest that the capacity for selective cholesteryl ester-uptake from high density lipoprotein by malignant and normal epithelial cells from the liver depends on expression of the scavenger receptor class B, type I.
Assuntos
Antígenos CD36/metabolismo , Ésteres do Colesterol/metabolismo , Hepatócitos/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana , Receptores de Lipoproteínas , Sialoglicoproteínas , Animais , Células CHO , Carcinoma Hepatocelular/metabolismo , Cricetinae , Células Epiteliais/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana Lisossomal , Receptores Depuradores , Receptores Depuradores Classe B , Células Tumorais CultivadasRESUMO
Fibrates are normolipidemic drugs used in atherogenic dyslipidemia because of their ability to raise high density lipoprotein (HDL) and decrease triglyceride levels. They exert multiple effects on lipid metabolism by activating the peroxisome proliferator-activated receptor-alpha (PPAR-alpha), which controls the transcriptional regulation of genes involved in hepatic fatty acid, cholesterol, and lipoprotein metabolism. The hepatic expression of the scavenger receptor class B type I (SR-BI) plays a critical role in lipoprotein metabolism, mainly due to its ability to mediate selective cholesterol uptake. Because fibrates and PPAR-alpha agonists up-regulate SR-BI expression in human and murine macrophages, we tested whether fibrates raised a similar regulatory response on hepatic SR-BI expression in mice. Surprisingly, fibrate treatment suppressed SR-BI protein expression in the liver without changing steady state SR-BI mRNA levels. Decreased hepatic SR-BI protein expression correlated with enlarged HDL particle size. This effect was concomitant with down-regulation of CLAMP, a putative SR-BI-stabilizing protein found in the hepatic plasma membrane, which was also not associated to changes in CLAMP mRNA levels. The post-transcriptional regulatory effect of fibrates over hepatic SR-BI protein levels was dependent on PPAR-alpha expression, because it was absent in PPAR-alpha-deficient mice. Restoring hepatic SR-BI expression in fibrate-treated mice by recombinant adenoviral gene transfer abolished fibrate-mediated HDL particle size enlargement. This study describes a novel effect of fibrates on hepatic SR-BI expression providing an alternative mechanism by which this drug family modulates HDL metabolism in vivo.
Assuntos
Antígenos CD36/metabolismo , Regulação para Baixo/efeitos dos fármacos , Hipolipemiantes/farmacologia , Proteínas de Membrana , Receptores Imunológicos , Receptores de Lipoproteínas , Adenoviridae/genética , Animais , Antígenos CD36/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Depuradores , Receptores Depuradores Classe B , TransfecçãoRESUMO
Hypochlorous acid/hypochlorite (HOCl/OCl(-)), a potent oxidant generated in vivo by the myeloperoxidase-H(2)O(2)-chloride system of activated phagocytes, alters the physiological properties of high density lipoprotein (HDL) by generating a proatherogenic lipoprotein particle. On endothelial cells lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) and scavenger receptor class B, type I (SR-BI), act in concert by mediating the holoparticle of and selective cholesteryl ester uptake from HOCl-HDL. We therefore investigated the ligand specificity of HOCl-HDL to SR-BI-overexpressing Chinese hamster ovary cells. Binding of HOCl-HDL was saturable, and the degree of HOCl modification was the determining factor for increased binding affinity to SR-BI. Competition experiments further confirmed that HOCl-HDL binds with increased affinity to the same or overlapping domain(s) of SR-BI as does native HDL. Furthermore, SR-BI-mediated selective HDL-cholesteryl ester association as well as time- and concentration-dependent cholesterol efflux from SR-BI overexpressing Chinese hamster ovary cells were, depending on the degree of HOCl modification of HDL, markedly impaired. The most significant findings of this study were that the presence of very low concentrations of HOCl-HDL severely impaired SR-BI-mediated bidirectional cholesterol flux mediated by native HDL. The colocalization of immunoreactive HOCl-modified epitopes with apolipoprotein A-I along with deposits of lipids in serial sections of human atheroma shown here indicates that the myeloperoxidase-H(2)O(2)-halide system contributes to oxidative damage of HDL in vivo.